INTRODUCTION

Programmed cell death protein 1 (PD-1) is a major inhibitor of T cell responses expressed on activated T cells. It is also expressed on natural killer cells, B cells, regulatory T cells, T follicular helper cells, and myeloid cells (1). The current model supports that a key mechanism dampening antitumor immune responses is the up-regulation of PD-1 ligands in cancer cells and antigen-presenting cells (APCs) of the tumor microenvironment (TME), which mediate ligation of PD-1 on tumor-infiltrating CD8+ T cells, leading to the development of T incapable of generating antitumor responses (2). Therapeutic targeting of the PD-1 pathway with antibodies blocking the PD-1 receptor or its ligands induces expansion of oligoclonal CD8+ tumor-infiltrating lymphocytes that recognize tumor neoantigens (3). Thus, in the context of cancer, PD-1 is considered a major inhibitor of T effector cells, whereas on APC and cancer cells, emphasis has been placed on the expression of PD-1 ligands. PD-1 ligand-1 expression in the TME is often a prerequisite for patient enrollment to clinical trials involving blockade of the PD-1 pathway. However, responses do not always correlate with PD-L1 expression, and it remains incompletely understood how the components of the PD-1:PD-L1/2 pathway suppress antitumor immunity.

Recent studies indicated that PD-1 can be induced by Toll-like receptor (TLR) signaling in macrophages (M) and negatively correlates with M1 polarization (4). PD-1 expression in macrophages plays a pathologic role by suppressing the innate inflammatory response to sepsis (5) and inhibiting Mycobacterium tuberculosis phagocytosis in active tuberculosis (6). Our knowledge about the function of PD-1 on myeloid cells in the context of cancer is very limited. However, similar to its role in infections, PD-1 expression inversely correlates with M1 polarization and phagocytic potency of tumor-associated M (TAM) against tumor (7, 8). The mechanisms of PD-1 expression in myeloid cells and the role of PD-1expressing myeloid cells in tumor immunity remain unknown.

The rapid increase in myeloid cell output in response to immunologic stress is known as emergency myelopoiesis. Terminally differentiated myeloid cells are essential innate immune cells and are required for the activation of adaptive immunity. Strong activation signals mediated by pathogen-associated molecular pattern or danger-associated molecular pattern molecules lead to a transient expansion and subsequent differentiation of myeloid progenitors to mature monocytes and granulocytes to protect the host. In contrast, during emergency myelopoiesis mediated by continuous low-level stimulation mediated by cancer-derived factors and cytokines, bone marrow common myeloid progenitors (CMPs) but, predominantly, granulocyte/macrophage progenitors (GMPs) undergo modest expansion with hindered differentiation, and a fraction of myeloid cells with immunosuppressive and tumor-promoting properties, named myeloid-derived suppressor cells (MDSCs), accumulates. MDSCs suppress CD8+ T cell responses by various mechanisms (9). In the mouse, MDSCs consist of two major subsets, CD11b+Ly6ChiLy6G (thereafter named CD11b+Ly6C+) monocytic (M-MDSC) and CD11b+Ly6CloLy6G+ (hereafter named CD11b+Ly6G+) polymorphonuclear (PMN-MDSC) (10). These cells have similar morphology and phenotype to normal monocytes and neutrophils but distinct genomic and biochemical profiles (9). In humans, in addition to M-MDSC and PMN-MDSC, a small subset of early-stage MDSC has been identified (10).

Although PMN-MDSCs represent the major subset of circulating MDSC, they are less immunosuppressive than M-MDSC when assessed on a per cell basis (1113). Current views support the two-signal requirement for MDSC function. The first signal controls MDSC generation, whereas the second signal controls MDSC activation, which depends on cues provided by the TME and promotes MDSC differentiation to TAM (14). Proinflammatory cytokines and endoplasmic reticulum stress response in the TME contribute to pathologic myeloid cell activation that manifests as weak phagocytic activity, increased production of reactive oxygen species and nitric oxide (NO) and expression of arginase-1 (ARG1), and convert myeloid cells to MDSC (9). MDSCs are associated with poor outcomes in many cancer types in patients and negatively correlate with response to chemotherapy, immunotherapy, and cancer vaccines (1519).

In the present study, we examined how PD-1 regulates the response of myeloid progenitors to cancer-driven emergency myelopoiesis and its implications on antitumor immunity. We determined that myeloid progenitors, which expand during cancer-driven emergency myelopoiesis, express PD-1 and PD-L1. PD-L1 was constitutively expressed on CMPs and GMPs, whereas PD-1 expression displayed a notable increase on GMPs that arose during tumor-driven emergency myelopoiesis. PD-1 was also expressed on tumor-infiltrating myeloid cellsincluding M-MDSCs and PMN-MDSCs, CD11b+F4/80+ M, and CD11c+major histocompatibility complex class II-positive (MHCII+) dendritic cells (DCs) in tumor-bearing miceand on MDSCs in patients with refractory lymphoma. Ablation of PD-1 signaling in PD-1 knockout (KO) mice prevented GMP accumulation and MDSC generation and resulted in increase of Ly6Chi effector monocytes, M and DC. We generated mice with conditional targeting of the Pdcd1 gene (PD-1f/f) and selectively eliminated PD-1 in myeloid cells or T cells. Compared with T cellspecific ablation of PD-1, myeloid-specific PD-1 ablation more effectively decreased tumor growth in various tumor models. At a cellular level, only myeloid-specific PD-1 ablation skewed the myeloid cell fate commitment from MDSC to effector Ly6Chi monocytes M and DC and induced T effector memory (TEM) cells with improved functionality. Our findings reveal a previously unidentified role of the PD-1 pathway and suggest that skewing of myeloid cell fate during emergency myelopoiesis and differentiation to effector APCs, thereby reprogramming T cell responses, might be a key mechanism by which PD-1 blockade mediates antitumor function.

For our studies, we selected the murine B16-F10 melanoma tumor model because it has been informative in dissecting mechanisms of resistance to checkpoint immunotherapy (20). First, we examined whether B16-F10 induces tumor-driven emergency myelopoiesis similarly to the MC17-51 fibrosarcoma, a mouse tumor model well established to induce cancer-driven emergency myelopoiesis (21). We assessed the expansion of myeloid progenitors in the bone marrow and the increase of CD11b+CD45+ myeloid cells in the spleen and tumor (figs. S1 and S2). Both tumor types induced increase of myeloid progenitors in the bone marrow and systemic increase of CD45+CD11b+ myeloid cells (fig. S3), providing evidence that B16-F10 melanoma is an appropriate tumor model to study tumor-driven emergency myelopoiesis and its consequences in tumor immunity. In the spleen of nontumor-bearing mice, few myeloid cells constitutively expressed very low levels of PD-L1, whereas PD-1 was very low to undetectable (Fig. 1, A and B). In B16-F10 tumor-bearing mice, expression of PD-1 and PD-L1 was up-regulated on myeloid cells of the spleen (Fig. 1, C to F). PD-1 and PD-L1 were also expressed on myeloid cells at the tumor site (Fig. 1, G to I). All subsets of myeloid cells expanding in tumor-bearing mice including M-MDSCs, PMN-MDSCs, CD11b+F4/80+ Ms, and CD11c+MHCII+ DCs expressed PD-1 (Fig. 1, D and G). Kinetics studies of PD-1 expression on myeloid cells in the spleen of tumor-bearing mice showed a gradual increase over time (Fig. 1, J to M).

(A and B) Expression of PD-1 and PD-L1 on CD11b+Ly6C+ monocytes and CD11c+MHCII+ DC in the spleen of nontumor-bearing C57BL/6 mice. FMO, fluorescence minus one. (C) C57BL/6 mice were inoculated with B16-F10 mouse melanoma, and at the indicated time points, expression of PD-1 was examined by flow cytometry in the spleen after gating on the indicated myeloid populations; contour plots depicting the percentage of positive cells are shown. On day 16 after tumor inoculation, expression of PD-1 and PD-L1 was assessed in the spleen (D) and the tumor site (G) after gating on the indicated myeloid populations. (D and G) Fluorescence-activated cell sorting (FACS) histograms and contour plots depicting the percentage of positive cells and bar graphs (E, F, H, and I) of mean SEM positive cells. Results are representative of 12 independent experiments with six mice per group. (J to M) Kinetics of PD-1 up-regulation on CD11b+Ly6C+, CD11b+Ly6G+, CD11b+F4/80+, and CD11c+MHCII+ of the spleen after tumor inoculation. **P < 0.01, ***P < 0.005, ****P < 0.001.

Because myeloid cells that give rise to MDSC and TAM are generated from myeloid progenitors in the bone marrow during tumor-driven emergency myelopoiesis, we examined PD-1 and PD-L1 expression in these myeloid progenitors. In nontumor-bearing mice, PD-1 was detected at very low levels on GMPs (Fig. 2A), whereas PD-L1 was constitutively expressed in CMPs but mostly on GMPs (Fig. 2B). In tumor-bearing mice, PD-L1 was up-regulated in CMPs and GMPs, and its expression levels remained elevated during all assessed time points (Fig. 2, F to J). PD-1 expression was induced on CMPs but more prominently on GMPs (Fig. 2, C to I). Kinetics studies showed that PD-1 expression on GMPs peaked early after tumor inoculation (Fig. 2, C, E, and I), at a time point when tumor growth was not yet measurable. Thus, induction of PD-1 expression in myeloid progenitors is an early event during tumor development.

(A and B) Expression of PD-1 and PD-L1 on CMPs and GMPs of nontumor-bearing mice. (C to J) C57BL/6 mice were inoculated with B16-F10 mouse melanoma, and expression of PD-1 and PD-L1 on CMPs and GMPs was examined on days 9, 12, 14, and 16 after implantation. FACS histograms (C and F) and contour plots (D, E, G, and H) indicating the percentage of positive cells and bar graphs of mean SEM positive cells (I and J) are shown. Results are representative of four independent experiments with six mice per group. (K and L) Kinetics of PD-1 (K) and PD-L1 (L) expression on CMPs (blue) and GMPs (orange) during tumor-driven emergency myelopoiesis. Results are representative of four separate experiments with six mice per group. *P < 0.05, ***P < 0.005, ****P < 0.001.

To determine whether PD-1 expression on GMPs was mediated by growth factors regulating emergency myelopoiesis, we cultured bone marrow cells from nontumor-bearing mice with granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony growth factor (GM-CSF), and the TLR4 ligand lipopolysaccharide. PD-1 that was constitutively expressed at low levels in GMPs was up-regulated by culture with each of these factors (fig. S4A), consistent with our findings that PD-1 expression was rapidly induced on GMPs of tumor-bearing mice in vivo (Fig. 2, C, E, and I). Quantitative polymerase chain reaction (qPCR) in purified Linneg bone marrow cells showed that PD-1 mRNA was constitutively expressed in myeloid progenitors and was up-regulated by culture with G-CSF or GM-CSF (fig. S4B). Together, these in vivo and in vitro studies provide evidence that PD-1 expression on myeloid progenitors is regulated by a direct cell-intrinsic effect of factors driving cancer-mediated emergency myelopoiesis.

To examine whether PD-1 was expressed in MDSCs in humans, we used samples from healthy donors and patients with malignant non-Hodgkins lymphoma (NHL) (figs. S5 and S6). A high level of PD-1expressing M-MDSCs was detected in the peripheral blood of three patients with treatment-refractory NHL but not in two patients who responded to treatment or five healthy donors (fig. S6). These results show that PD-1 expression is detected in human MDSCs and serve as a paradigm, suggesting that PD-1 expression in MDSCs of patients with cancer might be a clinically relevant event.

