A major barrier to successful antitumor vaccination is tolerance of high-avidity T cells specific to tumor antigens. In keeping with this notion, HER-2/neu (neu)-targeted vaccines, which raise strong CD8+ T cell responses to a dominant peptide (RNEU420-429) in WT FVB/N mice and protect them from a neu-expressing tumor challenge, fail to do so in MMTV-neu (neu-N) transgenic mice. However, treatment of neu-N mice with vaccine and cyclophosphamide-containing chemotherapy resulted in tumor protection in a proportion of mice. This effect was specifically abrogated by the transfer of neu-N–derived CD4+CD25+ T cells. RNEU420-429-specific CD8+ T cells were identified only in neu-N mice given vaccine and cyclophosphamide chemotherapy which rejected tumor challenge. Tetramer-binding studies demonstrated that cyclophosphamide pretreatment allowed the activation of high-avidity RNEU420-429-specific CD8+ T cells comparable to those generated from vaccinated FVB/N mice. Cyclophosphamide seemed to inhibit regulatory T (T reg) cells by selectively depleting the cycling population of CD4+CD25+ T cells in neu-N mice. These findings demonstrate that neu-N mice possess latent pools of high-avidity neu-specific CD8+ T cells that can be recruited to produce an effective antitumor response if T reg cells are blocked or removed by using approaches such as administration of cyclophosphamide before vaccination.

DNMT3a is a de novo DNA methyltransferase expressed robustly after T-cell activation that regulates plasticity of CD4 + T-cell cytokine expression. Here we show that DNMT3a is critical for directing early CD8 + T-cell effector and memory fate decisions. Whereas effector function of DNMT3a knockout T cells is normal, they develop more memory precursor and fewer terminal effector cells in a T-cell intrinsic manner compared with wild-type animals. Rather than increasing plasticity of differentiated effector CD8 + T cells, loss of DNMT3a biases differentiation of early effector cells into memory precursor cells. This is attributed in part to ineffective repression of Tcf1 expression in knockout T cells, as DNMT3a localizes to the Tcf7 promoter and catalyzes its de novo methylation in early effector WT CD8 + T cells. These data identify DNMT3a as a crucial regulator of CD8 + early effector cell differentiation and effector versus memory fate decisions.antigen-specific T-cell clone undergoes massive proliferation and clonal expansion, generating a heterogeneous population of daughter cells (1). Shortly after activation, CD8 + T cells down-regulate CD62L and CD127 and have been termed early effector cells. These further divide and differentiate into CD127 − killer cell lectin-like receptor G1 (KLRG1) + terminal effector and CD127 + KLRG1 − memory precursor cells (2-4). Several factors have been identified that influence the differentiation and polarization of early effector cells toward either terminal effector cells or memory precursor cells. The initial CD8 + T-cell clonal frequency (5, 6), inflammatory signals driving transcription factor expression (2, 7), cytokine stimulation (8, 9), and transcription factor expression levels (10, 11) all impact the fate of early effector CD8 + T cells. As these T cells are genetically identical, cellular processes of epigenetic regulation would also be predicted to play a key role in determining and perpetuating the fate decisions of individual CD8 + T cells.Epigenetic gene regulation encompasses the heritable covalent DNA and histone posttranslational modifications made in individual cells at specific gene loci that function to regulate the accessibility of these genes within chromatin to transcriptional activation (recently reviewed in ref. 12). Epigenetic regulation within T cells has been studied in detail for individual genes (13, 14) and more recently on the whole genome scale (15-17). These studies have identified patterns of histone marks and DNA methylation that differ across the genome between naïve, activated, and memory T cells and correlate with patterns of gene expression.DNA methylation on the cytosine of CpG dinucleotides in gene promoter regions is associated with silencing gene expression. Of the DNA methyltransferases, only DNA methyltransferase 3a (DNMT3a) and 3b (DNMT3b) are capable of adding de novo CpG methylation marks and thus may dynamically regulate gene silencing. We and others have previously shown that DNMT3a is the dominant DNA methyltransferase act...

