Payal Watchmaker

Type-1 polarized dendritic cells primed for high IL-12 production show enhanced activity as cancer vaccines.

While multiple pathways of dendritic cell (DC) maturation result in transient production of IL-12, fully mature DCs show reduced ability to produce IL-12p70 upon a subsequent interaction with Ag-specific T cells, limiting their in vivo performance as vaccines. Such “DC exhaustion” can be prevented by the presence of IFNγ during the maturation of human DCs (type-1-polarization), resulting in improved induction of tumor-specific Th1 and CTL responses in vitro. Here, we show that type-1 polarization of mouse DCs strongly enhances their ability to induce CTL responses against a model tumor antigen, OVA, in vivo, promoting the induction of protective immunity against OVA-expressing EG7 lymphoma. Interestingly, in contrast to the human system, the induction of mouse DC1s requires the participation of IL-4, a nominal Th2-inducing cytokine. The current data help to explain the previously reported Th1-driving and anti-tumor activities of IL-4, and demonstrate that type-1 polarization increases in vivo activity of DC-based vaccines.

Helper roles of NK and CD8+ T cells in the induction of tumor immunity. Polarized dendritic cells as cancer vaccines.

The work in our laboratory addresses two interrelated areas of dendritic cell (DC) biology: (1) the role of DCs as mediators of feedback interactions between NK cells, CD8+ and CD4+ T cells; and (2) the possibility to use such feedback and the paradigms derived from anti-viral responses, to promote the induction of therapeutic immunity against cancer. We observed that CD8+ T cells and NK cells, the classical “effector” cells, also play “helper” roles, regulating ability of DCs to induce type-1 immune immunity, critical for fighting tumors and intracellular pathogens. Our work aims to delineate which pathways of NK and CD8+ T cell activation result in their helper activity, and to identify the molecular mechanisms allowing them to induce type-1 polarized DCs (DC1s) with selectively enhanced ability to promote type-1 responses and anti-cancer immunity. The results of these studies allowed us and our colleagues to design phase I/II clinical trials incorporating the paradigms of DC polarization and helper activity of effector cells in cancer immunotherapy.

Independent Regulation of Chemokine Responsiveness and Cytolytic Function versus CD8+ T Cell Expansion by Dendritic Cells

The ability of cancer vaccines to induce tumor-specific CD8+ T cells in the circulation of cancer patients has been shown to poorly correlate with their clinical effectiveness. In this study, we report that although Ags presented by different types of mature dendritic cells (DCs) are similarly effective in inducing CD8+ T cell expansion, the acquisition of CTL function and peripheral-type chemokine receptors, CCR5 and CXCR3, requires Ag presentation by a select type of DCs. Both “standard” DCs (matured in the presence of PGE2) and type 1-polarized DCs (DC1s) (matured in the presence of IFNs and TLR ligands, which prevent DCs “exhaustion”) are similarly effective in inducing CD8+ T cell expansion and acquisition of CD45RO+IL-7R+IL-15R+ phenotype. However, granzyme B expression, acquisition of CTL activity, and peripheral tissue-type chemokine responsiveness are features exclusively exhibited by CD8+ T cells activated by DC1s. This advantage of DC1s was observed in polyclonally activated naive and memory CD8+ T cells and in blood-isolated melanoma-specific CTL precursors. Our data help to explain the dissociation between the ability of cancer vaccines to induce high numbers of tumor-specific CD8+ T cells in the blood of cancer patients and their ability to promote clinical responses, providing for new strategies of cancer immunotherapy.

Isocitrate dehydrogenase mutations suppress STAT1 and CD8 + T cell accumulation in gliomas