To examine whether PD-1 might have an active role in tumor-induced stress myelopoiesis, we used PD-1deficient (PD-1/) mice. PD-1 deletion, which resulted in decreased tumor growth (Fig. 3, A and B), substantially altered tumor-induced stress myelopoiesis (Fig. 3, C to E). Although accumulation of CMPs was comparable, accumulation of GMPs was significantly diminished in PD-1/ mice (Fig. 3, C and D), indicating that GMPs might be a key target on which PD-1 mediated its effects on myeloid progenitors (Fig. 3E). Kinetics studies showed sustained GMP expansion in wild-type (WT) tumor-bearing mice. In contrast, in PD-1/ tumor-bearing mice, GMPs displayed a rapid expansion and subsequent decline (fig. S7). In parallel, in PD-1/ mice, there was an increase of differentiated CD11b+Ly6Chi monocytic cells not only in the tumor (Fig. 3H) but also in the spleen and the small intestine, which also displayed an increase in CD11c+MHCII+ DCs (Fig. 3, F and G). Moreover, at these sites, there was a significant increase of the CD11b+Ly6C+/CD11b+Ly6G+ ratio (Fig. 3, I to K), indicating a shift of myelopoiesis output toward monocytic lineage dominance. These Ly6Chi monocytes, CD11b+F4/80+ Ms, and CD11c+MHCII+ DCs in PD-1/ tumor-bearing mice expressed interferon (IFN) regulatory factor 8 (IRF8), and all myeloid subsets had elevated expression of the retinoic acid receptor-related orphan receptor (RORC or ROR) (Fig. 3, L to N, and fig. S8). Similar results were observed in two additional tumor models, the MC38 colon adenocarcinoma and the MC17-51 fibrosarcoma model (fig. S9), both of which induced cancer-driven emergency myelopoiesis (fig. S3).

(A and B) WT and PD-1/ mice were inoculated with B16-F10 melanoma, and tumor size was monitored daily (A). Mice were euthanized on day 16, and tumor weight was measured (B). Data shown are means SEM of six mice per group and are representative of six independent experiments. (C) Mean percentages SEM of LSK (Linneg, Sca1pos, CD127neg, c-kitpos) and LK (Linneg, Sca1neg, CD127neg, c-kitpos) hematopoietic precursors, CMP, and GMP in the bone marrow of nontumor-bearing and tumor-bearing WT and PD-1/ mice. GMPs in PD-1/ mice were significantly lower compared with GMPs in WT mice (**P < 0.01). (D) Representative contour plots of FACS analysis for CMP and GMP in the bone marrow of tumor-bearing WT and PD-1/ mice. (E) Schematic presentation of myeloid lineage differentiation. The arrowhead indicates GMP, the key target population of PD-1 during emergency myelopoiesis. HSC, hematopoietic stem cells; MPP, multi-potent progenitor; MDP, monocyte/macrophages and DC precursors; CDP, common dendritic cell progenitors; CLP, common lymphoid progenitors. (F to H) Mean percentages of CD45+CD11b+, CD11b+Ly6C+, CD11b+Ly6G+, and CD11c+MHCII+ in the spleen (F), small intestine (G), and B16-F10 site (H) of tumor-bearing WT and PD-1/ mice. (I to K) Representative plots of FACS analysis for CD11b+Ly6Chi and CD11b+Ly6C+/CD11b+Ly6G+ ratio in the spleen (I), small intestine (J), and B16-F10 site (K). (L to N) Mean percentages SEM of RORC and IRF8 expressing CD11b+Ly6C+, CD11b+Ly6G+, CD11b+F4/80+, and CD11c+MHCII+ myeloid cells within the CD45+CD11b+ gate in the spleen (L), small intestine (M), and B16-F10 site (N). Data from one representative experiment of three independent experiments with six mice per group are shown. (O and P) Diminished suppressive activity (O) and NO production (P) of CD11b+Ly6C+ cells isolated from PD-1/ tumor-bearing mice. CD11b+Ly6C+ cells were isolated from tumor-bearing WT and PD-1/ mice and cultured at various ratios with OTI splenocytes stimulated with OVA257264. Data show means SEM of one representative of two experiments (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.001).

IRF8 regulates myeloid cell fate to monocyte/macrophage and DC differentiation versus granulocyte differentiation (22, 23), explaining the increase of CD11b+Ly6C+/CD11b+Ly6G+ ratio that we observed in tumor-bearing PD-1 KO mice. IRF8 is designated as one of the terminal selectors that control the induction and maintenance of the terminally differentiated state of these myeloid cells (22, 23). Moreover, IRF8 shifts the fate of myeloid cells away from immature MDSC, which are characterized by a restriction in IRF8 expression (24, 25). Retinoid-related orphan nuclear receptors not only are required for myelopoiesis and are mediators of the inflammatory response of effector Ly6Chi monocytes and macrophages (21, 26) but also can be expressed by MDSC (21). For these reasons, we examined the functional properties of CD11b+Ly6C+ cells in PD-1/ tumor-bearing mice. A key mechanism by which CD11b+Ly6C+ M-MDSCs mediate suppression of T cell responses involves the production of NO (27). We assessed the immunosuppressive function and found diminished NO production and diminished suppressor capacity of CD11b+Ly6C+ myeloid cells isolated from tumor-bearing PD-1/ mice compared with their counterparts isolated from tumor-bearing WT control mice (Fig. 3, O and P). Thus, PD-1 ablation switches the fate and function of myeloid cells away from immunosuppressive MDSC and promotes the generation of differentiated monocytes, M, and DC. The expansion of CD11b+Ly6Chi monocytes, the increase of the CD11b+Ly6C+/CD11b+Ly6G+ ratio, and the up-regulation of RORC in myeloid cells of the spleen of PD-1/ mice were already observed on day 9 after tumor inoculation, when tumors were not yet measurable, and on day 12, when tumors in WT and PD-1/ mice had comparable size (fig. S10). These results indicate that the effects of PD-1 ablation on the myeloid compartment of PD-1/ tumor-bearing mice preceded the differences in tumor growth.

To determine the potential therapeutic relevance of these findings, we examined whether changes in the myeloid compartment might be detected during treatment with PD-1blocking antibody. Compared with the control treatment group, mice receiving antiPD-1 antibody (fig. S11A) had diminished accumulation of GMP in the bone marrow (fig. S11B) and increased expansion of Ly6C+ monocytes and DC in the tumor site (fig. S11D), with effector features characterized by the expression of RORC, IRF8, and IFN- (fig. S11, E to G and I). In contrast, cells expressing interleukin-4 receptor (IL-4Ra), a marker of MDSC (10, 28), were significantly decreased (fig. S11H). Thus, treatment with antiPD-1blocking antibody promotes the differentiation of myeloid cells with effector features while suppressing expansion of MDSC in tumor-bearing mice.

To determine whether these changes on myeloid cell fate in PD-1/ mice were mediated by myeloid cellintrinsic effects of PD-1 ablation or by the effects of PD-1neg T cells on myeloid cells, we generated mice with conditional targeting of Pdcd1 gene (PD-1f/f) (fig. S12A) and crossed them with mice expressing cre recombinase under the control of the lysozyme (LysM) promoter to induce selective ablation of the Pdcd1 gene in myeloid cells (PD-1f/fLysMcre) or with mice expressing cre recombinase under the control of the CD4 promoter to induce selective ablation of the Pdcd1 gene in T cells (PD-1f/fCD4cre) (fig. S12, B and C). In PD-1f/fLysMcre mice, tumor growth was significantly diminished (Fig. 4, A and B), indicating that despite the preserved PD-1 expression in T cells, myeloid-specific PD-1 ablation in PD-1f/fLysMcre mice was sufficient to inhibit tumor growth. Tumor-driven emergency myelopoiesis was selectively affected in PD-1f/fLysMcre mice. Although myeloid-specific PD-1 ablation resulted in expansion of CMPs, accumulation of GMPs was prevented (Fig. 4C). In contrast, no change on cancer-driven emergency myelopoiesis was detected in PD-1f/fCD4cre mice, which had comparable expansion of CMP and GMP to PD-1f/f control mice (Fig. 5A).

(A and B) PD-1f/f, PD-1f/fLysMcre, and PD-1/ mice were inoculated with B16-F10 melanoma, and tumor size was monitored daily (A). After mice were euthanized, tumor weight was measured (B). (C) Mean percentages SEM of CMP and GMP in the bone marrow of tumor-bearing PD-1f/f and PD-1f/fLysMcre mice. (D) Mean percentages SEM of CD11b+CD45+ cells and CD11b+Ly6C+, CD11b+Ly6G+, CD11b+F4/80+, and CD11c+MHCII+ myeloid subsets in the spleen of tumor-bearing mice. (E) Mean percentages SEM of CD11b+CD45+, CD11b+Ly6C+, and CD11b+Ly6G+ cells and (F) representative contour plots of FACS analysis for CD11b+CD45+ and CD11b+Ly6C+ cells at the tumor site in PD-1f/f, PD-1f/fLysMcre, and PD-1/ mice. (G) Mean percentages SEM of CD16/CD32+, CD86+, CD88+, and CD80+ cells and IFN-expressing myeloid cell subsets within the CD45+CD11b+ gate in B16-F10 tumors from PD-1f/f, PD-1f/fLysMcre, and PD-1/ mice. (H) Mean percentages SEM and (I) FACS histograms of IL-4Ra, CD206, and ARG1 expression in CD11b+Ly6C+, CD11b+Ly6G+, CD11b+F4/80+, and CD11c+MHCII+ myeloid cells within the CD11b+CD45+ gate in the spleen of tumor-bearing PD-1f/f, PD-1f/fLysMcre, and PD-1/ mice. Data are from one representative of three independent experiments with six mice per group are shown in all the panels (*P < 0.05, **P < 0.01, ***P < 0.005, and ****P < 0.001).

PD-1f/f and PD-1f/fCD4cre mice were inoculated with B16-F10 melanoma. (A) On day 16, mice were euthanized, and bone marrow CMPs and GMPs were examined by flow cytometry. Mean percentages SEM of CMP or GMP are shown. (B and C) Tumor size was assessed every other day from inoculation (B). On the day of euthanasia, tumor weight was measured (C). (D) Mean percentages SEM of CD11b+CD45+ cells and CD11b+Ly6C+ and CD11b+Ly6G+ populations within the CD11+CD45+ gate in the spleen. (E) Mean percentages SEM of CD11b+CD45+ cells and CD11b+Ly6C+, CD11b+Ly6G+, CD11b+F4/80+, and CD11c+MHCII+ cells within the CD11b+CD45+ gate in the tumor site. (F) Mean percentages SEM of CD16/CD32+, CD86+, CD88+, CD80+, and IFN- expression in the indicated myeloid subsets (CD11b+Ly6C+, CD11b+Ly6G+, CD11b+F4/80+, and CD11c+MHCII+) within the CD11b+CD45+ gate in the tumor site. (G to J) Mean percentages SEM of CD4+ and CD8+ TCM and TEM (G), as well as IFN-, IL-2, and IL-17 (H to J) expression in CD4+ and CD8+ TEM and TCM at the tumor site, and respective contour plots (K to M). Results are from one representative of two independent experiments with six mice per group are shown (*P < 0.05 and **P < 0.01).

Myeloid-specific PD-1 ablation in PD-1f/fLysMcre mice not only shifted the differentiation of CD11b+Ly6C+ and CD11b+Ly6G+ myeloid subsets and increased the CD11b+Ly6C+/CD11b+Ly6G+ ratio in the spleen and tumor site as in PD-1/ mice (Fig. 4, D to F) but also resulted in a notably different immunological profile of CD11b+Ly6C+ monocytic myeloid cells, consistent with effector myeloid function as indicated by the expression of effector myeloid cell markers including CD80, CD86, CD16/32 (Fc receptor II/III), and CD88 (C5aR) (Fig. 4G). Consistent with the improved function of myeloid cells, PD-1f/fLysMcre mice also had higher levels of IFN-expressing CD11b+Ly6Chi monocytes and CD11b+F4/80+ Ms (Fig. 4G and fig. S13, A and B) and increase of IRF8+ and RORC+ CD11b+Ly6Chi monocytes (fig. S13, C and D). In contrast, cells expressing IL-4Ra, CD206, and ARG1which are markers of MDSC, immunosuppressive neutrophils, and tolerogenic DCs (2933)were diminished (Fig. 4, H and I). Thus, myeloid-intrinsic PD-1 ablation skews the fate of myeloid cells away from immunosuppressive MDSCs; promotes the differentiation of functional effector monocytes, Ms, and DCs; and has a decisive role in systemic antitumor immunity despite PD-1 expression in T cells.

