DNA methylation based glioblastoma subclassification is related to tumoral T-cell infiltration and patient survival

BackgroundHistologically classified glioblastomas (GBM) can have different clinical behavior and response to therapy, for which molecular subclassifications have been proposed. We evaluated the relationship of epigenetic GBM subgroups with immune cell infiltrations, systemic immune changes during radiochemotherapy, and clinical outcome.Methods450K genome-wide DNA methylation was assessed on tumor tissue from 93 patients with newly diagnosed GBM, treated with standard radiochemotherapy and experimental immunotherapy. Tumor infiltration of T cells, myeloid cells, and Programmed cell death protein 1 (PD-1) expression were evaluated. Circulating immune cell populations and selected cytokines were assessed on blood samples taken before and after radiochemotherapy.ResultsForty-two tumors had a mesenchymal, 27 a receptor tyrosine kinase (RTK) II, 17 RTK I, and 7 an isocitrate dehydrogenase (IDH) DNA methylation pattern. Mesenchymal tumors had the highest amount of tumor-infiltrating CD3+ and CD8+ T cells and IDH tumors the lowest. There were no significant differences for CD68+ cells, FoxP3+ cells, and PD-1 expression between groups. Systemically, there was a relative increase of CD8+ T cells and CD8+ PD-1 expression and a relative decrease of CD4+ T cells after radiochemotherapy in all subgroups except IDH tumors. Overall survival was the longest in the IDH group (median 36 mo), intermediate in RTK II tumors (27 mo), and significantly lower in mesenchymal and RTK I groups (15.5 and 16 mo, respectively).ConclusionsMethylation based stratification of GBM is related to T-cell infiltration and survival, with IDH and mesenchymal tumors representing both ends of a spectrum. DNA methylation profiles could be useful in stratifying patients for immunotherapy trials.     

Characterization and immunotherapeutic potential of γδ T-cells in patients with glioblastoma

Classical immunotherapeutic approaches to glioblastoma multiforme (GBM) have shown mixed results, and therapies focused on innate lymphocyte activity against GBM have not been rigorously evaluated. We examined peripheral blood lymphocyte phenotype, γδ T-cell number, mitogenic response, and cytotoxicity against GBM cell lines and primary tumor explants from GBM patients at selected time points prior to and during GBM therapy. Healthy volunteers served as controls and were grouped by age. T-cell infiltration of tumors from these patients was assessed by staining for CD3 and T-cell receptor γδ. Our findings revealed no differences in counts of mean absolute T-cells, T-cell subsets CD3⁺CD4⁺ and CD3⁺CD8⁺, and natural killer cells from healthy volunteers and patients prior to and immediately after GBM resection. In contrast, γδ T-cell counts and mitogen-stimulated proliferative response of γδ T-cells were markedly decreased prior to GBM resection and throughout therapy. Expanded/activated γδ T-cells from both patients and healthy volunteers kill GBM cell lines D54, U373, and U251, as well as primary GBM, without cytotoxicity to primary astrocyte cultures. Perivascular T-cell accumulation was noted in paraffin sections, but no organized T-cell invasion of the tumor parenchyma was seen. Taken together, these data suggest that γδ T-cell depletion and impaired function occur prior to or concurrent with the growth of the tumor. The significant cytotoxicity of expanded/activated γδ T-cells from both healthy controls and selected patients against primary GBM explants may open a previously unexplored approach to cellular immunotherapy of GBM.     

Dominance of CD4+ alpha/beta T-cells and inferior role of innate immune reaction in liver metastases

BACKGROUND: Tumor infiltrating lymphocytes (TIL) are frequently present in human tumors with CD8+(-)T-cells as effector and CD4+ T-cells as helper cells. Despite the well established knowledge about primary tumors, only little is known about metastatic disease, especially for liver metastases. The role of the innate immune system in the tumor defence is still enigmatic. MATERIALS AND METHODS: We performed a subtyping of TIL in 20 liver metastases. Using immunohistochemistry, CD20+, CD3+, CD56+, CD4+ and CD8+ lymphocytes, gamma/delta-T-cells and alpha/beta-T-cells in the tumor, the peritumoral region, portal tracts and lobules were investigated. RESULTS: The immune response was highly accentuated in the surroundings of the metastases with only few lymphocytes in the tumor itself. There was a dominance of CD3+(-)CD4+(-)alpha/beta-T-cells with a lower number of CD8+(-)T-cells. The CD4+/CD8+ ratio was 6:1. CD56+(-)NK/NKT-cells and gamma/delta-T-cells were rare. No differences were found between metastases from different primaries or according to the number or diameter of the metastases. CONCLUSION: TIL are part of an interaction between the metastatic tumor and the liver. Among them CD4+ T-cells seem to have a unique independent function in tumor response. The localization of the immune response in the tumor periphery might be a reason for insufficient tumor defense. A defect in the innate immune system could be a reason for the escape of the metastatic tumor cells from tumor surveillance.     

