PD-1 In Tumor-infiltrating Lymphocytes Research Articles

Driving the Chimeric Antigen Receptor: The Road Ahead for Cell Therapy

Driving the Chimeric Antigen Receptor: The Road Ahead for Cell Therapy

Highly efficient PD-1-targeted CRISPR-Cas9 for tumor-infiltrating lymphocyte-based adoptive T cell therapy

Adoptive T cell therapy (ACT) with expanded tumor-infiltrating lymphocytes (TIL) can induce durable responses in cancer patients from multiple histologies, with response rates of up to 50%. Antibodies blocking the engagement of the inhibitory receptor programmed cell death protein 1 (PD-1) have been successful across a variety of cancer diagnoses. We hypothesized that these approaches could be combined by using CRISPR-Cas9 gene editing to knock out PD-1 in TILs from metastatic melanoma and head-and-neck, thyroid, and colorectal cancer. Non-viral, non-plasmid-based PD-1 knockout was carried out immediately prior to the traditional 14-day TIL-based ACT rapid-expansion protocol. A median 87.53% reduction in cell surface PD-1 expression was observed post-expansion and confirmed at the genomic level. No off-target editing was detected, and PD-1 knockout had no effect on final fold expansion. Edited cells exhibited few phenotypic differences and matched control functionality. Pre-clinical-scale results were confirmed at a clinical scale by generating a PD-1-deficient TIL product using the good manufacturing practice facilities, equipment, procedures, and starting material used for standard patient treatment. Our results demonstrate that simple, non-viral, non-plasmid-based CRISPR-Cas9 methods can be feasibly adopted into a TIL-based ACT protocol to produce treatment products deficient in molecules such as PD-1, without any evident negative effects.

Monitoring PD-1 Phosphorylation to Evaluate PD-1 Signaling during Antitumor Immune Responses.

PD-1 expression marks activated T cells susceptible to PD-1-mediated inhibition but not whether a PD-1-mediated signal is being delivered. Molecular predictors of response to PD-1 immune checkpoint blockade (ICB) are needed. We describe a monoclonal antibody (mAb) that detects PD-1 signaling through the detection of phosphorylation of the immunotyrosine switch motif (ITSM) in the intracellular tail of mouse and human PD-1 (phospho-PD-1). We showed PD-1+ tumor-infiltrating lymphocytes (TILs) in MC38 murine tumors had high phosphorylated PD-1, particularly in PD-1+TIM-3+ TILs. Upon PD-1 blockade, PD-1 phosphorylation was decreased in CD8+ TILs. Phospho-PD-1 increased in T cells from healthy human donors after PD-1 engagement and decreased in patients with Hodgkin lymphoma following ICB. These data demonstrate that phosphorylation of the ITSM motif of PD-1 marks dysfunctional T cells that may be rescued with PD-1 blockade. Detection of phospho-PD-1 in TILs is a potential biomarker for PD-1 immunotherapy responses.

EVALUATION OF IMMUNOINHIBITORY CHECKPOINTS IN ORAL OSTEOSARCOMAS

Objective Cancers have been categorized into 4 different tumor microenvironments (TME) based on the presence of PD-L1 expression and tumor-infiltrating lymphocytes (TIL). However, oral osteosarcoma (OO) has not yet been studied. This research evaluated the immunoexpression of PD-L1 and TIL CD8+ and PD-1+ in a series of cases of OO. Study Design It is a multicenter, observational and cross-sectional study. The sample was composed of 20 cases of primary OO diagnosed in various centers of reference in oncology. Immunohistochemistry was used to identify PD-L1+, TIL CD8+, and PD-1+ cells. Analysis of positivity was assessed by the percentage of PD-L1+ neoplastic cells (1 = absent [< 10%], 2 = 10%-50%, and 3 = > 50%) and densities of TIL per mm2 (1 = absent [25 cells/mm2], 2 = low [25 a 130 cells/mm2], and 3 = high [130 cells/mm2]) adapted from Van Erp et al. (2017). Results From all cases studied, 75% showed overexpression of PD-L1 (score 3). On the other hand, all samples revealed absent or low TIL CD8+ (score 1 or 2) and absent TIL PD-1+ (score 1). Conclusions OO showed an immune profile characterized by high PD-L1/low CD8/absent PD-1, which may represent a TME type unresponsive to immunotherapy. Grant: CNPq 305897/2015-2. Cancers have been categorized into 4 different tumor microenvironments (TME) based on the presence of PD-L1 expression and tumor-infiltrating lymphocytes (TIL). However, oral osteosarcoma (OO) has not yet been studied. This research evaluated the immunoexpression of PD-L1 and TIL CD8+ and PD-1+ in a series of cases of OO. It is a multicenter, observational and cross-sectional study. The sample was composed of 20 cases of primary OO diagnosed in various centers of reference in oncology. Immunohistochemistry was used to identify PD-L1+, TIL CD8+, and PD-1+ cells. Analysis of positivity was assessed by the percentage of PD-L1+ neoplastic cells (1 = absent [< 10%], 2 = 10%-50%, and 3 = > 50%) and densities of TIL per mm2 (1 = absent [25 cells/mm2], 2 = low [25 a 130 cells/mm2], and 3 = high [130 cells/mm2]) adapted from Van Erp et al. (2017). From all cases studied, 75% showed overexpression of PD-L1 (score 3). On the other hand, all samples revealed absent or low TIL CD8+ (score 1 or 2) and absent TIL PD-1+ (score 1). OO showed an immune profile characterized by high PD-L1/low CD8/absent PD-1, which may represent a TME type unresponsive to immunotherapy. Grant: CNPq 305897/2015-2.