We studied antitumor responses in mice with T cellspecific PD-1 ablation and found that PD-1f/fCD4cre mice had diminished antitumor protection (Fig. 5, B and C). Consistent with the causative role of myeloid cellspecific PD-1 targeting in the differentiation and function of myeloid cells, T cellspecific PD-1 ablation did not induce expansion of CD11b+CD45+ leukocytes, CD11b+F4/80+ Ms, and CD11c+MHCII+ DCs and increase of CD11b+Ly6C+/CD11b+Ly6G+ ratio (Fig. 5, D and E) or immunological features of functional effector myeloid cells (Fig. 5F) in PD-1f/fCD4cre tumor-bearing mice, compared with control tumor-bearing mice. Moreover, despite PD-1 ablation, tumor-bearing PD-1f/fCD4cre mice did not have quantitative differences in tumor-infiltrating TEM cells compared with control tumor-bearing mice (Fig. 5G) or features of enhanced effector function as determined by assessment of cytokine-producing cells (Fig. 5, H to M).

Similar outcomes to those observed with B16-F10 tumor in the differentiation of myeloid cells toward myeloid effectors versus MDSC were obtained when PD-1f/fLysMcre and PD-1f/fCD4cre mice were inoculated with MC38 colon adenocarcinoma cells (Fig. 6, B to I). Moreover, PD-1f/fLysMcre but not PD-1f/f CD4cre mice inoculated with MC38 had functional differences in tumor-infiltrating TEM and T central memory (TCM) cells compared with control tumor-bearing mice (Fig. 6, J to L). In the context of this highly immunogenic tumor, PD-1 ablation in myeloid cells resulted in complete tumor eradication, whereas mice with PD-1 ablation in T cells showed progressive tumor growth (Fig. 6A). Together, these results suggest that by preventing the differentiation of effector myeloid cells and promoting generation of MDSC, myeloid-specific PD-1 expression has a decisive role on T cell function. Thus, although PD-1 is an inhibitor of T cell responses (2, 34, 35), ablation of PD-1 signaling in myeloid cells is an indispensable requirement for induction of systemic antitumor immunity in vivo.

(A) PD-1f/f, PD-1f/fCD4cre, and PD-1f/fLysMcre mice were inoculated with MC38 colon adenocarcinoma, and tumor size was monitored daily. Mice were euthanized on day 21, and mean percentages SEM of CD45+CD11b+ cells and CD11b+Ly6C+, CD11b+Ly6G+, CD11b+F4/80+, and CD11c+MHCII+ myeloid subsets in the spleen (B) and tumor site (C) were determined. (D) Mean percentages SEM of RORC- and IRF8-expressing CD11b+Ly6C+, CD11b+Ly6G+, CD11b+F/480+, and CD11c+MHCII+ myeloid cells and (E) mean percentages SEM of ARG1, IL-4Ra, CD88, and CD80 cells within the same myeloid subsets in the spleen. (F and G) Representative flow cytometry plots for RORC and IRF8 expression. (H) Mean percentages SEM and (I) representative flow cytometry plots of IFN- and ARG1-expressing CD11b+Ly6C+ and CD11b+Ly6G+ myeloid cells at the tumor site. (J to L) Mean percentages SEM of CD4+ and CD8+ TCM and TEM cells (J) and IFN-expressing CD4+ and CD8+ TEM and TCM at the tumor site (K) and respective contour plots (L). Data are from one representative of three experiments with six mice per group (*P < 0.05, **P < 0.01, and ***P < 0.001).

To further investigate the direct effects of PD-1 on myeloid cell fate in the absence of T cells, we used recombination activating gene 2 (RAG2) KO mice (lacking mature T cells and B cells). Treatment of RAG2 KO tumor-bearing mice with antiPD-1blocking antibody resulted in decreased accumulation of GMPs during tumor-driven emergency myelopoiesis (fig. S14A), myeloid cell expansion in the spleen and tumor site (fig. S14, B and C), and enhanced generation of effector myeloid cells (fig. S14, D to G), providing evidence that blockade of PD-1mediated signals skews myeloid lineage fate to myeloid effector cells in a myeloid cellintrinsic and T cellindependent manner. In RAG2 KO mice treated with antiPD-1 antibody, despite the absence of T cells, a decrease of tumor growth was also observed (fig. S14, H and I), suggesting that ablation of PD-1 signaling promotes myeloid-specific mechanisms that induce tumor suppression, one of which might involve increased phagocytosis (8).

To understand mechanisms that might be responsible for the significant differences of myeloid cell fate commitment induced by myeloid-specific PD-1 targeting, we examined whether PD-1deficient bone marrow myeloid progenitors might have distinct signaling responses to the key hematopoietic growth factors that mediate cancer-driven emergency myelopoiesis, which also induced PD-1 expression in GMP during in vitro culture. To avoid any potential impact of bone marrowresiding PD-1/ T cells or mature myeloid cells on the signaling responses of myeloid progenitors, we used Linneg bone marrow from PD-1f/fLysMcre mice because LysMcre is expressed in CMPs and GMPs (36), allowing us to take advantage of the selective deletion of PD-1 in these myeloid progenitors. PD-1deficient GMPs (fig. S15) had enhanced activation of extracellular signalregulated kinase 1/2 (Erk1/2), mammalian target of rapamycin complex 1 (mTORC1), and signal transducer and activator of transcription 1 (STAT1) in response to G-CSF, a main mediator of emergency myelopoiesis (37, 38). These results are notable because each of these signaling targets has a decisive role in the differentiation and maturation of myeloid cells while preventing the generation of immature immunosuppressive MDSC (3942). These findings indicate that PD-1 might affect the differentiation of myeloid cells by regulating the fine tuning of signaling responses of myeloid progenitors to hematopoietic growth factors that induce myeloid cell differentiation and lineage fate determination during emergency myelopoiesis.

Metabolism has a decisive role in the fate of hematopoietic and myeloid precursors. Stemness and pluripotency are regulated by maintenance of glycolysis (43). Switch from glycolysis to mitochondrial metabolism and activation of oxidative phosphorylation and trichloroacetic acid (TCA) cycle are associated with differentiation (44). This is initiated by glycolysis-mediated mitochondrial biogenesis and epigenetic regulation of gene expression (43). The structural remodeling of the mitochondrial architecture during differentiation is characterized by increased replication of mitochondrial DNA to support production of TCA cycle enzymes and electron transport chain subunits, linking mitochondrial metabolism to differentiation (45).

We examined whether PD-1 ablation, which promoted the differentiation of myeloid cells in response to tumor-mediated emergency myelopoiesis, might affect the metabolic properties of myeloid precursors. Linneg bone marrow myeloid precursors were cultured with the cytokines G-CSF/GM-CSF/IL-6 that drive tumor-mediated emergency myelopoiesis in cocktail (Fig. 7, A and B) or individually (Fig. 7, C and D). Hematopoietic stem cell differentiation was documented by decrease of Linneg, which was more prominent in the cultures of PD-1deficient bone marrow cells, and coincided with increase of CD45+CD11b+ cells (Fig. 7, A and B). Ly6C+ monocytic cells dominated in the PD-1f/fLysMcre cultures, whereas Ly6G+ granulocytes were decreasing compared with PD-1f/f control cultures (Fig. 7, C and D), providing evidence for a cell-intrinsic mechanism of PD-1deficient myeloid precursors for monocytic lineage commitment. Glucose uptake, but more prominently, mitochondrial biogenesis, was elevated in PD-1deficient CMP and GMP (Fig. 7, E and F). Bioenergetics studies showed that PD-1deficient cells developed robust mitochondrial activity (Fig. 7G) and increase of oxygen consumption rate (OCR)/extracellular acidification rate (ECAR) ratio during culture (Fig. 7H), indicating that mitochondrial metabolism progressively dominated over glycolysis. This bioenergetic profile is consistent with metabolism-driven enhanced differentiation of hematopoietic and myeloid precursors (45, 46).

(A and B) Linneg bone marrow from PD-1f/f and PD-1f/fLysMcre mice was cultured with GM-CSF, G-CSF, and IL-6 for the indicated time intervals. Mean percentages SEM of CD11b+CD45+ (A) and Linneg cells (B) are shown. (C and D) Bone marrow cells purified as in (A) and (B) were cultured with the indicated growth factors, and mean percentages SEM of CD11b+Ly6C+ and CD11b+Ly6G+ cells were examined after 48 hours of culture. (E to H) Bone marrow cells were prepared and cultured as in (A) and (B), and at 48 hours of culture, glucose uptake was assessed using 2-[N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino]-2-Deoxyglucose (2-NBDG) (E), and mitochondrial biogenesis was assessed by MitoGreen staining and flow cytometry (F). (G) At 24, 48, and 72 hours of culture, OCR and ECAR were measured by a Seahorse extracellular flux analyzer, and mitostress responses at each time point of culture were examined. (H) OCR/ECAR ratio was measured at these time points, and the increase of OCR/ECAR ratio during stimulation was calculated. (I) Linneg bone marrow cells from PD-1f/f and PD-1f/fLysMcre mice were cultured with G-CSF and GM-CSF for 48 hours, and metabolite analysis was performed by mass spectrometry. The unsupervised hierarchical clustering heat map of the top 50 metabolites is shown. (J) At 24, 48, and 72 hours of culture with G-CSF and GM-CSF, mRNA was extracted and analyzed for the expression of the indicated genes by qPCR. Results of the 48-hour culture are shown and are presented as the fold increase over the mRNA level expressed by PD-1f/f cells. Results are from one of three independent experiments. (K to M) At 24 hours of culture with GM-CSF, G-CSF, or IL-6, the content of neutral lipid droplets, including triglycerides and cholesterol esters, was assessed by flow cytometry using boron-dipyrromethene (BODIPY) 493/503. Mean percentages SEM (K) of BODIPY 493/503positive cells within the CD11b+CD45+ gate, representative contour plots (L), and histograms of FACS analysis (M) are shown. (N) PD-1f/f and PD-1f/fLysMcre DC were differentiated in the presence of B16-F10 tumor supernatant, and the content of neutral lipids was assessed. Mean percentage SEM of BODIPY 493/503positive DC within the CD45+CD11b+ gate is shown. Results are representative of three experiments. *P < 0.05, **P < 0.01, and ***P < 0.005.

We performed unbiased global metabolite analysis to determine whether PD-1deficient myeloid precursors developed a distinct metabolic program. Compared with control, PD-1deficient cells had elevated metabolic intermediates of glycolysis and pentose phosphate pathway (PPP), acetylcoenzyme A (coA), and the TCA cycle metabolites citrate and -ketoglutarate, but the most prominent difference was the elevated cholesterol (Fig. 7I, figs. S16 and S17, and table S1). Abundant cytosolic acetyl-coA can be used for fatty acid and cholesterol biosynthesis (fig. S17) (43). Moreover, mTORC1 activates de novo cholesterol synthesis via sterol regulatory element-binding protein 1 (SREBP1), which regulates transcription of enzymes involved in cholesterol synthesis (47, 48). Because acetyl-coA was elevated (Fig. 7I and fig. S17) and mTORC1 activation was enhanced in PD-1deficient myeloid progenitors in response to growth factors driving emergency myelopoiesis (fig. S15), we examined whether activation of the mevalonate pathway that induces cholesterol synthesis (fig. S18A) might be involved. In PD-1deficient myeloid progenitors cultured with growth factors of emergency myelopoiesis, mRNA of genes regulating cholesterol synthesis and uptake was increased, mRNA of genes promoting cholesterol metabolism was decreased (Fig. 7J and fig. S18B), whereas cellular cholesterol and neutral lipid content was elevated (Fig. 7, K to M). PD-1deficient DC not only differentiated in vitro in the presence of B16-F10 tumor supernatant but also had a significant increase of cholesterol and neutral lipids compared with similarly differentiated DC from control mice (Fig. 7N). Consistent with these in vitro findings, glucose uptake and content of cholesterol and neutral lipids were elevated in GMPs of tumor-bearing PD-1 KO mice compared with control mice at days 7 or 9 after tumor inoculation, respectively, when tumors were not yet detectable or tumors in WT and PD-1 mice had equal size (fig. S19). Thus, features associated with metabolism-driven differentiation of myeloid progenitors are enhanced early in tumor-bearing PD-1 KO mice.