T cell costimulation via OX40 is known to increase CD4+ T cell expansion and effector function and enhances the development of T cell memory. OX40 costimulation can also prevent, and even reverse, CD4+ T cell anergy. However, the role of OX40 in CD8+ T cell function is less well defined, particularly in the setting of immune tolerance. To determine the effects of OX40 costimulation on the induction of the host CD8+ T cell repertoire to an endogenous tumor Ag, we examined the fate of CD8+ T cells specific for the immunodominant rat HER-2/neu epitope, RNEU420–429, in FVB MMTV-neu (neu-N) mice, which express rat HER-2/neu protein in a predominantly mammary-restricted fashion. We show that the RNEU420–429-specific T cell repertoire in neu-N mice expands transiently after vaccination with a neu-targeted GM-CSF-secreting whole-cell vaccine, but quickly declines to an undetectable level. However, OX40 costimulation, when combined with GM-CSF-secreting tumor-targeted vaccination, can break established CD8+ T cell tolerance in vivo by enhancing the expansion, and prolonging the survival and effector function of CD8+ T cells specific for RNEU420–429. Moreover, we demonstrate that OX40 expression is up-regulated on both CD4+ and CD8+ T cells shortly after administration of a GM-CSF expressing vaccine. These studies highlight the increased efficacy of OX40 costimulation when combined with a GM-CSF-secreting vaccine, and define a new role for OX40 costimulation of CD8+ T cells in overcoming tolerance and boosting antitumor immunity.

Background: T cells undergo lineage commitment during an immune response and "remember" their lineage choice after the instructive signals cease. Results: T cells lacking a de novo DNA methyltransferase fail to silence the ifn␥ gene. Conclusion: DNMT3a opposes T cell trans-differentiation by epigenetically silencing "off-lineage" genes. Significance: The proper control of inflammatory cytokine gene expression is crucial for immune homeostasis.

Translational Relevance Treatment paradigm for sarcomas has been unchanged for the past four decades with survival outcomes plateauing and patients with HR disease facing an abysmal prognosis. SARC028, trial testing pembrolizumab in STS, demonstrated responses in UPS and dedifferentiated liposarcomas. Alliance A091401, trial combining ipilimumab and nivolumab, showed responses in more histologies, but OS rates similar to standard chemotherapy. Here, we compare the immune TME of two molecularly distinct sarcomas: the genetically complex, ICR, UPS, and the genetically simple, fusion-driven, poorly ICR, RMS, to identify factors that may contribute to their immunotherapy responsiveness. These two subtypes represent the main genomic aberrancies observed in sarcomas. Results show both tumors dominated by TAMs. T-cells in UPS are diffusely distributed, while T-cells in RMS cluster with B-cells near perivascular beds forming TLS. Our findings suggest targeting the myeloid compartment and tumor angiogenesis could overcome the immunosuppressive niche sustained by TAMs and lead to potential therapeutic targets.

