Gating strategy for MDSCs and sorting purity

TLR expression on MDSCs and viability

<p>T cells in MC38 tumors treated with MDSCs</p>

<p>Tumor conditioned medium and GM-CSF contribute to features of I-MDSCs</p>

Abstract Myeloid-derived suppressor cells (MDSC) promote colorectal cancer by several mechanisms, including suppression of antitumor T cells and production of tumorigenic factors. We previously showed that an intermediate MDSC subset (I-MDSC) is expanded in an intestinal tumor model (ApcMin/+ mice), but the importance of this subset in promoting tumors is unclear. Here, we show that I-MDSCs are a distinct heterogeneous subset due to differential and reduced expression of the monocytic marker, Ly6C, and granulocytic marker, Ly6G. Besides causing necroptotic cell death, receptor-interacting protein kinase 3 (RIPK3) has an alternate function as a signaling component inducing cytokine synthesis. We evaluated whether RIPK3 regulates inflammatory cytokines in I-MDSCs to assess the nonimmunosuppressive function of I-MDSCs in promoting tumors. Inhibition of RIPK3 with the commercially available small-molecule inhibitor GSK 872 showed that RIPK3-mediated inflammation promoted intestinal tumors in two intestinal tumor models, ApcMin/+ mice and an MC38 transplantable tumor model. Mechanistically, RIPK3 signaling in I-MDSC increased tumor size by expanding IL17-producing T cells in MC38 tumors. Collectively, these data suggest RIPK3 signaling as a potential therapeutic target in colorectal cancer. Significance: The specific role of RIPK3 in intestinal tumors and MDSC function sheds light on a key inflammatory mechanism driving tumorigenesis and allows for possible therapeutic intervention.

Suppressive function and surface marker analysis of BM-MDSCs

<p>Ripk3 inhibition alters intestinal stem cells and T cells in Apc min/+ mice</p>

<p>Surface marker analysis of Apc min/+ and MC38 tumor myeloid cells</p>

One of the mechanisms causing resistance to checkpoint inhibitor treatment is accumulation of suppressive immune cells including myeloid-derived suppressor cells (MDSCs). In CRC patients, accumulation of MDSCs correlates with poor overall survival. Among a subset of CRC patients with high mutation burden, 31.1% respond to checkpoint inhibitor treatment. This implies that targeting resistance mechanisms such as MDSCs in these CRC patients could boost the efficacy of this treatment. B7-H3 (CD279), a member of the B7 family, is expressed on myeloid cells and suppresses T-cell proliferation. Therefore, we hypothesize that B7-H3 on MDSCs could promote CRC tumor growth by suppressing cytotoxic CD8 T cells. We used polyps from ApcMin/+ mice, an intestinal tumor model and a transplantable CRC tumor model such as MC38 colon carcinoma to assess the role of B7-H3. Our findings show that B7-H3 is upregulated in ApcMin/+ polyps and is expressed on in vitro derived monocytic MDSCs (M-MDSCs). The frequency of MDSCs increased with tumor growth, indicating the close correlation between MDSCs and disease burden. Blocking B7-H3 with an antibody in MC38 tumor model significantly reduced tumor volume (control group receiving isotype antibody: 1672.2 ± 121 mm3; B7-H3 antibody treated group: 892.7 ± 36.4 mm3; p Citation Format: Asha Jayakumar, Alfred Bothwell. B7-H3 promotes antitumor T-cell suppression via MDSCs in colorectal cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A84.

Myeloid-derived suppressor cells (MDSCs) are immature suppressive cells found in tumors and immunological niches. In this article, we highlight the ability of MDSCs to promote IL-17–producing T cells (Th17) and regulatory T cells in addition to suppressing cytotoxic T cells in different tumor models. These interactions between MDSCs and T cells support tumor growth because IL-17 is tumorigenic in many cancer types and regulatory T cells suppress antitumor T cells. Besides T cells, MDSCs promote regulatory B cells and suppress overall B cell function; however, tumor-evoked regulatory B cells also regulate MDSC function, suggesting cross-regulation between MDSCs and B cells. These multiple functions shed light on how MDSCs dysregulate several arms of host immune response. Moreover, MDSCs promote tumor cell survival and angiogenesis to support tumors. Therefore, the multifunctional feature of MDSCs make them attractive immunotherapeutic targets.