In addition to cholesterol synthesis, mevalonate also leads to the synthesis of isoprenoids, including geranylgeranyl pyrophosphate (GGPP) (fig. S17), which is required for protein geranylgeranylation catalyzed by geranylgeranyltransferase and has an active role in the up-regulation of RORC expression (49). Our metabolite analysis showed increased GGPP (Fig. 7I), providing a mechanistic explanation for the up-regulation of RORC in PD-1deficient myeloid cells. Cholesterol accumulation is associated with skewing of hematopoiesis toward myeloid lineage and monocytosis, induces a proinflammatory program in monocytes/macrophages and DC, and amplifies TLR signaling (5052). Together, these results unravel a previously unidentified role of PD-1 targeting in regulating myeloid lineage fate commitment and proinflammatory differentiation of monocytes, macrophages, and DC during tumor-driven emergency myelopoiesis, through metabolic reprogramming.

Previously, it was determined that monocyte/macrophage terminal differentiation is controlled by the combined actions of retinoid receptors and the nuclear receptor peroxisome proliferatoractivated receptor (PPAR), which is regulated by cholesterol and promotes gene expression and lipid metabolic processes, leading to terminal macrophage differentiation (26, 53). Because our in vitro studies showed that PD-1deficient myeloid progenitors developed a distinct metabolic program with elevated cholesterol metabolism, we examined whether PD-1 ablation might alter the expression of PPAR in addition to RORC. We found that the expression of PPAR was elevated in CD11b+Ly6C+ monocytic cells and M isolated from tumors of PD-1/ and PD-1f/fLysMcre mice (Fig. 8, A to C). Because PD-1deficient myeloid progenitors developed robust mitochondrial activity during culture in vitro (Fig. 7, G and H) and PPAR is involved in mitochondrial function (53), we examined whether myeloid cells in tumor-bearing mice have improved mitochondrial metabolism, a feature that has an important role in supporting antitumor function of other immune cells (54). Monocytes, M, and DC isolated from tumor of PD-1/, and PD-1f/fLysMcre mice had increased mitochondrial membrane potential compared with myeloid cells from control tumor-bearing mice, consistent with enhanced mitochondrial metabolism (Fig. 8, D to G).

(A to C) Expression of PPAR in myeloid cells at the B16-F10 site in PD-1f/f, PD-1f/fLysMcre, and PD-1/ mice was examined by flow cytometry. Mean percentages SEM (A), representative histograms (B), and contour plots (C) of PPAR-expressing CD11b+Ly6C+, CD11b+F4/80+, and CD11c+MHCII+ subsets. (D to G) Mitochondrial metabolic activity of myeloid cells at the B16-F10 tumor site in PD-1f/f, PD-1f/fLysMcre, and PD-1/ mice was examined by assessing mitochondrial membrane potential using MitoRed. Mean fluorescence intensity (MFI) SEM of MitoRedpositive CD11b+Ly6C+, CD11b+F4/80+, and CD11c+MHCII+ subsets within the CD45+CD11b+ gate (D to F) and representative plots of FACS analysis (G) are shown. (H to L) In parallel, expression of IFN-, IL-17A, IL-2, IL-10, RORC, and ICOS in CD8+ TCM and TEM isolated from B16-F10bearing PD-1f/f and PD-1f/fLysMcre mice was assessed by flow cytometry. Representative histograms (H), contour plots (I and K), and mean percentages SEM (J, L, and M) within the CD44hiCD62Lhi gate (for TCM) and CD44hiCD62lo gate (for TEM) cells are shown. Data are from one representative of four independent experiments (*P < 0.05, **P < 0.01, and ***P < 0.005).

We investigated whether these significant immunometabolic changes of myeloid cells, induced by myeloid-specific PD-1 targeting, affected immunological properties of T cells that have key roles in their antitumor function. Compared with control PD-1f/f tumor-bearing mice, PD-1f/fLysMcre tumor-bearing mice had no quantitative differences in CD4+ or CD8+ TEM and TCM cells (fig. S20A) but had significant functional differences. There was an increase of IFN-, IL-17, and IL-10producing CD8+ TEM cells and IL-2producing CD8+ TCM cells (Fig. 8, H to J). Inducible T cell costimulator (ICOS) and lymphocyte-activation gene 3 (Lag3) were elevated in T cells from PD-1f/fLysMcre tumor-bearing mice but cytotoxic T-lymphocyte-associated protein 4 (CTLA4), T cell immunoglobulin and mucin domain 3 (Tim3), CD160, and PD-1/PD-L1 were comparable in T cells from PD-1f/f and PD-1f/fLysMcre tumor-bearing mice (Fig. 8, K to M, and fig. S20B). These findings are significant because IL-17producing T helper cell 17 (TH17)/ T cytotoxic cell 17 (Tc17) cells have enhanced antitumor function and mediate durable tumor growth inhibition (55). Moreover, T cells with a hybrid phenotype producing both IFN- and IL-17 might have superior antitumor properties by combining the enhanced effector function of TH1/Tc1 and the longevity and stemness of TH17/Tc17 cells (56). In our studies, these properties of TEM cells correlated with improved antitumor function in PD-1f/fLysMcre mice.

To examine experimentally whether PD-1deficient myeloid cells differentiated in tumor-bearing mice in vivo have improved capacity of inducing antigen-specific T cell responses, we assessed responses of the same primary CD4+ or CD8+ T cells to antigen-loaded DCs isolated from PD-1/ or control mice bearing B16-F10 tumors (fig. S21A). DCs isolated from the spleen of tumor-bearing WT and PD-1/ mice were pulsed with ovalbumin (OVA) and cocultured with OVA-specific CD4+ or CD8+ T cells from OTI or OTII T cell receptor (TCR)transgenic mice. DCs from tumor-bearing PD-1/ mice had superior ability to induce OTI and OTII T cell proliferation and IFN- expression (fig. S21, B and C). Together, our data provide evidence that myeloid cellintrinsic PD-1 ablation induces potent antitumor immunity by decreasing accumulation of MDSC and promoting proinflammatory and effector monocytic/macrophage and DC differentiation, thereby leading to enhanced effector T cell responses.

Our present studies reveal a previously unidentified role of the PD-1 pathway in regulating lineage fate commitment and function of myeloid cells that arise from tumor-driven emergency myelopoiesis. These outcomes are mediated by myeloid-intrinsic effects of PD-1 ablation, leading to altered signaling and metabolic reprogramming of myeloid progenitors characterized by enhanced differentiation and elevated cholesterol synthesis. Consequently, the accumulation of immature immunosuppressive and tumor-promoting MDSC is diminished, and the output of differentiated, inflammatory effector monocytes, M, and DC is enhanced. These immunometabolic changes of myeloid cells promote the differentiation of TEM cells and systemic antitumor immunity in vivo despite preserved PD-1 expression in T cells.

We found that PD-1deficient myeloid progenitors had enhanced activation of Erk1/2 and mTORC1 in response to G-CSF. These results indicate that Erk1/2 and mTORC1, a downstream mediator of phosphatidylinositol 3-kinase (PI3K)/Akt signaling, which are major targets of PD-1 in T cells (2), are subjected to PD-1mediated inhibition in myeloid cells. These results are revealing because Erk1/2 phosphorylation subverts MDSC-mediated suppression by inducing M-MDSCs differentiation to APC (39). Erk and PI3K regulate glycolysis in response to G-CSF (57). PI3K/Akt/mTORC1 signaling is critical in myeloid lineage commitment. Expression of constitutively active Akt in CD34+ cells induces enhanced monocyte and neutrophil development, whereas a dominant negative Akt has the opposite effect (58). mTORC1 is necessary for the transition of hematopoietic cells from a quiescent state to a prepared alert state in response to injury-induced systemic signals (59), for G-CSFmediated differentiation of myeloid progenitors (40), and for M-CSFmediated monocyte/macrophage generation (41). mTORC1 stimulates translation initiation through phosphorylation of 4E (eIF4E)binding protein 1 (4E-BP1) and ribosomal S6 kinases and has a decisive role in the expression of glucose transporters and enzymes of glycolysis and PPP (47). Consistent with these, our studies showed that PD-1deficient myeloid progenitors had elevated expression of glycolysis and PPP intermediates after culture with emergency cytokines in vitro and enhanced monocytic differentiation in tumor-bearing mice in vivo. Together, our findings indicate that PD-1 might affect the differentiation of myeloid cells by regulating the fine tuning of signaling responses of myeloid progenitors to hematopoietic growth factors that induce myeloid cell differentiation and lineage fate determination during emergency myelopoiesis. Further studies will identify how receptor-proximal signaling events mediated by hematopoietic growth factors are targeted by PD-1 in a manner comparable to PD-1mediated targeting of signaling pathways in T cells (2, 34, 35).

Our metabolite analysis showed that a notable difference of PD-1deficient myeloid progenitors was the increased expression of mevalonate metabolism enzymes and the elevated cholesterol. mTORC1 activates SREBP1, which induces transcription of enzymes involved in fatty acid and cholesterol synthesis (48), thereby leading to glycolysis-regulated activation of the mevalonate pathway. Our signaling studies showing enhanced mTORC1 activation and our metabolic studies showing enhanced mitochondrial metabolism and increased cholesterol content in PD-1deficient myeloid cells provide a mechanistic link between the altered differentiation of PD-1deficient myeloid progenitors and the altered immunophenotypic and functional program of PD-1deficient monocytes, M, and DC in tumor-bearing mice. Cholesterol drives myeloid cell expansion and differentiation of macrophages and DC (50, 51, 60) and promotes antigen-presenting function (61). These properties are consistent with the metabolic profile and the increased cholesterol of PD-1deficient myeloid progenitors; the inflammatory and effector features of differentiated monocytes, M, and DC; and the enhanced T effector cell activation in tumor-bearing mice with myeloid-specific PD-1 ablation that we identified in our studies. By such mechanism, PD-1 might centrally regulate antitumor immunity, independently of the expression of PD-1 and its ligands in the TME. Our studies showed that PD-1 expression on myeloid progenitors is an early event during tumor-mediated emergency myelopoiesis and indicate that PD-1 blockade at early stages of cancer might have a decisive effect on antitumor immunity by preventing MDSC generation from myeloid progenitors and inducing the systemic output of effector myeloid cells that drive antitumor T cell responses.

In addition to its expression in myeloid progenitors, in the bone marrow, we found that PD-1 is expressed in all myeloid subsets including M-MDSC, PMN-MDSC, CD11b+F4/80+ M, and CD11c+MHCII+ DC in the tumor and the spleen of tumor-bearing mice, albeit at different levels. This difference might be related to gradient of tumor-derived factors responsible for PD-1 induction such as G-CSF and GM-CSF that we found to induce PD-1 transcription in myeloid progenitors. This possibility would be consistent with the gradual up-regulation of PD-1 expression in splenic myeloid cells, determined by our kinetics studies, which correlates with tumor growth that might be responsible for the increase of systemic levels of tumor-derived soluble factors that induce PD-1. Other cues of the TME known to mediate the activation step of MDSC (14) might also be responsible for the induction of higher PD-1 expression level in the tumor versus the splenic myeloid cells. Our findings unravel a previously unidentified role of PD-1 in myeloid cell fate commitment during emergency myelopoiesis, a process that is involved not only in antitumor immunity but also in the control of pathogen-induced innate immune responses and sterile inflammation (62).

An additional important finding of our studies is that the nuclear receptors RORC and PPAR are up-regulated in myeloid cells by PD-1 ablation. RORs were initially considered retinoic acid receptors but were subsequently identified as sterol ligands. RORC not only is induced by sterols and isoprenoid intermediates (49) but also serves as the high-affinity receptor of the cholesterol precursor desmosterol (63, 64), a metabolic intermediate of cholesterol synthesis via the mevalonate pathway that regulates inflammatory responses of myeloid cells (52, 60). Desmosterol and as sterol sulfates function as endogenous RORC agonists and induce expression of RORC target genes (63, 64). Our studies showed that, in addition to cholesterol, the mevalonate metabolism product GGPP that has an active role in the up-regulation of RORC expression (49) was elevated in PD-1deficient myeloid cells, providing a mechanistic basis for our finding of the elevated RORC expression. Retinoid receptors and PPAR together regulate monocyte/macrophage terminal differentiation (26). Although initially thought to be involved in proinflammatory macrophage differentiation, it was subsequently understood that PPAR predominantly promotes macrophage-mediated resolution of inflammation by inducing expression of the nuclear receptor liver X receptor and the scavenger receptor CD36, thereby regulating tissue remodeling (65). PPAR also regulates macrophage-mediated tissue remodeling by efferocytosis and production of proresolving cytokines (66), which can suppress cancer growth (67). The combined actions of RORC and PPAR induced by myeloid-specific PD-1 ablation might be involved in the antitumor function by promoting both proinflammatory and tissue remodeling properties of myeloid cells. Future studies will dissect the specific role of each of these nuclear receptors on the antitumor immunity induced by myeloid cellspecific ablation of PD-1.