T-cell subset analysis of Lewis lung carcinoma tumor rejection: heterogeneity of effectors and evidence for negative regulatory lymphocytes correlating with metastasis.

We have analyzed the phenotypes of the T-cell subsets generated in response to Lewis lung carcinoma clones in C57BL/6J recipients. The metastatic derivative, which expresses low levels of H-2Kb gene, predominantly elicited CD8, V beta 8, and V beta 9+ T-cells. The nonmetastatic clone expressing high levels of H-Kb gene triggered a more heterogeneous response of V beta-5, -6, -8, -9, and -11 CD8+ T-cells. Comparison of the T-cell receptor (TCR) expression of the T-cells infiltrating the tumor site with the lymphocytes in the periphery of tumor-bearing animals revealed a pattern of homing of CD4+ T-cells bearing V beta-5, -6, and -11 TCR chains and CD8+ T-cells bearing V beta-5, -6, -9, and -11. Depletion of V beta 5 or V beta 6+ T-cells correlated with accelerated tumor growth, implying their protective role as tumor-specific effectors and consistent with the cytotoxicity of T-cells with this TCR phenotype. V beta 11 TCR expression in the tumor-infiltrating lymphocytes increased with the tumor size. Depletion of V beta 11+ T-cells enhanced resistance to primary tumor growth and conferred protection from metastasis in recipients cleared of V beta 5 and V beta 6 T-cell subsets. Those results suggest that tumor-specific effectors as well as negative regulator T-cells home, infiltrate, and coexist in the tumor site.     

CD4+ T cells inhibit the neu-specific CD8+ T-cell exhaustion during the priming phase of immune responses against breast cancer

Studies conducted in animal model of infectious diseases or H-Y antigen model suggest a crucial role for CD4+ T cells in providing help for CD8+ T-cell memory responses. This concept suggests that inclusion of T helper epitopes in vaccine formulation will result in improved CD8+ T-cell responses. Although this concept has been applied to cancer vaccine design, the role of CD4+ T cells in the memory differentiation of CD8+ T cells and retention of their anti-tumor function have never been tested in breast cancer model. Using the FVB mouse model of neu-positive breast carcinoma we report for the first time that helpless T cells showed cytostatic or tumor inhibitory effects during primary tumor challenge whereas, helped T cells showed cytotoxic effects and resulted in complete tumor rejection. Such differential effects, in vivo, were associated with higher frequency of CD8+PD-L1+ and CD8+PD-1+ T cells in animals harboring helpless T cells as well as higher titer of IL-2 in the sera of animals harboring helped T cells. However, depletion of CD4+ T cells did not alter the ability of neu-specific CD8+ T cells to differentiate into memory cells and to retain their effector function against the tumor during recall challenge. These results suggest the inhibitory role of CD4+ T cells on CD8+ T-cell exhaustion without substantial effects on the differentiation of memory T cells during priming phase of the immune responses against breast cancer.     

Infiltrating T-cell markers in cervical carcinogenesis: a systematic review and meta-analysis

Background The host adaptive immune response helps determine which cervical HPV infections persist and progress to precancer and cancer, and systematic characterisation of T-cell infiltration would help inform key steps in cervical carcinogenesis.Methods A systematic review and meta-analysis were conducted of infiltrating T-cells in normal cervix, low-grade lesions, high-grade lesions, and invasive cancers including epithelial, stromal, and total tissue and the following markers: CD3, CD4, CD8, FoxP3, CD25, and the CD4:CD8 ratio. An additional qualitative review summarised longitudinal data on associations between infiltrating T-cells and cervical disease persistence, regression, progression, or prognosis.Results There were fewer CD3+, CD4+, and CD8+ cells in cervical lesions and more cells in cancers compared to normal epithelium. FoxP3 and CD25+ regulatory T-cell infiltration is high in persistent and precancerous lesions, and longitudinal data show improved outcomes with lower regulatory T-cell levels.Conclusions Successful immune evasion may reduce T-cell infiltration in HPV infected and precancerous epithelium, while invasive cancers are highly immunogenic, and regulatory T-cell infiltration increases with cervical disease progression. Understanding these factors may have prognostic value and could aid in novel treatment development and clinical guidelines, but published data are highly heterogeneous and leave important gaps to be filled by future studies.Subject terms: Adaptive immunity, Tumour immunology, Cervical cancer, Cancer epidemiology, Cancer epidemiology     