BackgroundCurrent therapy for osteosarcoma pulmonary metastases (PMs) is ineffective. The mechanisms that prevent successful immunotherapy in osteosarcoma are incompletely understood. We investigated the tumor microenvironment of metastatic osteosarcoma with the goal of harnessing the immune system as a therapeutic strategy.Methods66 osteosarcoma tissue specimens were analyzed by immunohistochemistry (IHC) and immune markers were digitally quantified. Tumor-infiltrating lymphocytes (TILs) from 25 specimens were profiled by functional cytometry. Comparative transcriptomic studies of distinct tumor-normal lung ‘PM interface’ and ‘PM interior’ regions from 16 PMs were performed. Clinical follow-up (median 24 months) was available from resection.ResultsIHC revealed a statistically significantly higher concentration of TILs expressing immune checkpoint and immunoregulatory molecules in PMs compared with primary bone tumors (including programmed cell death 1 (PD-1), programmed death ligand 1 (PD-L1), lymphocyte-activation gene 3 (LAG-3), T-cell immunoglobulin and mucin domain-containing protein 3 (TIM-3), and indoleamine 2,3-dioxygenase (IDO1). Remarkably, these lymphocytes are excluded at the PM interface compared with PM interior. TILs from PMs exhibited significantly higher amounts of PD-1 and LAG-3 and functional cytokines including interferon-γ (IFNγ) by flow cytometry. Gene expression profiling further confirmed the presence of CD8 and CD4 lymphocytes concentrated at the PM interface, along with upregulation of immunoregulatory molecules and IFNγ-driven genes in the same region. We further discovered a strong alternatively activated macrophage signature throughout the entire PMs along with a polymorphonuclear myeloid-derived suppressor cell signature focused at the PM interface. Expression of PD-L1, LAG-3, and colony-stimulating factor 1 receptor (CSF1R) at the PM interface was associated with significantly worse progression-free survival (PFS), while gene sets indicative of productive T cell immune responses (CD8 T cells, T cell survival, and major histocompatibility complex class 1 expression) were associated with significantly improved PFS.ConclusionsOsteosarcoma PMs exhibit immune exclusion characterized by the accumulation of TILs at the PM interface. These TILs produce effector cytokines, suggesting their capability of activation and recognition of tumor antigens. Our findings suggest cooperative immunosuppressive mechanisms in osteosarcoma PMs including immune checkpoint molecule expression and the presence of immunosuppressive myeloid cells. We identify cellular and molecular signatures that are associated with patient outcomes, which could be exploited for successful immunotherapy.

The composition of tumor infiltrating lymphocytes (TIL) is heterogeneous. In addition, the ratio of various subpopulations in the tumor microenvironment is highly dependent on the nature of the host's immune response. Here, we characterize Foxp3-expressing CD8 1 T cells in the tumor that demonstrate effector function and accumulate in the context of an effective antitumor response. CD8 1 Foxp3 1 T cells are induced in TIL in regressing tumors of FVB/N mice treated with a GM-CSF secreting HER-2/neu targeted whole cell vaccine. Foxp3 expression in tumor antigen-specific CD8 T cells is restricted to the tumor microenvironment and influenced by cues in the tumor. Interestingly, Foxp3 1 and Foxp3 2 CD8 1 T cells have similar IFN-c production and antigen-specific degranulation after stimulation with RNEU 420-429 , the immunodominant HER-2/neu (neu) epitope in this model. Adoptive transfer studies, using RNEU (420-429) -specific effector T cells into neu-N mice (a model that results in immune tolerance to neu), confirm that CD8 1 Foxp3 1 T cells are present in tumors only if there is an existing pool of tumor-rejecting effector T cells. CD8 1 Foxp3 1 TILs mark the presence of tumor-rejecting antigen-specific T cells and their accumulation serves as a marker for an effective T cell response.

Most effector CD8+ T cells die, while some persist and become either “effector” (TEM) or “central” (TCM) memory T cells. Paradoxically, effector CD8+ T cells with greater memory potential have higher levels of the pro-apoptotic molecule Bim. Here, we report, using a novel Bim-mCherry knock-in mouse, that cells with high levels of Bim preferentially develop into TCM cells. Bim levels remained stable and were regulated by DNA methylation at the Bim promoter. Notably, high levels of Bcl-2 were required for Bimhi cells to survive. Using Nur77-GFP mice as an indicator of TCR signal strength, Nur77 levels correlated with Bim expression and Nur77hi cells also selectively developed into TCM cells. Altogether, these data show that Bim levels and TCR signal strength are predictive of TEM- vs. TCM-cell fate. Further, given the many other biologic functions of Bim, these mice will have broad utility beyond CD8+ T-cell fate.