In conclusion, our results provide multiple levels of evidence that myeloid-specific PD-1 targeting mediates myeloid cellintrinsic effects, which have a decisive role on systemic antitumor responses. This might be a key mechanism by which PD-1 blockade induces antitumor function. Recapitulating this immunometabolic program of myeloid cells will improve the outcome of cancer immunotherapy.

immunology.sciencemag.org/cgi/content/full/5/43/eaay1863/DC1

Materials and Methods

Fig. S1. Gating strategy of hematopoietic and myeloid precursors in the bone marrow.

Fig. S2. Gating strategy of myeloid subsets in the spleen and tumor site.

Fig. S3. Cancer-induced emergency myelopoiesis in three different mouse tumor models.

Fig. S4. PD-1 expression is induced on myeloid progenitors by emergency cytokines.

Fig. S5. Gating strategy for identification of MDSC in human blood samples.

Fig. S6. PD-1 expression in human MDSC.

Fig. S7. PD-1 ablation alters tumor-driven emergency myelopoiesis.

Fig. S8. PD-1 ablation induces expression of RORC and IRF8 in myeloid cells expanding in response to tumor-driven emergency myelopoiesis.

Fig. S9. PD-1 ablation induces expression of RORC and IRF8 in myeloid cells expanding in mice-bearing MC38 or MC17-51 tumors.

Fig. S10. PD-1 ablation increases the output of RORChi effector-like myeloid cells at early stages of tumor growth.

Fig. S11. Therapeutic targeting of PD-1 increases effector features of myeloid cells and decreases tumor growth.

Fig. S12. Myeloid-specific and T cellspecific PD-1 deletion.

Fig. S13. Myeloid-specific PD-1 ablation promotes expansion of IRF8hi and RORChi monocytes and IFN-producing monocytes and macrophages in the tumor site.

Fig. S14. Tumor-induced emergency myelopoiesis and myeloid effector differentiation in Rag2-deficient mice treated with PD-1 antibody.

Fig. S15. PD-1 ablation reduces the threshold of growth factormediated signaling in GMP.

Fig. S16. Myeloid-specific PD-1 ablation induces a distinct metabolic profile characterized by elevated cholesterol.

Fig. S17. Metabolic pathways linking glycolysis to PPP, fatty acid, and cholesterol synthesis.

Fig. S18. Schematic presentation of the mevalonate pathway.

Fig. S19. Increase of glucose uptake and neutral lipid content in PD-1deficient myeloid progenitors early after tumor implantation.

Fig. S20. Myeloid-specific PD-1 deletion alters the immunological profile of CD8+ TEM cells.

Fig. S21. PD-1 ablation enhances antigen presentation ex vivo by tumor-matured DC.

Table S1. List of significantly different metabolites.

Table S2. List of antibodies used for surface staining.

Table S3. List of antibodies used for intracellular staining.

Table S4. List of antibodies used for phenotype of human MDSC.

Table S5. Raw data in Excel spreadsheet.

References (6871)

Acknowledgments: Funding: This work was supported by NIH grants CA183605, CA183605S1, and AI098129-01 and by the DoD grant PC140571. Author contribution: L.S. participated in the conceptualization of the project and experimental design, performed experiments and the analysis and validation of the data, prepared figures, and participated in the preparation of the manuscript. M.A.A.M. performed experiments and the analysis and validation of the data, prepared figures, and participated in the preparation of the manuscript. J.D.W., N.M.T.-O., A.C., R.P., Q.W., and M.Y. participated in various steps of the experimental studies. J.A. participated in the experimental design of metabolite studies and the formal analysis and the validation of the data and participated in the preparation of the manuscript. N.P. participated in the conceptualization of the project, designed and performed the bioenergetics studies, and participated in experiments, the analysis and validation of the data, and the preparation of the manuscript. V.A.B. had the overall responsibility of project conceptualization, experimental design, investigation, data analysis and validation, and preparation of the manuscript and figures. Competing interests: V.A.B. has patents on the PD-1 pathway licensed by Bristol-Myers Squibb, Roche, Merck, EMD-Serono, Boehringer Ingelheim, AstraZeneca, Novartis, and Dako. The authors declare no other competing interests. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper or the Supplementary Materials.

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Targeted deletion of PD-1 in myeloid cells induces antitumor immunity - Science

Dr. Howard Grey, former president and scientific director of theLa Jolla Institute for Immunology, recently died in Denver. He was 87 years old.

Grey, a highly respected biochemist and a pioneering immunologist with a reputation for offering unadorned insight, defined how T cells recognize their targets. He took over as scientific director and third president of LJI after the retirement of Dr. Kimishige Ishizaka in 1996. Over the next seven years, Grey would strengthen LJI's ties with business partners, expand the faculty from six to 14 members, and initiate plans to move the Institute to LJI's current home in UC San Diegos Research Park. When he retired as CEO in 2003, he continued to be involved in immunological research at LJI until 2015.

"In the '60s, at a time before proteins could be sequenced, Howard was a pioneer in studying the structure of B cell immunoglobulins," said LJI President and Chief Scientific Officer Mitchell Kronenberg, whom Grey recruited as a faculty member to LJI in 1997. "Later, seminal and widely acclaimed work showed how the T cell receptor recognized fragments of proteins or peptides. His formidable biochemical skills drove achievements in both areas."

Born in New York City, Grey earned a B.A. in chemistry in 1953 from the University of Pennsylvania and then attended medical school, earning an M.D. from New York University and interning at Johns Hopkins. In 1958, however, he moved from the clinic to fundamental research and began a six-year research fellowship studying antigen-antibody interactions, first at the University of Pittsburgh and then at the Scripps Clinic and Research Foundation in La Jolla.

He was mentored in those early years by Dr. Frank Dixon, who later became well known for leading the Scripps Research Institute. He then relocated to Rockefeller University as an investigator and assistant professor in the mid-60's and then back to Scripps in 1967, investigating the structure of gamma-globulins.

In 1970, Grey joined the faculty of the University of Colorado Medical Center in Denver, where he began two decades of high impact research, served as head of the Basic Immunology Division from 1978-1988, and expanded his interests to include T cell activation. With his lab housed in the Department of Medicine at Denver's National Jewish Hospital and Research Center, a mecca for immunology research, Grey was one of a group of trail-blazing immunologists who postulated that for T cells to respond to a pathogen, a T cell receptor must bind short fragments of pathogen proteins that presented or captured by so-called major histocompatibility (MHC) proteins expressed on the surface of the target cells.

In the late '70s, those ideas were controversial, in part because the structure of the T cell receptor was unclear. Some even proposed there were two distinct T cell receptors, one recognizing MHC proteins and the other binding antigen. By 1983, however, Grey, collaborating with next-door lab neighbors John Kappler and Philippa Marrack, had proven that protein antigens must be chopped up or "processed" into short fragments, or peptides, to activate T cells. And in 1986 and 1987, respectively, Grey's group published seminal papers in the Proceedings of the National Academy of Sciences and Science leaving no doubt that peptide antigens must be presented in the grasp of MHC proteins to activate what was now known to be a single T cell receptor.

Grey then moved on to answer multiple biochemical questions relevant to the "ligand end" of T cell activation, for example showing in a 1988 Science paper how a single MHC molecule can present multiple types of peptide antigens in a specific manner. Grey's contributions over this highly productive period of his career would earn him prestigious awards in the late '80s and '90s, including being named co-winner of the William B Coley Award for distinguished research in basic and tumor immunology and culminating in membership in the National Academy of Sciences in 1999.

In 1988, Grey left Colorado to co-found the San Diego biotechnology company Cytel, in part to realize his work's clinical potential by creating and testing novel peptide drugs that modulate the immune system, some to build better vaccines and others intended to dampen immune responses in autoimmune disease. Grey remained the companys vice president for research and development until 1994, when he moved to LJI to become division head of Immunochemistry and, in 1996, LJI president.

His accomplishments at LJI prove that a successful scientist can also have business acumen. "In addition to recruiting outstanding up and coming faculty, Howard initiated important discussions with our long-term business partners at Kirin, encouraging them to support our move in 2006 to a state-of-the-art facility on the UCSD campus," said Kronenberg. "Howard solidified our relationship by fostering interactions of Kirin's scientists with our own, strengthening our partnership and securing support for our research."

Grey's communication style, however, was hardly that of a smooth-operating corporate executive. He was renowned among some as being taciturn to the point of intimidating. "I'd give him 90 out of 100 on the laconic scale," says Kronenberg, but what he did say, was almost always highly important.

LJI professor Alessandro Sette, Dr. Biol. Sci., who did postdoctoral work in Greys lab and later followed him to Cytel, agrees but says that Grey's no-nonsense demeanor was something that attracted excellent scientists into his orbit. "He was a man of few words and valued direct communication in a work environment over niceties and political correctness," says Sette, but when Howard said something, you could hang your hat on it.

Most who knew Grey agree that his occasional brusqueness reflected very high standards he set for himself and others. He expected you to drop a hypothesis if it was not supported by experiments, something he was equally ready to do, says Sette.

Grey stepped down as LJI CEO in 2003 and at age 71 assumed a part-time position in LJI's Division of Vaccine Development working with Sette, his former post-doc who had since joined the faculty. "Howard remained in my lab another 10 years as a partner and key contributor in driving grants and mentoring young students and postdocs," says Sette, who worked with Grey for almost 30 years. "He was my most influential mentor and one of the smartest people I ever met."

Grey served as LJI President Emeritus until his death.

Howard Grey is survived by his wife Hilda, two of his three children, Allen and Stuart Grey, and seven grandchildren.

A service will be held at 1 pm on January 4, 2020, at Olinger Chapel Hill, 6601 South Colorado Boulevard, Denver, Colorado 80129.

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Howard Grey distinguished immunologist and former LJI leader dies - Dr. Howard Grey former president and scientific director of the La Jolla Institute...

If you're considering investing in the gene editing sector, it's worth taking some time to look through all the main players in this small but promising biotech market. At the moment, there are just a few noteworthy companies in this space, all of them still at early clinical stages despite commanding market valuations well into the billions of dollars.

CRISPR Therapeutics (NASDAQ:CRSP) is likely the first gene editing stock to come to mind, and it's considered by many to be the leading company in the market, if only by market cap. However, smaller companies, like Sangamo Therapeutics (NASDAQ:SGMO), also have plenty of promise.

If you're wondering which of these two stocks is the better buy, then read below to find out all the details.

Image source: Getty Images.

While different gene editing companies target their own specific conditions, investors will notice that many tend to coalesce around the area of blood disorders. Both CRISPR and Sangamo are working on drug candidates that target sickle cell disease and transfusion-dependent beta thalassemia (TDT), which are disorders that hinder the ability of hemoglobin to carry oxygen around the body.

CRISPR is working on CTX001, which has been used to treat two different patients, one with sickle cell disease and the other with beta thalassemia. Both patients have shown a complete reversal of all key symptoms, with more patients now undergoing CTX001 treatment.

Unlike CTX001, Sangamo has two separate drug candidates, each targeting only one of the blood disorders mentioned above. ST-400 is Sangamo's beta thalassemia drug, while BIVV003 is its sickle cell candidate. Both are being developed alongside Sanofi, which has partnered with Sangamo to develop these drugs.

While BIVV003 is still undergoing early clinical testing, with investors still waiting to see the preliminary results, ST-400 has proven to be an early success so far. Sangamo released data in early December regarding the first three patients treated with ST-400 for TDT, with all of them showing encouraging results with few side effects. Further results are expected to come out in 2020.

Sangamo has a pretty diverse portfolio of drug candidates that are either in preclinical or clinical stages of development, totaling 15 separate projects in comparison to CRISPR's nine. Five of those are in early phase 1/2 trials. Besides Sangamo's sickle cell and beta thalassemia treatments, Sangamo is working on treatments for Fabry disease, Hemophilia A, and Hunter syndrome (also known as mucopolysaccharidosis type 2 or MPS II).