Mutations in the isocitrate dehydrogenase genes IDH1 and IDH2 are among the first genetic alterations observed during the development of lower-grade glioma (LGG). LGG-associated IDH mutations confer gain-of-function activity by converting &agr;-ketoglutarate to the oncometabolite R-2-hydroxyglutarate (2HG). Clinical samples and gene expression data from The Cancer Genome Atlas (TCGA) demonstrate reduced expression of cytotoxic T lymphocyte–associated genes and IFN-&ggr;–inducible chemokines, including CXCL10, in IDH-mutated (IDH-MUT) tumors compared with IDH-WT tumors. Given these findings, we have investigated the impact of IDH mutations on the immunological milieu in LGG. In immortalized normal human astrocytes (NHAs) and syngeneic mouse glioma models, the introduction of mutant IDH1 or treatment with 2HG reduced levels of CXCL10, which was associated with decreased production of STAT1, a regulator of CXCL10. Expression of mutant IDH1 also suppressed the accumulation of T cells in tumor sites. Reductions in CXCL10 and T cell accumulation were reversed by IDH-C35, a specific inhibitor of mutant IDH1. Furthermore, IDH-C35 enhanced the efficacy of vaccine immunotherapy in mice bearing IDH-MUT gliomas. Our findings demonstrate a mechanism of immune evasion in IDH-MUT gliomas and suggest that specific inhibitors of mutant IDH may improve the efficacy of immunotherapy in patients with IDH-MUT gliomas.

Memory CD8+ T Cells Protect Dendritic Cells from CTL Killing

CD8+ T cells have been shown to be capable of either suppressing or promoting immune responses. To reconcile these contrasting regulatory functions, we compared the ability of human effector and memory CD8+ T cells to regulate survival and functions of dendritic cells (DC). We report that, in sharp contrast to the effector cells (CTLs) that kill DCs in a granzyme B- and perforin-dependent mechanism, memory CD8+ T cells enhance the ability of DCs to produce IL-12 and to induce functional Th1 and CTL responses in naive CD4+ and CD8+ T cell populations. Moreover, memory CD8+ T cells that release the DC-activating factor TNF-α before the release of cytotoxic granules induce DC expression of an endogenous granzyme B inhibitor PI-9 and protect DCs from CTL killing with similar efficacy as CD4+ Th cells. The currently identified DC-protective function of memory CD8+ T cells helps to explain the phenomenon of CD8+ T cell memory, reduced dependence of recall responses on CD4+ T cell help, and the importance of delayed administration of booster doses of vaccines for the optimal outcome of immunization.

Novel and shared neoantigen derived from histone 3 variant H3.3K27M mutation for glioma T cell therapy

The median overall survival for children with diffuse intrinsic pontine glioma (DIPG) is less than one year. The majority of diffuse midline gliomas, including more than 70% of DIPGs, harbor an amino acid substitution from lysine (K) to methionine (M) at position 27 of histone 3 variant 3 (H3.3). From a CD8+ T cell clone established by stimulation of HLA-A2+ CD8+ T cells with synthetic peptide encompassing the H3.3K27M mutation, complementary DNA for T cell receptor (TCR) &agr;- and &bgr;-chains were cloned into a retroviral vector. TCR-transduced HLA-A2+ T cells efficiently killed HLA-A2+H3.3K27M+ glioma cells in an antigen- and HLA-specific manner. Adoptive transfer of TCR-transduced T cells significantly suppressed the progression of glioma xenografts in mice. Alanine-scanning assays suggested the absence of known human proteins sharing the key amino acid residues required for recognition by the TCR, suggesting that the TCR could be safely used in patients. These data provide us with a strong basis for developing T cell–based therapy targeting this shared neoepitope.

Single-cell profiling of human gliomas reveals macrophage ontogeny as a basis for regional differences in macrophage activation in the tumor microenvironment

BackgroundTumor-associated macrophages (TAMs) are abundant in gliomas and immunosuppressive TAMs are a barrier to emerging immunotherapies. It is unknown to what extent macrophages derived from peripheral blood adopt the phenotype of brain-resident microglia in pre-treatment gliomas. The relative proportions of blood-derived macrophages and microglia have been poorly quantified in clinical samples due to a paucity of markers that distinguish these cell types in malignant tissue.ResultsWe perform single-cell RNA-sequencing of human gliomas and identify phenotypic differences in TAMs of distinct lineages. We isolate TAMs from patient biopsies and compare them with macrophages from non-malignant human tissue, glioma atlases, and murine glioma models. We present a novel signature that distinguishes TAMs by ontogeny in human gliomas. Blood-derived TAMs upregulate immunosuppressive cytokines and show an altered metabolism compared to microglial TAMs. They are also enriched in perivascular and necrotic regions. The gene signature of blood-derived TAMs, but not microglial TAMs, correlates with significantly inferior survival in low-grade glioma. Surprisingly, TAMs frequently co-express canonical pro-inflammatory (M1) and alternatively activated (M2) genes in individual cells.ConclusionsWe conclude that blood-derived TAMs significantly infiltrate pre-treatment gliomas, to a degree that varies by glioma subtype and tumor compartment. Blood-derived TAMs do not universally conform to the phenotype of microglia, but preferentially express immunosuppressive cytokines and show an altered metabolism. Our results argue against status quo therapeutic strategies that target TAMs indiscriminately and in favor of strategies that specifically target immunosuppressive blood-derived TAMs.