Immune activation status of CD8+ T cells infiltrating non-small cell lung cancer

In a variety of human cancers, the presence of tumor-infiltrating T lymphocytes (TILs) is associated with tumor regression and favorable prognosis. Local interferon (IFN)-gamma secretion from activated T cells is supposed to induce a specific immune response leading to tumor-specific cytotoxicity. Nonetheless, significance and properties of TILs still remains controversial in lung cancer patients. We determined CD8+ T cell counts in 31 patients with non-small cell lung cancer (NSCLC) by immunohistochemistry, and assessed T-cell immune activation status in a subset of patients by measuring IFN-gamma mRNA expression by quantitative PCR (TaqMan). Semi-quantitative immunohistochemical analysis revealed significantly higher CD8+ T cell counts within the tumor as when compared to the invasive margin. CD8+ T cells immune activation status, represented in the IFN-gamma/CD8 mRNA ratio, correlated with the median number of CD8+ T cells presented at the tumor-host interface. Neither tumor histology and grade, nor CD8+ T cell counts and IFN-gamma/CD8 ratio could demonstrate an influence on overall survival in these patients. Our results indicate that CD8+ T cells infiltrating the tumor cell nests may be inadequately activated and thus incapable of mounting an effective anti-tumor immune response.     

干细胞样CD8+T细胞在抗肿瘤免疫治疗中的地位与演进效应

以抗细胞程序性死亡-1/细胞程序性死亡-配体1(programmed cell death-1/programmed cell death-ligand 1,PD-1/PD-L1)为代表的免疫检查点抑制剂(immune checkpoint inhibitors,ICIs)治疗显现了CD8⁺T细胞在治疗和可能治愈恶性肿瘤方面的潜力。但仅约20%的患者对ICIs治疗获益,因此阐明CD8⁺T细胞在免疫微环境中的有效抗肿瘤功能,及其分子和空间的决定因素尤为重要。具有自我更新、增殖分化和杀伤肿瘤细胞能力的干细胞样CD8⁺T细胞亚群,在介导抗肿瘤免疫效应方面扮演极为重要的角色。本文就干细胞样CD8⁺T细胞在抗肿瘤免疫循环中的地位与演进效应进行综述。     

Immune surveillance: both CD3+ CD4+ and CD3+ CD8+ T cells control in vivo growth of P815 mastocytoma.

The aim of this study was to examine whether a spontaneous immune response controls neoplastic growth in P815-bearing DBA/2 mice, and to characterize the cells involved in tumor resistance in vivo. Several cell lineages such as T-cell-receptor (TcR)-bearing T cells, NK cells and macrophages mediate some anti-tumor activity in vitro. P815 was chosen as a model because it is weakly immunogenic and is a good target both for tumor-specific, MHC-restricted CTL-mediated lysis and for MHC-unrestricted lysis exerted by long-term cultured lymphocytes or activated macrophages. Since most "NK-like activity" in freshly isolated populations appears to be associated with CD3- cells, whereas antigen-specific, MHC-restricted T cells mostly express CD3 determinants, CD3 was a good marker for evaluating the role of T cells and "NK" cells in tumor resistance in vivo. The survival of anti-CD3-treated animals that were inoculated with tumor cells was strongly reduced (mean survival time: 17 days vs. 40 days for the control group) and was associated with increased tumor growth rate. We followed the same approach to define the T-cell subset(s) that mediate(s) this immune response. Both CD4+ and CD8+ T cells were required for induction of immune control on neoplastic growth. The approach used has revealed the important role of CD4+ T cells in immune responses that control in vivo growth of a class-I-positive, class-II-negative tumor and suggests that these cells may play a central role in tumor resistance. Since CD4+ cells are activated by soluble, exogenous proteins, this finding may have important implications for immunotherapy.     