The Hemophilia A treatment, SB-525, showed strong results in its phase 1/2 study earlier this year. Patients with this blood disorder, who experience a lack of a key blood-clotting factor, showed significant improvements in levels of this clotting factor after taking SB-525.

Even patients with severe cases of hemophilia A, which is extremely hard to treat, showed impressive improvements in the levels of this clotting factor. Pfizer, which is partnered with Sangamo to develop SB-525, is now moving toward a new phase 3 trial, which is expected to begin sometime in 2020.

Sangamo's Fabry treatment, ST-920, is still undergoing its own early-stage clinical trials, with little information available at present. The only setback for Sangamo has been in its MPS II drug, SB-913, which ended up failing to significantly help patients with the rare genetic disorder. While the company hasn't given up on SB-913 yet, it's definitely the weak link in an otherwise strong drug portfolio.

CRISPR's drug portfolio is a bit narrower, with only two drugs in clinical testing in comparison to Sangamo's five. Besides the previously mentioned CTX001, CRISPR has a fairly strong cancer immunology lineup. CTX110, CTX120, and CTX130 are its selection of immunology candidates, although CTX110 is the only one in clinical testing at the moment.

Cancer immunology is a massive market that's estimated to reach $127 billion by 2026, and a home run in this area would be a major win for CRISPR. CTX110 is a CAR-T (chimeric antigen receptor T-cell) therapy, a type of treatment in which immune cells are extracted from a patient, retrained outside the body, and later reintroduced into the patient's system in hopes they will perform better. While it's not the only CAR-T therapy being developed, CRISPR's treatment could prove to be much cheaper than current treatments, which cost hundreds of thousands of dollars for a single patient.

CRISPR has had a strong fiscal third quarter, reporting $138.4 million in net income on revenues of $211.9 million. But in 2019, CRISPR has so far only reported $36.3 million in net income, as the earlier quarters reported losses. While it's nice that CRISPR is reporting a profit, something very few early-stage biotech companies can boast, it's still a very small figure considering CRISPR's $3.9 billion market cap.

Sangamo's financials look a lot different. Besides being a fraction of CRISPR's size with a market cap of $970 million, Sangamo's Q3 2019 revenues came in at $21.9 million, while reporting a net loss of $27.4 million for the quarter. However, the company has an impressive $408.3 million in cash and equivalents, enough to last for around four years at the current rate of expenses.

In terms of traditional valuation metrics, it's hard to evaluate clinical-stage biotech stocks by looking at ratios, as their financial figures can change dramatically if a drug candidate receives approval. Currently, CRISPR trades at 16.7 its price to sales (P/S) ratio, but in July, the company was trading at an astronomical 1,800 P/S ratio, meaning that investors were willing to pay extraordinarily high amounts for what little revenue it was making that quarter. In comparison, Sangamo is more moderately priced, with a 12.2 P/S ratio.

Both companies are compelling investments if you're looking for exposure to the gene editing sector. While Sangamo has a broader pipeline of projects, I still think CRISPR is the better choice if you had to pick just one of these companies. CRISPR has not only shown positive clinical results for CTX001 and CTX110, but is also reporting a profit for this most recent quarter, which is pretty rare for early-stage biotech stocks. Meanwhile, Sangamo isn't expected to turn a profit anytime soon.

However, gene-editing drugs are still at an early stage of clinical development, and plenty of things can change over the coming years. Both CRISPR and Sangamo are promising investments for someone who's comfortable buying into early-stage biotech stocks.

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Better Buy: CRISPR Therapeutics vs. Sangamo Therapeutics - The Motley Fool

Meet the inflammation and immunity researcher studying the fundamental cellular mechanisms behind uncontrolled inflammatory responses to allergens

As the prevalence of allergic disease continues to rise worldwide, the work of immunologist Dr. Adam MacNeil has never been more important. By identifying novel targets in allergic inflammation to enable the development of innovative therapies, MacNeil and his team are pushing toward a healthier future. Were interested in allergic inflammation from two different branches, firstly, how the cells that contribute to inflammation emerge from the bone marrow, and secondly, how mature mast cells contribute to inflammatory mechanisms at the site of exposure, explains MacNeil, associate professor in the interdisciplinary Health Sciences department at Brock University, Canada.

Dr. Adam J. MacNeil, Associate Professor of Immunologyat Brock University's Department of Health Sciences.Pictured from left to rightare;Melissa Rouillard, Aindriu Maguire, Rob Crozier, Adam MacNeil, Jeremia Coish, Katie Hunter, Colton Watson, and Natalie Hicks. Image courtesy of theMacNeil Lab.

The MacNeil Lab investigates mechanisms in hematopoietic stem cells directing the maturation of the most well-known allergic mediator cellsmature mast cellsthat drive allergic inflammation. A key research goal for the team is to identify how an allergen activates a mast cell to create an inflammatory response.

Seeking to understand the signals that stimulate a progenitor cell to become a mast cell in different tissues, this research looks to determine the signaling pathways directing the epigenetic, and ultimately proteomic, profile of these cells1-3. To do this, cells are isolated and matured from bone marrow to create functional, phenotypical mast cells, which are primed with allergen-specific IgE molecules before addition of the allergen to activate the cells. The inflammatory response to the allergen, and the cell signaling processes that contribute to the inflammatory mechanisms, can then be measured through the secretion of histamines in degranulation mechanisms, or release of pro-inflammatory mediators such as cytokines, chemokines, and lipid metabolites.

Brock University

Being able to identify and sort cells with a specific immune profile requires tools capable of precision sorting of heterogeneous populations of cells. MacNeil expands: Were working with a heterogeneous population of cells in the bone marrow and trying to take only the stem cells out. So, it's a very small population within the total population of cells. Many of the assays that we want to do with that small population of cells are very well-suited to being sorted directly onto a 96-well plate where we can then actually conduct the experiment directly, knowing exactly how many cells are in each well and what the particular profile of those cells is. That makes the Sony SH800S a really strong tool for our lab.

When it comes to optimizing and streamlining the lab's work, Sony technology offers advantages over traditional methods. The traditional flow cytometer or cell sorter in any core lab is operated by a technician, and they're the only one allowed to touch it. That doesn't make for great learning opportunities for graduate students, and it's much better if they can actually interface with the instrument themselves, says MacNeil. The software and automation really allow for that to happen, but also adds to the robustness of the instrument. The way in which it has been designed means that it's pretty difficult to break it.

With an epigenetic approach to understanding how mast cells differentiate, and the effect of inhibiting specific signaling pathways in those cells, the MacNeil Lab uses sorted cells in functional assays such as immune cell profiling and cytokine secretion. Also, the cells can be sorted into plate-based assays for ChIP or RNA-Seq to assess their genetic profile. We're not only interested in sorting. We bought the device because it's robustly dynamic, explains MacNeil, referring to the Sony SH800S. You can look at data acquisition and not have to even use the sorting function at all in certain scenarios. There are many times that were simply interested in looking at the phenotype of our cells and not worried about sorting necessarily. Weve found this instrument to be very easy to use and to give us robust data in terms of the immune profile of our cells.

In addition, the SH800S microfluidic sorting chip helps to automate key stages of instrument setup and demonstrates versatility with a wide range of chip sizes, ranging from 70130 m, for sorting a variety of cells. The chip ultimately gets to the robustness of the instrument, explains MacNeil. Because of the chip, we have such peace of mind about how the instrument functions that we don't even worry about clogging of the instrument and all of the problems that the chip ultimately solves. If we do run into a problem, we can just change the chip. I certainly find the chip technology to be really well suited to our type of lab environment.

For MacNeil, the Sony SH800S Cell Sorter is a great fit for the lab, with a seamless software interface and great overall instrument design and modularity for easy plate-based sorting.MacNeil lab logocourtesy of the MacNeil Lab.

Working within the diverse multidisciplinary department at Brock University opens unique and fascinating research avenues not available to all immunologists and has led MacNeil to interesting collaborations and knowledge exchange on transdisciplinary projects.

As part of these broader research avenues, working with sociologist Prof. Terrance Wade and cardiovascular biologist Prof. Deborah OLeary, MacNeil also studies adverse experiences in childhood. The team is investigating whether such events may set the immunological stage for dysregulated inflammation in later life, through mechanisms involving stress-stimulated cortisol release that can shape how the immune system is responding4.

In another stream of collaborative immunological research, MacNeil collaborates with psychologist Prof. Anthony Bogaert to look at the role of the immune system in shaping sexual orientation as part of the fraternal birth order effect. This research looks at how early pregnancies stimulate the immune system to make antibodies against brain proteins in fetal males that may then affect their social behaviors in later life5. Its something I may not have expected to ever work on, says MacNeil. But when you come to a diverse department with a wide lens on health, these kinds of opportunities emerge. Were now interested in using the SH800S to test hypotheses for particular mechanisms underlying this phenomenon.

Looking ahead, MacNeil expects tissue heterogeneity to be a key issue to tackle in the field of immunology. Cell populations simply aren't uniform, he says. Mast cells in different locations in the body don't have exactly the same phenotype, and so, as our research proceeds and we continue to probe the role of the mast cell in allergic inflammation, we're very conscious that tissue heterogeneity is going to be a factor. But with such challenges come opportunities. Were ultimately interested in going into those tissues and trying to pull mast cells out. To do this, we would require an instrument like a cell sorter. Once the cells are sorted, we can interrogate their functional phenotype, including how they degranulate, secrete cytokines and metabolize lipids etc. toward one day potentially modulating their phenotype for the hundreds of millions affected by this inappropriate immune response, MacNeil concludes.

Originally posted here:
Innovative therapies: Novel targets in allergic inflammation - SelectScience

In one photo, the babys head is turned away from the camera, as someone holds his arm up to show the pink area on his back. In another, a cluster of red bumps ring the area where the babys arm and back meet, and a third, of the childs chest, shows what looks like a bumpy red rash near the belly button.

Hi all does this look like an allergic reaction? asks the poster in a Facebook allergy parent group.

Have you tried a naturopath or chiropractor? And adding probiotics and vitamin D to hid [sic] diet? reads one response.

You might think this social media post, presented by Dr. David Stukus to a room full of experts at the annual meeting of the American College of Allergy, Asthma, and Immunology, would cause an uproar. Why would a parent turn to Facebook with such a severe reaction?Who has the nerve to respond as an expert and give such misguided advice? Instead, the post elicits familiar groans. Every single person I talked to after my presentation has seen this in his or her practice, says Stukus, an associate professor of pediatrics and associate director of the Pediatric Allergy & Immunology Fellowship Program at The Ohio State University College of Medicine. But whats a room full of immunologists to do? Fighting promises of quick fixes with clear science has always been an uphill battle when it comes to the health of kids. Increasingly, parents ofkids with food allergies have seen this first-hand, thanks to the rise of parenting groups who are taking a page from anti-vaxxers and offering medically dubious advice and promoting conspiracies. For worried parents, its disorienting and dangerous. Fortunately, experts are speaking up, looking to nip this trend in the bud before it does real, extensive harm.

Does preschool or preschool-age childcare put a financial strain on your family?

No

Yes, but we can handle it

Yes, but were freaking out a bit

Yes. Its a serious problem

Thanks for the feedback!

The fact is that there is no cure for food allergies, which affect more than 4 million kids, or 5 percent of children in the U.S.

If parents believed everything they read online about food allergies, theyd worry that smelly feces could signal a gluten intolerance. Theyd shell out $250 for at-home food allergy tests and would ban charcoal briquettes from their grills. Theyd think a detoxifying elixir might cure allergies and that the body can reverse allergies with the help of vitamin B5, probiotics and crystallized sulfur. Theyd make a child having an anaphylactic allergic reaction drink activated charcoal and hope for the best. They would blame the government for the rise of peanut allergies among kids because they started putting peanut oil in vaccines in the 1960s.

Many parents of kids with food allergies correctly understand that theres no scientific evidence supporting the above claims. But a sizable portion missed the lessons and are all too happy to share unsubstantiated clickbait containing dubious health claims via myriad online podiums that offer the misinformed a megaphone. CountlessFacebook groups for allergy parents have cropped up, many of which have tens of thousands of members. People offer anecdotal advice on allergy blogs and YouTube videos, and, to a lesser degree, on allergy-related Instagram accounts (there are more than 50,000 Instagram posts with the tag #allergymom.).