Myeloid-derived suppressor cells (MDSC) promote colorectal cancer by several mechanisms, including suppression of antitumor T cells and production of tumorigenic factors. We previously showed that an intermediate MDSC subset (I-MDSC) is expanded in an intestinal tumor model (ApcMin/+ mice), but the importance of this subset in promoting tumors is unclear. Here, we show that I-MDSCs are a distinct heterogeneous subset due to differential and reduced expression of the monocytic marker, Ly6C, and granulocytic marker, Ly6G. Besides causing necroptotic cell death, receptor-interacting protein kinase 3 (RIPK3) has an alternate function as a signaling component inducing cytokine synthesis. We evaluated whether RIPK3 regulates inflammatory cytokines in I-MDSCs to assess the nonimmunosuppressive function of I-MDSCs in promoting tumors. Inhibition of RIPK3 with the commercially available small-molecule inhibitor GSK 872 showed that RIPK3-mediated inflammation promoted intestinal tumors in two intestinal tumor models, ApcMin/+ mice and an MC38 transplantable tumor model. Mechanistically, RIPK3 signaling in I-MDSC increased tumor size by expanding IL17-producing T cells in MC38 tumors. Collectively, these data suggest RIPK3 signaling as a potential therapeutic target in colorectal cancer. SIGNIFICANCE: The specific role of RIPK3 in intestinal tumors and MDSC function sheds light on a key inflammatory mechanism driving tumorigenesis and allows for possible therapeutic intervention.

KRAS mutant p53 deficient (KP) non-small cell lung carcinoma (NSCLC) lacks targeted therapies. Existing treatments for lung cancer cause resistance and result in toxicities requiring novel effective therapies. By targeting mechanisms causing resistance such as myeloid derived suppressor cells (MDSCs), KP tumors could be inhibited. MDSCs are functionally diverse and suppress T cells in many cancers. RIPK3, a cell death inducing enzyme, also functions as a signaling component producing cytokines that mediate the suppressive function of MDSCs. Partial deletion of RIPK3 in myeloid cells reduced KP tumor growth. This also reduced accumulation of MDSCs with a consistent increase in antigen specific IFNγ producing CD8 T cells. Inhibiting RIPK3 with a small molecule inhibitor such as GSK 872 effectively reduces RIPK3 activity in myeloid cells including MDSCs and reduces growth of small and large KP tumors. GSK 872 in combination with checkpoint inhibitors such as anti PD-1 and anti CTLA-4 further decreased KP tumor size. Together, our findings show that inhibiting RIPK3 in MDSCs is effective in inhibiting KP NSCLC and is a viable therapeutic option for improving existing immunotherapeutic treatments.

Intestinal tumorigenesis in the ApcMin/+ model is initiated by aberrant activation of Wnt pathway. Increased IL-4 expression in human colorectal cancer tissue and growth of colon cancer cell lines implied that IL-4–induced Stat6-mediated tumorigenic signaling likely contributes to intestinal tumor progression in ApcMin/+ mice. Stat6 also appears to promote expansion of myeloid-derived suppressor cells (MDSCs) cells. MDSCs promote polyp formation in the ApcMin/+ model. Hence, Stat6 could have a broad role in coordinating both polyp cell proliferation and MDSC expansion. We found that IL-4–induced Stat6-mediated proliferation of intestinal epithelial cells is augmented by platelet-derived growth factor–BB, a tumor-promoting growth factor. To determine whether polyp progression in ApcMin/+ mice is dependent on Stat6 signaling, we disrupted Stat6 in this model. Total polyps in the small intestine were fewer in ApcMin/+ mice lacking Stat6. Furthermore, proliferation of polyp epithelial cells was reduced, indicating that Stat6 in part controlled polyp formation. Stat6 also promoted expansion of MDSCs in the spleen and lamina propria of ApcMin/+ mice, implying regulation of antitumor T-cell response. More CD8 cells and reduced PD-1 expression on CD4 cells correlated with reduced polyps. In addition, a strong CD8-mediated cytotoxic response led to killing of tumor cells in Stat6-deficient ApcMin/+ mice. Therefore, these findings show that Stat6 has an oncogenic role in intestinal tumorigenesis by promoting polyp cell proliferation and immunosuppressive mediators, and preventing an active cytotoxic process.