In Situ Hybridization of HBZ and Tax in FFPE Samples from ATLL Patients and Its Association with Clinicopathological Characteristics

IntroductionAdult T-cell leukemia/lymphoma (ATLL) is a mature T-cell neoplasm caused by human T-cell leukemia virus type 1 (HTLV-1). Many studies on HTLV-1-related mRNA including HTLV-1 bZIP factor (HBZ) and Tax have been performed, mainly using cell lines, patient-derived cells, and mice. However, there are scant data concerning HTLV-1-related mRNA in formalin-fixed, paraffin-embedded (FFPE) tissue samples. We detected HBZ and Tax mRNA on FFPE tissue samples using in situ hybridization (ISH), and investigated the association with clinicopathological characteristics.Materials and methodsEighty-seven biopsy samples from newly diagnosed ATLL patients were examined. The same samples were used in a previous study (Miyoshi et al. Blood, 2016). Use of patient materials and clinical information was approved by the Ethical Committee of Kurume University, in accordance with the Declaration of Helsinki.ISH was performed on FFPE tissue samples using RNAscope 2.5 HD Reagent Kit-BROWN (Advanced Cell Diagnostics, Hayward, CA). HBZ-and Tax-specific probes were used. MT-4 (HTLV-1 immortalized cell line) and Jurkat (T-cell acute lymphoblastic leukemia cell line) were used as positive and negative controls, respectively. Dot-like signals were counted at high magnification (40 diameters) on 10 randomly selected fields and the number of signals per 1,000 ATLL cells were calculated. The results were reviewed by two experienced hematopathologists. High expression was indicated when more than the median value of HBZ or Tax signals was stained. The antibodies for immunohistochemistry targeted CD4, CD30, Ki-67(MIB-1), CCR4, FoxP3, GATA3, IRF4, HLA class I, β2-microglobulin (β2M), PD-1, and PD-L1.Clinicopathological characteristics of ATLL patients were compared by Fisher's exact test (2-sided), Mann-Whitney's U test, and Spearman's rank correlation analysis. Overall survival was estimated by the Kaplan-Meier method and compared by the Log-rank test. P< .05 was considered statistically significant. EZR ver. 1.32 was used for all statistical analyses.ResultsIn the histogram and scatter plot of HBZ-ISH and Tax-ISH, the median values of HBZ signals and Tax signals were 806/1000 ATLL cells (range 0.4 - 4013.1) and 5.0/1000 ATLL cells (range 0.1 - 891.2), respectively (Figure 1). Representative samples of HBZ-ISH and Tax-ISH are presented in Figure 2A and 2B, respectively.Notably, the high-expression group of HBZ displayed significant reductions in skin lesions (P = .025), Ann Arbor stage (P = .021), and peripheral blood involvement (P = .028). The high-expression group of Tax displayed significant increases in lactate dehydrogenase activity (P = .0020), splenomegaly (P = .0070), CD 30 (P = .015), and PD-1 in tumor-infiltrating lymphocytes (PD-1_TIL; P <.0010).Weak but significant positive correlation was observed between HBZ and IRF4 (ρ = .33; P = .029), between HBZ and PD-L1 (ρ = -. 28; P = .010), between Tax and CD4 (ρ = .41; P = .0074), between Tax and CD30 (ρ = .26; P = .016), between Tax and GATA 3 (ρ = .26; P = .014), between Tax and PD-1 in ATLL cells (ρ = .25; P = .0074), between Tax and PD-L1 in ATLL cells (ρ =. 32; P = .0026), and between Tax and PD-1_TIL (ρ = .37; p = .0026) (Figure 3). There was almost no correlation between HBZ and Tax (ρ = .052; P = .63). There were no significant differences between HBZ or Tax and OS (Log-rank P = .21 and .95, respectively).ConclusionThe RNA scope assay is useful for detecting low expressing mRNA in FFPE tissue samples. Our data suggest that ISH of HBZ and Tax can detect differences in clinicopathological characteristics of ATLL patients. In clinical and cell samples, HBZ and Tax might be involved in the pathogenesis of ATLL.DisclosuresNo relevant conflicts of interest to declare. [Display omitted] DisclosuresAsano:Takeda: Honoraria; Chugai Pharma: Honoraria; Celgene: Honoraria.