Adoptive transfer via immune T-lymphocytes of effective anti-tumor immunity against a malignant rat glioma in the brain

The aim of the present study was to develop an animal model to test the therapeutic potential of purified CD4 and CD8 T-lymphocytes against the intracerebrally implanted rat glioma cell line TZ363. Peripheral immunization of donor rats was performed by subcutaneous injection of viable TZ363 tumor cells while control animals received buffer injection. Donor splenic T-lymphocytes were prepared 14 days later and enriched by immune-bead MACS sorting. FACScan analysis revealed that of the pooled and sorted cells 91% of the tumor immune group were T-lympocytes and from the control animals 96%. The purified immune CD4/CD8 T-lymphocytes (1.2 to 5x10(7) cells) were injected intraperitoneally into 12 adult rats (three groups; each four animals), which were challenged five days later by an intracerebral injection of 5x10(4) TZ363 glioma cells. Four rats received 1.4x10(7) T-cells from control animals. While 3 of 4 animals developed a brain tumor and died in the control group, all animals, which received 5x10(7) immune T-cells survived the intracerebral tumor challenge. In the other groups survival rate depended on the amount of T-cells given. All other rats were sacrificed 32 days after intracerebral grafting. No tumor was found in these animals. Our data demonstrate that an anti-tumor T-cell response can be raised against the malignant rat glioma TZ363 and that purified CD4 and CD8 T-lymphocytes from tumor immunized donors can transfer protective immunity across the blood-brain barrier into recipient rats which are tumor challenged intracerebrally.     

Intratumoral delivery of encapsulated IL-12, IL-18 and TNF-α in a model of metastatic breast cancer

Intratumoral (i.t.) cytokine release through the use of poly-lactic acid microspheres (PLAM) holds tremendous potential for the immunotherapy of breast cancer as it harnesses the immunologic potential of autologous tumor in a clinically feasible and minimally toxic manner. We examined the potential of combinations of i.t. IL-12, IL-18 and TNF-α PLAM to generate a tumor-specific immune response and improve outcome in a model of metastatic breast cancer. Balb/c mice with established 4T1 mammary carcinomas were treated with a single injection of BSA, IL-12, IL-18 or TNF-α-loaded PLAM alone or in combination after spontaneous metastases occurred. Combined treatment with IL-12 and TNF-α PLAM was superior to all other treatments, including the triple combination of IL-12, IL-18 and TNF-α in ablation of the primary tumor, eradicating distant disease and enhancing survival. Simultaneous delivery of IL-12 and TNF-α was superior to sequential delivery of IL-12 followed by TNF-α, but not TNF-α followed by IL-12. In vivo lymphocyte depletion studies established that the effects of IL-12 alone are mediated primarily by NK cells, while the combination of IL-12 and TNF-α is dependent upon CD8+ T-cells. Only the combination of IL-12 and TNF-α results in an increase in both CD4+ and CD8+ T-cells and a reduction in CD4+CD25+ cells. While there was no change in the dendritic cell population, IL-12 and TNF-α resulted in a dramatic increase in DC maturation and antigen presentation. Neoadjuvant immunotherapy with simultaneous intratumoral delivery of IL-12 and TNF-α PLAM augments DC antigen presentation and increases cytotoxic T-cells without increasing regulatory T-cells, resulting in a T-cell based anti-tumor immune response capable of eradicating disseminated disease. The addition of IL-18 did not improve the efficacy.     

Requirements for the generation of a Lyt-2+ T-cell proliferative response to a syngeneic tumor in the absence of L3T4+ T-cells