Abstract 3767: Identification of a novel and a shared H3.3K27M mutation derived neoantigen epitope and H3.3K27M specific TCR engineered T cell therapy for glioma

Brain cancers are the leading cause of cancer related mortality in children and young adults with median overall survival of 9-10 months and hence represents a significant unmet medical need. Genome-wide sequencing efforts of pediatric gliomas have identified a recurrent and shared missense mutation in the gene encoding the replication-independent variant of histone 3, H3.3. Approximately 70% of diffuse intrinsic pontine gliomas (DIPG) and 50% of thalamic and other midline gliomas harbor the amino acid substitution from lysine (K) to methionine (M) at the position 27 of H3.3 gene. Tumor specific missense mutations are not subjected to self-tolerance and can be suitable targets (neoantigens) for cancer immunotherapy. Herein, we evaluated whether the H3.3K27M mutation can induce specific cytotoxic T lymphocyte (CTL) response in HLA-A2+ T cells. In vitro stimulation of HLA-A2+ donor derived CD8+ T cells with a synthetic peptide encompassing the H3.3K27M mutation (H3.3K27M epitope) induced CTL lines which recognized not only T2 cells loaded with the synthetic H3.3K27M epitope peptide but also lysed the HLA-A2+ DIPG cells which endogenously harbor the H3.3K27M mutation. On the other hand, the CTL lines did not react to either HLA-A2+ but H3.3K27M- negative DIPG cell lines or H3.3K27M positive but HLA-A2 negative DIPG cells. The H3.3K27M epitope peptide but not the non-mutant counterpart indicated an excellent binding affinity (Kd 151nM) to HLA-A2 based on competitive binding inhibition assay. From CTL clones with high and specific affinities to HLA-A2-H3.3K27M-tetramer, cDNAs for T cell receptor (TCR) alpha and beta chains were cloned into a retroviral vector. Human HLA-A2+ T cells transduced with the TCR demonstrated antigen specific reactivity as well as anti-glioma responses in vitro. Peptide titration assays suggested that the H3.3K27M specific TCR had the half-maximal reactivity for peptide recognition of around 100nM. Furthermore, critically important for safety of clinical application, alanine scanning demonstrated that the key amino acid sequence motif in the epitope of the TCR reactivity is not shared by any known human protein. Finally, intravenous administration of T cells transduced with H3.3K27M specific TCR significantly inhibited the growth of intracranial HLA-A2+ H3.3K27M positive glioma xenografts in immune deficient NSG mice. These data provide us with a strong basis for developing peptide based vaccines as well as adoptive transfer therapy using autologous T cells transduced with the H3.3K27M specific TCR. Acknowledgements: This study is supported by the NIH/NINDS (1RO1NS096954), V Foundation and Parker Institution for Cancer Immunotherapy. Citation Format: Zinal Chheda, Gary Kohanbash, John Sidney, Kaori Okada, Naznin Jahan, Diego Carrera, Payal Watchmaker, Kira Downey, Shuming Liu, Shruti Shrivastav, Sabine Mueller, Ian F. Pollack, Angel M. Carcaboso, Alessandro Sette, Yafei Hou, Hideho Okada. Identification of a novel and a shared H3.3K27M mutation derived neoantigen epitope and H3.3K27M specific TCR engineered T cell therapy for glioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3767. doi:10.1158/1538-7445.AM2017-3767