The fact is that there is no cure for food allergies, which affect more than 4 million kids, or 5 percent of children in the U.S., according to the Asthma and Allergy Foundation of America. And although the Food and Drug Administration is close to approving a new peanut allergy treatment, currently, the only available treatments for food allergies are avoiding the allergens and possibly medication and immunotherapy. Sadly, however, many parents get their hopes up chasing spurious and often expensive allergy fixes discouraged by their allergists and that turn out to be useless.

Its not just well-meaning but misinformed parents spreading bad food allergy advice. Irresponsible bloggers and companies selling supplements, herbs, treatment programs, DIY allergy tests and chiropractic services based on junk science prey on parents dealing with the anxiety-inducing new world of severe child food allergies. In addition, even well-informed parents might sometimes click on a promise of some new treatment or remedy that at best is a waste of time and at worst, could lead to dangerous medical decisions affecting their childs health.

One Facebook allergy group member, a father of a 15-month-old son who has an anaphylactic allergic reaction to sesame seeds, peanuts, cashew nuts, and pistachios, offered his story as evidence: Im pretty skeptical, says the man who asked to remain anonymous.He and his wife follow the doctors instructions and do their own research when it comes to allergy treatments or restaurant menu tips they read online. A lot of that research starts for them in Facebook groups for allergy parents which sometimes offer well-cited information that they then verify. But there are also plenty of too-good-to-be-true posts and ads that, he admits, can be hard to resist. I have to say, as a dad with an allergic son, I really wish I could believe the headlines and wish I could think Oh, hes going to be OK, theyve found a cure.Since allergies are still somewhat a science mystery, he says, its ripe territory for clickbait and false information posing as science.

This is what fortune-tellers do: they cast a wide net until they find something that may have some application to somebodys life and go with it.

Its no surprise that parents are vulnerable targets for all sorts of allergy quackery. Its difficult enough to keep kids safe as they navigate the world but can be overwhelming having to worry that a piece of cake containing hidden allergens at a birthday party might kill them. But the volume of targeting this vulnerable population is subject to from modern-day snake-oil salesmen is shocking.

Stukus studied six years worth of allergy-related posts on social media and presented his findings at the American College of Allergy, Asthma & Immunology annual meeting in October. What he saw was alarming, he says, and no surprise to any of his colleagues at the meeting.

There are companies as well as different types of medical providers that deliberately target the food allergy community and peddle pseudoscience as a way to make a profit for their services, such as home food allergy sensitivity testing, which is not an accurate way to diagnose anything, he says.

One branch of quackery aimed at food allergy parents involves dubious means of diagnosing food allergies, such as chiropractic adjustments, muscle testing, and hair analysis, Stukus says. Websites peddling food allergy home tests often are loose with the terms allergy and sensitivity and use them interchangeably, even though food allergies and food intolerances are wildly different things. (Stukus goes so far as to say food sensitivities arent real.)

These bogus online [food intolerance] quizzes basically keep asking about every common symptom until you say yes, Stukus says. This is what fortune-tellers do: they cast a wide net until they find something that may have some application to somebodys life and go with it.

More alarming than persuading someone that they have a nonexistent food allergy, however, is that allergy misinformation can feed a mistrust of mainstream medicine that can endanger kids health. Some Facebook and YouTube videos feature doctors of chiropractic or alternative medicine offering advice that your traditional allergists wont tell you, or point out that avoidance of an allergen isnt a cure and frame their dangerous or useless remedy as more proactive than recommendations from a board-certified allergist.

Scrolling through the comments on some of these videos reveals viewers who enthuse that the advice in the video saved them a trip to the doctor for a diagnosis or ask for a virtual diagnosis of an allergic reaction. Describing big red bleeding bumps, sharp stomach pains and swells around their lips, a sufferer on one video commented, I was just wondering if I should go to the doctor or I should just put cream on it and hope for the best.

The lack of an effective cure means that were a big, ripe target for every medical quack and health scammer out there, including the anti-vaxxers.

The prevalence of food allergies among children has increased, and speculation about the reasons for the spike veers into conspiracy-theory territory with, perhaps unsurprisingly, some crossover from the anti-vaxxer movement.

Heated arguments abound in the parent allergy community over the theory that the government began adding peanut oil to vaccines decades ago and is to blame for the increase in peanut allergies in children. This is a debunked claim that even some anti-vaxxers say is false. Yet many parents believe it and might not vaccinate their children for fear theyll develop a life-threatening peanut allergy.

If not getting vaccinated prevented food allergies then unvaccinated kids should not have food allergies, but they do, says Melanie Carver, vice president of community health services and marketing for the Asthma and Allergy Foundation of America. Delaying vaccination because of a fear of allergies poses health risks to children, she says.

The lack of an effective cure (as opposed to a few treatments still in development), means that were a big, ripe target for every medical quack and health scammer out there, including the anti-vaxxers, says Laurel, an author of an allergy blog and member of several Facebook groups for allergy parents who asked to remain anonymous. Laurel says she recently was kicked out of an allergy group after flagging an anti-vax post to a mod. It turned out that the anti-vax poster was the moderator, and Laurelwas booted.

The hundreds, if not thousands, of Facebook groups for allergy parents, vary widely in terms of the quality of information and how well theyre policed for misleading and dangerous posts, Laurel says. Plenty of good, responsible Facebook groups and blogs help parents understand scientific studies related to allergies. Allergy parents are often anxious and overwhelmed, and the support they can get online from other parents who understand what theyre going through can be invaluable.

But gauging the reliability of Facebook allergy groups is time-consuming. In general, its safer to think of social media as one step in evidence-gathering about allergies and evaluate each article about a study or tip about allergy-free restaurant independently, says Nicole Smith, a longtime allergy parent blogger in Colorado Springs, Colorado.

If anything is claimed to be a cure, run in the opposite direction, Smith says. Parents need to be cautious and discuss even innocuous-seeming herbal supplements with their childs allergist before trying them, she says, because you dont know what else could be in one that could set off the system.

Instead of looking at blogs and less reliable information portals, turn to nonprofit or medical society resources for parents such as FARE, the AAAI and the ACAAI, recommends allergy researcher Thomas Casale, MD, former head of the ACAAI and professor of pediatrics at the University of South Florida.

Remember that allergies are so individual that your childs allergist will always be the most informed source of information.Keep a file of research, remedies, and recommendations you see online and bring the list to appointments to discuss with your allergist. They know your child and are a better source of information than a stranger with a kid whose condition could have little bearing on your childs condition.

Its dangerous to take another persons online anecdote and apply it to your own situation, not recognizing there are many nuances never discussed that can vastly impact whether the anecdote even applies to [your child], Stukus says.

The scariest part of all this is people with a child whos having active symptoms and posts a picture of a rash asking their group, What should I do? And other people with no training whatsoever offer their opinions, he continues. Thats how I see someone might die, and that really scares the hell out of me.

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Food Allergy Treatments and Cures Are Cropping Up Everywhere Online. Parents Beware. - Fatherly

NeoImmuneTechs Chief Business Officer, Samuel Zhang, Ph.D., MBA, will review NeoImmuneTechs recent progress and future directions, including an update on the companys lead drug candidate, Hyleukin-7. NeoImmuneTech is currently conducting several clinical trials of Hyleukin-7 in different types of cancer and multiple pre-IND and non-clinical studies in both solid tumors and hematologic malignancies.

Presentation details:Date: Tuesday, January 14Time: 3:00pm PTTrack: Franciscan B (Ballroom Level)Location: Hilton Hotel, Downtown San Francisco, CA

About Hyleukin-7

Hyleukin-7, the only clinical-stage long-acting human IL-7, is uniquely positioned to address unmet medical needs in immuno-oncology. IL-7 is a fundamental cytokine for T-cell development and for sustaining immune response to chronic antigens (as in cancer). Hyleukin-7's favorable PK/PD and safety profiles make it an ideal combination partner for immunotherapy standard of care (SOC) such as Checkpoint Inhibitor and CAR-T therapies. Hyleukin-7 is being studied in multiple clinical trials in solid tumors, and is being planned for testing in hematologic malignancies, additional solid tumors and other immunology-focused indications.

About NeoImmuneTech

NeoImmuneTech, Inc. (NIT) is a clinical-stage T cell-focused biopharmaceutical company dedicated to expanding the immuno-oncology frontier with Hyleukin-7 and beyond. NIT is partnering with industry and academic leaders to investigate Hyleukin-7 in combination with various immunotherapeutics. For more information, please visit http://www.neoimmunetech.com.

View source version on businesswire.com: https://www.businesswire.com/news/home/20200103005008/en/

Excerpt from:
NeoImmuneTech to Present at the 2020 Biotech Showcase - BioSpace

Scientists piece together the inflammation mechanism in IgG4-related disease, an autoimmune condition with no current cure, revealing possible therapeutic targets

IgG4-related disease is an autoimmune disorder affecting millions and has no established cure. Previous research indicates that T cells, a major component of the immune system, and the immunoglobulin IgG4 itself are key causative factors, but the mechanism of action of these components is unclear. Now, Scientists from Tokyo University of Science have meticulously explored this pathway in their experiments, and their research brings to light new targets for therapy.

Autoimmune diseases are a medical conundrum. In people with these conditions, the immune system of the body, the designated defense system, starts attacking the cells or organs of its own body, mistaking the self-cells for invading disease-causing cells. Often, the cause for this spontaneous dysfunction is not clear, and hence, treatment of these diseases presents a major and ongoing challenge.

One recently discovered autoimmune disease is the IgG4-related disease (or IgG4-RD), which involves the infiltration of plasma cells that are specific to the immunoglobulin (antibody) IgG4 into the body tissue, resulting in irreversible tissue damage in multiple organs. In most patients with IgG4-RD, the blood levels of IgG4 also tend to be higher than those in healthy individuals. Previous studies show that T cellswhich are white blood cells charged with duties of the immune responseplay a key role in the disease mechanism. In particular, special T cells called cytotoxic T lymphocytes, or CTLs, were found in abundance from the inflamed or affected pancreas of patients, along with IgG4. But what was the exact role of CTLs?

In a new study published in International Immunology, a team of scientists from Tokyo University of Science decided to find the answer to this question. Prof. Masato Kubo, a member of this team, states that their aim was twofold. We planned to explore how IgG4 Abs contributes to the CTL-mediated pancreas tissue damage in IgG4-RD, and also to evaluate the pathogenic function of human IgG4 Abs using the mouse model that we have established. The latter is especially important, as IgG4 is not naturally present in mice, meaning that there is a severe lack of adequate animal models to explore this disease.

With these aims, they selected mice that have been genetically programmed to express a protein called ovalbumin (the major protein in egg white) in their pancreas. Then, they injected IgG4 that specifically targets ovalbumin into the mice. Their assumption was that IgG4 would target the pancreas and bring about IgG-4-RD-like symptoms. However, what they found was surprising. No inflammation or any other symptom typical of IgG4-RD appeared. This convinced the researchers that IgG4 alone was not the causative factor of IgG4-RD.

Next, to check if it was the CTLs that were perhaps the villain of the story, the scientists injected both IgG4 specific against ovalbumin as well as CTLs. Now, the pancreas of the mice showed tissue damage and inflammation. Thus, it was established that the presence of CTLs and IgG4 was necessary for pancreatic inflammation.

When they probed further, they found that another variation of T cells, known as T follicular helper or TFH cells, which develop from the natural T cells of the mice, produce self-reactive antibodies like IgG4, which induce inflammation in combination with CTLs.

Once the puzzle was pieced together, the scientists now had the opportunity to zero in on the target step for intervention; after all, if one of these steps is disrupted, the inflammation can be prevented. After much deliberation, they propose that Janus kinase, or JAK, can be a suitable target. JAK is a key component of the JAK-STAT cellular signaling pathway, and this pathway is an integral step in the conversion of natural T cells of the mice to TFH cells. If this JAK is inhibited, this conversion will not take place, meaning that even the presence of CTLs will not be able to induce inflammation.

Prof. Kubo also suggests a broader outlook, not limited to the therapeutic option explored in the study. He states, based on our findings, the therapeutic targets for IgG4-related diseases can be the reduction of TFH cell responses and the auto-antigen specific CTL responses. These can also provide the fundamental basis for developing new therapeutic applications.

These proposed therapeutic targets need further exploration, but once developed, they have the potential to improve the lives of millions of patients with IgG4-RD worldwide.