Tumors may contain immunogenic antigens that are only recognizable in the context of class I, and not of class II, MHC molecules. Therefore, methods were developed to analyze the capacity of Lyt-2+ T-cells to respond to a syngeneic tumor in the absence of a contribution by L3T4+ T-cells. Conditions were defined in which purified Lyt-2+ T-cell populations, as well as L3T4+ T-cell populations, isolated from immune B6 spleen cells, could be induced to proliferate specifically in response to FBL, a retrovirally induced syngeneic tumor, without the addition of exogenous lymphokines. The purity of the subset responses was documented functionally by selective inhibition of the proliferative response of only the appropriate subset following addition of anti-Kb/Db or anti-I-Ab. The antigen and accessory cell (AC) requirements for triggering immune Lyt-2+ and L3T4+ T-cell populations were examined. The response of L3T4+ populations was predominantly specific for retrovirus envelope gp70, whereas Lyt-2+ populations predominantly recognized tumor antigens other than gp70, consistent with the hypothesis that some tumor antigens may be preferentially recognized by only class I- or class II-restricted T-cells. The FBL-stimulated proliferative response of each T-cell subset was dependent upon the presence of syngeneic AC. However, exogenous interleukin 1 was able to replace AC during the response of Lyt-2+ populations, whereas L3T4+ populations required AC also to biochemically process tumor-derived antigen and present it in the context of class II MHC molecules. The results suggest that under some conditions only the presence of AC or interleukin 1 may be limiting for the induction of antitumor responses by Lyt-2+ populations. These studies analyzed the ability to trigger purified Lyt-2+ T-cells in vitro following in vivo priming to tumor, and it remained possible that L3T4+ T-cells made an essential contribution during in vivo priming. Therefore, L3T4(+)-deficient mice were primed with FBL in vivo, and the Lyt-2+ T-cell response was assessed. Although priming was clearly less efficient in the absence of L3T4+ T-cells, Lyt-2+ T-cells from L3T4(+)-deficient mice proliferated and became cytolytically active following stimulation with FBL. Thus, under appropriate conditions, Lyt-2+ T-cells can generate an effective antitumor response in the absence of L3T4+ T-cells or exogenous lymphokines.     

CD4+T细胞与肿瘤免疫

在肿瘤免疫中细胞免疫发挥着重要作用,其中T细胞介导的特异性免疫应答反应更为重要.近年来CD4+T细胞在抗肿瘤免疫中的作用越来越受到重视.在肿瘤免疫中CD4+T细胞启动后可以通过多种机制启动细胞毒性T淋巴细胞(CTL),维持和加强CTL的抗肿瘤反应,并且可以作为效应细胞发挥抗肿瘤作用,CD4+T细胞中的一个亚群细胞CD4+ CD25+T调节细胞对肿瘤免疫有抑制作用.     

CD4+T细胞与肿瘤免疫

在肿瘤免疫中细胞免疫发挥着重要作用,其中T细胞介导的特异性免疫应答反应更为重要.近年来CD4+T细胞在抗肿瘤免疫中的作用越来越受到重视.在肿瘤免疫中CD4+T细胞启动后可以通过多种机制启动细胞毒性T淋巴细胞(CTL),维持和加强CTL的抗肿瘤反应,并且可以作为效应细胞发挥抗肿瘤作用,CD4+T细胞中的一个亚群细胞CD4+ CD25+T调节细胞对肿瘤免疫有抑制作用.     

基于免疫抑制性受体的血液肿瘤靶向免疫治疗研究

T细胞免疫功能缺陷导致多数血液肿瘤患者不能产生有效的抗肿瘤免疫应答,从而逃避宿主免疫系统的攻击,即肿瘤细胞免疫逃逸.近年来认为肿瘤免疫抑制最为重要的机制之一是CD8+T细胞的功能耗竭,主要为免疫细胞异常表达程序性死亡分子1(PD-1)和细胞毒性T淋巴细胞相关抗原(CTLA-4)等免疫抑制所介导,而阻断此通路可以使T细胞部分或全部恢复功能.文章介绍近年来血液肿瘤中PD-1和CTLA-4及二者在介导T细胞免疫耐受中的作用及其在靶向治疗研究中的进展,为血液肿瘤的免疫靶向治疗提供新的思路.     

Recombinant vesicular stomatitis virus targeted to Her2/neu combined with anti-CTLA4 antibody eliminates implanted mammary tumors

Vesicular stomatitis virus (VSV) is being developed for cancer therapy. We created a recombinant replicating VSV (rrVSV) that preferentially infected Her2/neu expressing breast cancer cells. We now used this rrVSV to treat macroscopic peritoneal tumor implants of a mouse mammary tumor cell line stably transfected to express Her2/neu. rrVSV therapy alone prolonged survival but did not cure any animals. rrVSV therapy combined with antibody to TGFb or antibody to IL-10 receptor (IL-10R) each produced cure in one of six animals. Strikingly, rrVSV therapy combined with anti-CTLA4 monoclonal antibody (MAb) produced cure in four of five animals. Anti-CTLA4 MAb was only effective when administered within one day of rrVSV therapy. Cure required CD4 T-cells early (<7 days) and late (>7 days) after rrVSV therapy whereas CD8 T-cells were required only late (>7 days) after rrVSV therapy. Surviving animals were resistant to re-challenge with D2F2/E2 suggesting a memory immune response. Histopathologic analysis demonstrated a dense inflammatory infiltrate of tumor nodules within days of therapy and foamy histiocytes replacing the tumor nodules 2 weeks following therapy. These studies demonstrate that targeted rrVSV combined with anti-CTLA4 MAb can eliminate established macroscopic tumor implants by eliciting an anti-tumor CD4 and CD8 T-cell immunologic response.     