BACKGROUND:Leishmania major infection induces robust interleukin-12 (IL12) production in human dendritic cells (hDC), ultimately resulting in Th1-mediated immunity and clinical resolution. The surface of Leishmania parasites is covered in a dense glycocalyx consisting of primarily lipophosphoglycan (LPG) and other phosphoglycan-containing molecules (PGs), making these glycoconjugates the likely pathogen-associated molecular patterns (PAMPS) responsible for IL12 induction. METHODOLOGY/PRINCIPAL FINDINGS:Here we explored the role of parasite glycoconjugates on the hDC IL12 response by generating L. major Friedlin V1 mutants defective in LPG alone, (FV1 lpg1-), or generally deficient for all PGs, (FV1 lpg2-). Infection with metacyclic, infective stage, L. major or purified LPG induced high levels of IL12B subunit gene transcripts in hDCs, which was abrogated with FV1 lpg1- infections. In contrast, hDC infections with FV1 lpg2- displayed increased IL12B expression, suggesting other PG-related/LPG2 dependent molecules may act to dampen the immune response. Global transcriptional profiling comparing WT, FV1 lpg1-, FV1 lpg2- infections revealed that FV1 lpg1- mutants entered hDCs in a silent fashion as indicated by repression of gene expression. Transcription factor binding site analysis suggests that LPG recognition by hDCs induces IL-12 in a signaling cascade resulting in Nuclear Factor κ B (NFκB) and Interferon Regulatory Factor (IRF) mediated transcription. CONCLUSIONS/SIGNIFICANCE:These data suggest that L. major LPG is a major PAMP recognized by hDC to induce IL12-mediated protective immunity and that there is a complex interplay between PG-baring Leishmania surface glycoconjugates that result in modulation of host cellular IL12.

Leishmania major-infected human dendritic cells (DCs) exhibit a marked induction of IL-12, ultimately promoting a robust Th1-mediated response associated with parasite killing and protective immunity. The host cell transcription machinery associated with the specific IL-12 induction observed during L. major infection remains to be thoroughly elucidated. In this study, we used Affymetrix GeneChip (Affymetrix) to globally assess the host cell genes and pathways associated with early L. major infection in human myeloid-derived DCs. Our data revealed 728 genes were significantly differentially expressed and molecular signaling pathway revealed that the type I IFN pathway was significantly enriched. Addition of a neutralizing type I IFN decoy receptor blocked the expression of IRF7 and IL-12p40 during DC infection, indicating the L. major-induced expression of IL-12p40 is dependent upon the type I IFN signaling pathway. In stark contrast, IL-12p40 expression is not elicited by L. donovani, the etiological agent of deadly visceral leishmaniasis. Therefore, we examined the gene expression profile for several IFN response genes in L. major versus L. donovani DC infections. Our data revealed that L. major, but not L. donovani, induces expression of IRF2, IRF7, and IFIT5, implicating the regulation of type I IFN-associated signaling pathways as mediating factors toward the production of IL-12.

Background: T-cell tolerance of allergic cutaneous contact sensitivity (CS) induced in mice by high doses of reactive hapten is mediated by suppressor cells that release antigen-specific suppressive nanovesicles.Objective: We sought to determine the mechanism or mechanisms of immune suppression mediated by the nanovesicles.Methods: T-cell tolerance was induced by means of intravenous injection of hapten conjugated to self-antigens of syngeneic erythrocytes and subsequent contact immunization with the same hapten. Lymph node and spleen cells from tolerized or control donors were harvested and cultured to produce a supernatant containing suppressive nanovesicles that were isolated from the tolerized mice for testing in active and adoptive cell-transfer models of CS.Results: Tolerance was shown due to exosome-like nanovesicles in the supernatants of CD8 1 suppressor T cells that were not regulatory T cells. Antigen specificity of the suppressive nanovesicles was conferred by a surface coat of antibody light chains or possibly whole antibody, allowing targeted delivery of selected inhibitory microRNA (miRNA)-150 to CS effector T cells. Nanovesicles also inhibited CS in actively sensitized mice after systemic injection at the peak of the responses. The role of antibody and miRNA-150 was established by tolerizing either panimmunoglobulin-deficient JH(-/-) or miRNA-150(-/-) mice that produced nonsuppressive nanovesicles. These nanovesicles could be made suppressive by adding antigen-specific antibody light chains or miRNA-150, respectively.Conclusions: This is the first example of T-cell regulation through systemic transit of exosome-like nanovesicles delivering a chosen inhibitory miRNA to target effector T cells in an antigen-specific manner by a surface coating of antibody light chains.