Evaluation of programmed cell death protein 1 (PD-1) expression as a prognostic biomarker in patients with clear cell renal cell carcinoma

ABSTRACT Programmed cell death protein 1 (PD-1) immune checkpoint inhibitors have shown activity in patients with advanced renal cell carcinoma (RCC). However, the role of PD-1 expression in tumor-infiltrating lymphocytes (TILs) as a biomarker for poor outcome is not clear. In this study, we evaluated the prognostic value of TIL PD-1 expression in patients with clear cell RCC (ccRCC). 82 patients who underwent nephrectomy for localized or metastatic ccRCC and followed up for at least four years were searched from our database and retrospectively enrolled. Their fixed primary tumor specimens were stained with anti-PD-1 (NAT105). The specimens were classified as negative or positive for PD-1 expression, and the positive specimens were further scored in 10% increments. 37 (45.12%) patients were negative (<1% stained), 26 (31.71%) patients were low (<10 and 10%), and 19 (23.17%) patients were high (20–50%) for PD-1 expression. The prognostic value of TIL PD-1 expression was evaluated by univariate Cox proportional hazards regression on overall and recurrence-free survivals. Higher TIL PD-1 expression was not associated with increased risk of death (P = 0.336) or with increased risk of recurrence (P = 0.572). Higher primary tumor stage was associated with increased risk of recurrence (P = 0.003), and higher Fuhrman nuclear grade was associated with increased risk of death (P <0.001) and with increased risk of recurrence (P <0.001). Our study shows that TIL PD-1 expression by immunohistochemistry (IHC) does not correlate with poor clinical outcome in patients with ccRCC and is inferior to established prognosticating tools.

Abstract 3989: Different expression and prognostic effect of PD-L1/PD-1 in SCC and non-SCC of non-small cell lung cancer

Abstract Although checkpoint inhibitors targeting PD-1/PD-L1 pathway have demonstrated similar clinical activity in treatment of NSCLC both for squamous(SCC) and non-squamous carcinoma (non-SCC) patients, the predictive role of PD-L1 expression is distinct between two histologies in clinical trials. In order to investigated the expression and prognostic roles of PD-1/PD-L1, data from TCGA and GEO database were analyzed and further validated with our original data by IHC in 133 resected NSCLC tumor tissue(49 SCC and 84 non-SCC). Through the analysis of online data, we concluded that PD-L1 expression in SCC was significantly higher than that of non-SCC. In addition, in non-SCC, PD-L1 was associated with immune response and indicated worse prognosis while no such results were observed in SCC. Further, our original data demonstrated PD-L1 expression in tumor cells(TC) and immune cells(IC) were significantly more often and more intense in SCC(P&amp;lt;0.001). In survival analysis, PD-L1 expression in TC and IC were associated with shorter overall survival in non-SCC (P=0.019 and 0.003, respectively). However, in SCC, PD-L1 did not demonstrate any significant effect neither in TC or IC (P=0.526 and 0.674, respectively) which consistent with online data. Contrary to PD-L1, no significant differences of PD-1 expression in tumor infiltrating lymphocytes(TILs) were showed between two histologies. PD-1 in TILs of SCC correlated with worse prognosis (P=0.025) while in non-SCC there was no correlation(P=0.986). Taken together, these findings suggest distinct regulation mechanisms and clinical significance of PD-L1/PD-1 pathway in NSCLC subgroups. Citation Format: Yunpeng Liu, Shuo Wang, Xiujuan Qu, Zhi Liu, Xiaofang Che, Xu Ling, Kezuo Hou, Yibo Fan, Na Song, Ti Wen, Ce Li. Different expression and prognostic effect of PD-L1/PD-1 in SCC and non-SCC of non-small cell lung cancer [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 3989. doi:10.1158/1538-7445.AM2017-3989