Tumor-reactive CD8+ T-cell clones in patients after NY-ESO-1 peptide vaccination

A major objective of peptide vaccination is the induction of tumor-reactive CD8+ T-cells. We have shown that HLA-A2 positive cancer patients frequently develop an antigen-specific CD8+ T-cell response after vaccination with NY-ESO-1 peptides p157-165/p157-167. These T-cells are highly reactive with the peptides used for vaccination, but only rarely recognize HLA-matched, NY-ESO-1 expressing tumor cell lines. To address the apparent lack of tumor recognition of vaccine-induced CD8+ T-cell responses, we used autologous tumor cells for in vitro stimulation and expansion of pre- and postvaccine CD8+ T-cells. In contrast to standard presensitization methods with peptide-pulsed antigen-presenting cells, mixed lymphocyte tumor culture favored the selective expansion of low-frequency tumor-reactive T-cells. In four patients, we were able to demonstrate that antigen-specific and tumor-reactive T-cells are detectable and are indeed elicited as a result of NY-ESO-1 peptide vaccination. Further analyses of postvaccine antigen-specific T-cells at a clonal level show that vaccine-induced antigen-specific T-cells are heterogeneous in functional activity. These results suggest that the methods of immunomonitoring are critical to identify the proportion of tumor-reactive T-cells within the population of vaccine-induced antigen-specific effector cells. Our results show that immunization with NY-ESO-1 peptides leads to strong tumor-reactive CD8+ T-cell responses. Our findings suggest that approaches to peptide vaccination may be improved to induce higher numbers of antigen-specific T-cells and to selectively increase the proportion of CD8+ T-cells that have the capacity to recognize and eliminate tumor cells.     

Validity of the in vitro system as a correlate of the in vivo model of RadLV lymphomagnesis

Adult BL/6 mice are highly sensitive to lymphomagnesis by the radiation leukemia virus variant A-RadLV (80-100% T-cell lymphoma incidence after a latency of 70-110 days). This study shows that the in vivo elimination of T-cell subsets (including suppressor and cytotoxic T-cells) achieved by the repeated administration of cyclophosphamide, cyclosporin A, anti CD4 or anti CD8 mAb shortly after virus infection did not interfere with the lymphomagenic pathway. No reduction in the high lymphoma incidence or tumor latency was observed following the different treatments. Thus the suggestion on the basis of in vitro studies that the early phase of A-RadLV lymphomagenesis is associated with suppressor T-cells which abrogate a potential anti-tumor immune response has not been confirmed in these studies.     

mTOR and MEK1/2 inhibition differentially modulate tumor growth and the immune microenvironment in syngeneic models of oral cavity cancer

We investigated the effects of mTOR and MEK1/2 inhibition on tumor growth and the tumor microenvironment in immunogenic and poorly immunogenic models of murine oral cancer. In vitro, rapamycin and PD901 inhibited signaling through expected downstream targets, but only PD901 reduced viability and altered function of MOC cells. Following transplantation of MOC cells into immune-competent mice, effects on both cancer and infiltrating immune cells were characterized following rapamycin and/or PD901 treatment for 21 days. In vivo, both rapamycin and PD901 inhibition reduced primary growth of established MOC tumors on treatment. Following withdrawal of PD901, rapid rebound of tumor growth limited survival, whereas durable tumor control was observed following rapamycin treatment in immunogenic MOC1 tumors despite more robust inhibition of oncogenic signaling by PD901. Characterization of the immune microenvironment revealed diminished infiltration and activation of antigen-specific CD8+ T-cells and other immune cells following PD901 but not rapamycin in immunogenic tumors. Subsequent in vitro T-cell assays validated robust inhibition of T-cell expansion and activation following MEK inhibition compared to mTOR inhibition. CD8 cell depletion abrogated rapamycin-induced primary tumor growth inhibition in MOC1 mice. These data have critical implications in the design of combination targeted and immune therapies in oral cancer.