Leishmania (Viannia) parasites present particular challenges, as human and murine immune responses to infection are distinct from other Leishmania species, indicating a unique interaction with the host. Further, vaccination studies utilizing small animal models indicate that modalities and antigens that prevent infection by other Leishmania species are generally not protective.Using a newly developed mouse model of chronic L. (Viannia) panamensis infection and the heterologous DNA prime - modified vaccinia virus Ankara (MVA) boost vaccination modality, we examined whether the conserved vaccine candidate antigen tryparedoxin peroxidase (TRYP) could provide protection against infection/disease.Heterologous prime - boost (DNA/MVA) vaccination utilizing TRYP antigen can provide protection against disease caused by L. (V.) panamensis. However, protection is dependent on modulating the innate immune response using the TLR1/2 agonist Pam3CSK4 during DNA priming. Prime-boost vaccination using DNA alone fails to protect. Prior to infection protectively vaccinated mice exhibit augmented CD4 and CD8 IFNγ and memory responses as well as decreased IL-10 and IL-13 responses. IL-13 and IL-10 have been shown to be independently critical for disease in this model. CD8 T cells have an essential role in mediating host defense, as CD8 depletion reversed protection in the vaccinated mice; vaccinated mice depleted of CD4 T cells remained protected. Hence, vaccine-induced protection is dependent upon TLR1/2 activation instructing the generation of antigen specific CD8 cells and restricting IL-13 and IL-10 responses.Given the general effectiveness of prime-boost vaccination, the recalcitrance of Leishmania (Viannia) to vaccine approaches effective against other species of Leishmania is again evident. However, prime-boost vaccination modality can with modulation induce protective responses, indicating that the delivery system is critical. Moreover, these results suggest that CD8 T cells should be targeted for the development of a vaccine against infection caused by Leishmania (Viannia) parasites. Further, TLR1/2 modulation may be useful in vaccines where CD8 T cell responses are critical.

In visceral leishmaniasis, the draining LN (DLN) is the initial site for colonization and establishment of infection after intradermal transmission by the sand fly vector; however, little is known about the developing immune response within this site. Using an intradermal infection model, which allows for parasite visceralization, we have examined the ongoing immune responses in the DLN of BALB/c mice infected with Leishmania infantum. Although not unexpected, at early times post-infection there is a marked B-cell expansion in the DLN, which persists throughout infection. However, the characteristics of this response were of interest; as early as day 7 post-infection, polyclonal antibodies (TNP, OVA, chromatin) were observed and the levels appeared comparable to the specific anti-leishmania response. Although B-cell-deficient J(h)D BALB/c mice are relatively resistant to infection, neither B-cell-derived IL-10 nor B-cell antigen presentation appear to be primarily responsible for the elevated parasitemia. However, passive transfer and reconstitution of J(h)D BALB/c with secretory immunoglobulins, (IgM or IgG; specific or non-specific immune complexes) results in increased susceptibility to L. infantum infection. Further, J(h)D BALB/c mice transgenetically reconstituted to secrete IgM demonstrated exacerbated disease in comparison to WT BALB/c mice as early as 2 days post-infection. Evidence suggests that complement activation (generation of C5a) and signaling via the C5a receptor (CD88) is related to the disease exacerbation caused by IgM rather than cytokine levels (IL-10 or IFN-gamma). Overall these studies indicate that polyclonal B-cell activation, which is known to be associated with human visceral leishmaniasis, is an early and intrinsic characteristic of disease and may represent a target for therapeutic intervention.