###

Reference

Journal:

International Immunology

About The Tokyo University of Science

Tokyo University of Science (TUS) is a well-known and respected university, and the largest science-specialized private research university in Japan, with four campuses in central Tokyo and its suburbs and in Hokkaido. Established in 1881, the university has continually contributed to Japans development in science through inculcating the love for science in researchers, technicians, and educators.

With a mission of Creating science and technology for the harmonious development of nature, human beings, and society, TUS has undertaken a wide range of research from basic to applied science. TUS has embraced a multidisciplinary approach to research and undertaken intensive study in some of todays most vital fields. TUS is a meritocracy where the best in science is recognized and nurtured. It is the only private university in Japan that has produced a Nobel Prize winner and the only private university in Asia to produce Nobel Prize winners within the natural sciences field.Website: https://www.tus.ac.jp/en/mediarelations/

About Professor Masato Kubo from Tokyo University of Science

Dr Masato Kubo is a Professor at the Tokyo University of Science. A respected and senior researcher in his field, he has more than 226 publications to his credit. He is also the corresponding author of this study. His research interests include Immunology and Allergology. He is the team leader at the Laboratory for Cytokine Regulation, RIKEN Center for Integrative Medical Sciences.

Funding information

This study was supported by grants from JSPS KAKENHI (grant no. 19H03491), Japan Agency for Medical Research and Development (AMED), AMED-CREST, and Toppan Printing CO., LTD.

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Solving the puzzle of IgG4-related disease, the elusive autoimmune disorder - QS WOW News

BEIJING A court in China on Monday sentenced He Jiankui, the researcher who shocked the global scientific community when he claimed that he had created the worlds first genetically edited babies, to three years in prison for carrying out illegal medical practices.

In a surprise announcement from a trial that was closed to the public, the court in the southern city of Shenzhen found Dr. He guilty of forging approval documents from ethics review boards to recruit couples in which the man had H.I.V. and the woman did not, Xinhua, Chinas official news agency, reported. Dr. He had said he was trying to prevent H.I.V. infections in newborns, but the state media on Monday said he deceived the subjects and the medical authorities alike.

Dr. He, 35, sent the scientific world into an uproar last year when he announced at a conference in Hong Kong that he had created the worlds first genetically edited babies twin girls. On Monday, Chinas state media said his work had resulted in a third genetically edited baby, who had been previously undisclosed.

Dr. He pleaded guilty and was also fined $430,000, according to Xinhua. In a brief trial, the court also handed down prison sentences to two other scientists who it said had conspired with him: Zhang Renli, who was sentenced to two years in prison, and Qin Jinzhou, who got a suspended sentence of one and a half years.

The court held that the defendants, in the pursuit of fame and profit, deliberately violated the relevant national regulations on scientific and medical research and crossed the bottom line on scientific and medical ethics, Xinhua said.

Dr. Hes declaration made him a pariah among scientists, cast a harsh light on Chinas scientific ambitions and embroiled other scientists in the United States who were connected to Dr. He. Though Dr. He offered no proof and did not share any evidence or data that definitively proved he had done it, his colleagues had said it was possible that he had succeeded.

American scientists who knew of Dr. Hes plans are now under scrutiny. Dr. Hes former academic adviser, Stephen Quake, a star Stanford bioengineer and inventor, is facing a Stanford investigation into his interaction with his former student. Rice University has been investigating Michael Deem, Dr. Hes Ph.D. adviser, because of allegations that he was actively involved in the project.

Dr. Quake has said he had nothing to do with Dr. Hes work. Mr. Deem has said he was present for parts of Dr. Hes research but his lawyers have denied that he was actively involved.

During the Hong Kong conference, Dr. He said he used in vitro fertilization to create human embryos that were resistant to H.I.V., the virus that causes AIDS. He said he did it by using the Crispr-Cas9 editing technique to deliberately disable a gene, known as CCR, that is used to make a protein H.I.V. needs to enter cells.

The international condemnation from the scientific community that followed Dr. Hes announcement came because many nations, including the United States, had banned such work, fearing it could be misused to create designer babies and alter everything from eye color to I.Q.

Although China lacks laws governing gene editing, the practice is opposed by many researchers there. Dr. Hes work prompted soul-searching among the countrys scientists, who wondered whether many of their peers had overlooked ethical issues in the pursuit of scientific achievement.

Many of them said it was long overdue for China to enact tough laws on gene editing. Chinas vice minister of science and technology said last year that Dr. Hes scientific activities would be suspended, calling his conduct shocking and unacceptable. A group of 122 Chinese scientists called Dr. Hes actions crazy and his claims a huge blow to the global reputation and development of Chinese science.

I think a jail sentence is the proper punishment for him, said Wang Yuedan, a professor of immunology at Peking University. It makes clear our stance on the gene editing of humans that we are opposed to it.

This is a warning effect, signaling that there is a bottom line that cannot be broken.

Despite the outcry, Dr. He was unrepentant. A day after he made his announcement on the genetically edited babies, he defended his actions, saying they were safe and ethical, and he was proud of what he had done.

Dr. He faced a maximum penalty of more than 10 years in prison if his work had resulted in death. In cases that have caused serious damage to the health of the victims, the punishment is three to 10 years in prison.

The court said the trial had to be closed to the public to guard the privacy of the people involved.

Dr. Hes whereabouts had been something of a mystery for the past year. After his announcement, he was placed under guard in a small university guesthouse in Shenzhen and he has made no statements since. But his conviction was a foregone conclusion after the government said its initial investigation had found that Dr. He had seriously violated state regulations.

After Dr. Hes announcement, Bai Hua, the head of Baihualin, an AIDS advocacy group that helped Dr. He recruit the couples, said that he regretted doing so and was deeply worried about the families. In a statement posted on his organizations official WeChat account, Mr. Bai, who uses a pseudonym, said he felt deceived.

When reached by phone, Mr. Bai said he had no idea where the babies were now and declined to say whether he was assisting the government with its investigation.

One H.I.V.-infected man Dr. Hes team tried to recruit said he was not told of the ethical concerns about editing human embryos, according to Sanlian Weekly, a Chinese newsmagazine. The man said a researcher had told him that the probability of his having an unhealthy baby was low and that the team had achieved a high success rate in testing with animals.

The announcement captured the attention of many Chinese people who had not seen or heard from Dr. He in the past year. The hashtag Sentencing in the Genetically Edited Babies Case was trending on Weibo, Chinas version of Twitter.

He violated medical ethics, disrespected life and let three poor children bear the consequences, all for his fame and fortune, one user wrote. I think this punishment is too light.

Elsie Chen contributed research.

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Chinese Scientist Who Genetically Edited Babies Gets 3 Years in Prison - The New York Times

Johnson & Johnson(NYSE:JNJ) enjoyed an excellent run from 2010 to 2017, climbing from $63 to $140 before entering a more volatile period over the past two years. Since then, the stock has bounced between $120 and $147, and it sits closer to the top of that range as 2019 comes to an end. Investors who rode that wave have probably felt frustrated with the ups and downs in recent years, and they're hoping the stock will fare better next year.

The branded company's pharmaceutical segment generates 53% of total company revenue and 67% of its operating profit. This portion of the business currently markets dozens of drugs, but Stelara, Remicade, Imbruvica, Zytiga, Invega, and Darzalex are the current best sellers. Potential regulatory changes and competitionthreatenthe segment, and heavy investments in research and development or acquisitions are required to maintain arobust pipelineto replace drugs with expiring patents. Previous top-seller Remicade is experiencing declining sales due to competition, and pipeline products fromAbbVie(NYSE:ABBV) could threaten Johnson & Johnson's immunology group with pending U.S. Food and Drug Administration approvals.

Image Source: Johnson & Johnson

Medical devices are roughly 30% of total sales and 21% of operating profits. This segment is driven by numerous products for orthopedic, surgical, vision, and interventional applications. The acquisition ofAuris Healthcould hasten Johnson & Johson's entry to the robotic surgery market, which it was already targeting through a partnership withAlphabet. This part of the company has struggled to produce sales growth, with the top line declining nearly 4% year-to-date.

Finally, Johnson & Johnson has a consumer health products segment that contributes 17% of revenue and 12% of total company operating profit. These products include well-known brands such as Neutrogena, Tylenol, Aveeno, Motrin, Zyrtec, Benadryl, Visine, Nicorette, Listerine, and Band-Aid. Johnson & Johnson's consumer division will grow through acquiring and developing promising brands moving forward, but this segment is best characterized as a mature, stable, and slow-moving cash flow generator.

Johnson & Johnson's valuation is somewhat complicated by its combination of businesses because no other health stock offers a direct comparison. Conducting a sum-of-parts analysis and backing into weighted average metrics can be illuminating.

Johnson & Johnson trades at 15.6 times forward earnings, which is somewhat lower than the 18.7 weighted average of major drugmakers, consumer staples companies, and medical device makers. This figure is somewhat less exciting when adjusting for the growth outlook, which results in a relatively high 2.6 PEG ratio. The stock trades at a similar discount based on its 19.6 price-to-free-cash-flow, though the above growth rate caveat is relevant here as well. Finally, Johnson & Johnson's 16.4 EV/EBITDA is roughly in-line with the weighted average, indicating that the company's relatively high financial leverage is partially driving the apparent discount.

For income investors, the stock pays a mediocre 2.6% dividend yield. This number is fine, but there are much higher alternatives elsewhere, and Johnson & Johnson has shown dedication to a buyback program that returns value in the form of anti-dilution to stimulate appreciation rather than income.

Analysts are forecasting below 3% growth for 2020, so the investment community does not seem to recognize massive drivers in the future. Expansion into robotic surgery could help bolster growth in the device segment. Tremfya and Spravato are two drugs that could turn into blockbusters to buoy growth in the medium term. However, Johnson & Johnson is simplyso large and diversifiedthat these positive items are likely only sufficient to maintain a moderately positive growth rate.

Major regulatory changes or issues stemming from its role in theopioid crisiscould certainly send shares tumbling, but there's very little about the current growth prospects or valuation metrics to suggest Johnson & Johnson has a substantial room to the upside. It is likely more prudent to buy this stock when it trades closer to the bottom of its recent range.

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Can Johnson & Johnson Break Out In 2020? - The Motley Fool

This is the fourth of several posts written by students at the North Carolina School of Science and Math as part of an elective about science communication with Dean Amy Sheck.

Dr. Giny Foudas research focuses oninfant immune responses to infection and vaccination.

Her curiosity about immunology arose during her fourth year of medical school in Camaroon, when she randomly picked up a book on cancer immunotherapy and was captivated. Until then, she conducted research on malaria and connected it to her interest in pediatrics by studying the effects of the parasitic disease on the placentas of mothers.

As a postdoctoral fellow at Duke, shethen linked pediatrics and immunology to begin examining mother to childtransmission of disease and immunity.

Today she is an M.D. and a Ph.D. and amember of the Duke Human Vaccine Institute. Shes an assistant professor inpediatrics and an assistant research professor in the Department of Molecular Geneticsand Microbiology at Duke University School of Medicine.

Based on the recent finding that children of HIV-positive mothers are more susceptible to inheriting the disease, Fouda believes that it is important to understand how to intervene in passive immunity transmissions in order to limit them. Children and adults recover from diseases differently and uncovering these differences is important for vaccine development.

This area of research is personally important to her, because she learned from her service in health campaigns in Central Africa that it is much easier to prevent disease than to treat.

However, she believes that it is important to recognize that research is a collaborative experience with a team of scientists. Each discovery is not that of an individual, but can be accredited to everyones contribution, especially those whose roles may seem small but are vital to the everyday operations of the lab.

At the Duke Human Vaccine Institute, Fouda enjoys collaborating as a team and contributing her time as a mentor and trainer of young scientists in the next generation.

Outside of the lab, Fouda likes to spend time reading books with her daughter, traveling, decorating and gardening. If there was one factor that improve how science in immunology is conducted, she would stress that preventing disease is significantly cheaper than treating those that become infected by it.

Dr. Fouda has made some remarkable progress in the field of disease treatment with her hard working and optimistic personality, and I know that she will continue to excel in her objectives for years to come.

Post by Vandanaa Jayaprakash NCSSM 2020

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Infants, Immunity, Infections and Immunization - Duke Today