Case-control study of PD-1, PD-L1, and B7-H3 expression in human immunodeficiency virus infected (HIV+) and uninfected patients with head and neck cancers.

e23194 Background: HIV+patients are typically excluded from immunotherapy trials but there are limited data on co-signaling molecule expression in these patients, including in head and neck squamous cell carcinoma (HNSCC). Methods: A case control study was conducted to evaluate tumor tissue sections for PD-L1, PD-1, and B7-H3 expression in HIV+HNSCC patients (n = 12) and HNSCC controls (n = 12). Cases and controls were matched for age at diagnosis, race, gender, TNM stage, and primary site. Smoking status, HIV RNA viral load (VL), and CD4 count were also recorded. Immunostained tumor sections were analyzed for percent of tumor cells expressing PD-L1 (PD-L1%) and B7-H3(B7-H3%) (Abcam), and percent of tumor infiltrating lymphocytes (TIL) expressing PD-1 (TIL-PD-1%) and PD-L1 (TIL-PD-L1%) (Abcam). Statistical analysis used the non-parametric Mann-Whitney test, chi-square test and Spearman’s rank correlation, Rs. Results: The 12 HIV+ HNSCC cases were predominantly male (67%), black race (67%) and cigarette smokers (100%), with a median age of 50.4 years, median viral load (VL) of 52399 copies/mL, median CD4 count (CD4) of 236cells/uL, predominantly locally advanced (75% Stage III/IVa/IVb), and oropharyngeal primary site (42%). Defining positive expression as &gt; 5%, 42% of HIV+ HNSCC patients tumors were positive for PD-L1, 100% for B7-H3, and 92% had TILs that expressed PD-1 and PD-L1. HIV+ patients had significantly higher B7-H3% (Median 60% vs. 20%, p = 0.03) and TIL-PD-L1% (Median 15% vs. 10%, p = 0.045) compared to controls. There were no differences in PD-L1% or TIL-PD-1%. In HIV+ cases, increased VL correlated with increased TIL-PD-1% (Rs = 0.73, p = 0.011) and increased CD4 count correlated with increased tumor PD-L1% (Rs = 0.62, p = 0.04). There were no other significant correlations between CD4 count or VL and co-signaling molecule expression. Conclusions: HIV+ HNSCC patients had significantly higher tumor B7-H3 and TIL PD-L1 expression, with similar tumor PD-L1 and TIL PD-1 expression, compared to HIV negative HNSCC control patients. These findings support inclusion of HIV+ HNSCC patients in immunotherapy trials with checkpoint inhibitors.

Abstract PD6-07: PD-L1 is highly expressed in tumor infiltrating lymphocytes in pregnancy associated breast cancer

Abstract Background: Pregnancy associated breast cancer (PABC), diagnosed during or after gestation, is typically triple negative, and is associated with a poor prognosis. Those diagnosed within two years have an even worse outcome. We previously assessed the immune microenvironment of invasive breast carcinomas in young women and reported that tumor infiltrating lymphocytes (TILs) were more prominent in PABC. Programmed cell death protein 1 (PD-1) is upregulated following activation of lymphocytes, while programmed death ligand 1 (PD-L1) is one of the primary ligands that it interacts with to inhibit T-cell activation and proliferation. PD-L1 may also be constitutively expressed on tumor cells as a result of oncogenic signaling or epithelial-mesenchymal transition. Emerging evidence suggests that the effect of the local immune system, particularly the interactions between PD-1 and PD-L1, is also key in breast cancer progression and in breast tumor responses to chemotherapy and targeted therapy. In this study, we assessed expression of PD-1 and PD-L1 in both TILs and tumor cells in PABC and in age-/stage-/grade-matched nulliparous women, and correlated their expression with clinicopathologic characteristics in this aggressive type of breast carcinomas. Design: 21 patients diagnosed with PABC within two years of pregnancy (mean age=35.7, range=26-48) and 15 matched controls (mean age=37.5, range=29-51) were evaluated. Slides were reviewed and pathologic tumor characteristics, including TILs, were noted. Immunohistochemical stains for PD-1 and PD-L1 were performed. Extent (1=1-25% positive tumor cells, 2=26-50%, 3=51-75%, 4=76-100%) and intensity (1=weak, 2=moderate or 3=strong) of staining were assessed. A composite score (CS) was calculated by multiplying the extent by intensity (range=0-12; weak=1-3; moderate=4-8 and strong=9-12). Results: The mean CS for PD-L1 in TILs was significantly higher in PABC (5.86) compared to controls (3.07), p=0.03. Further, strong expression of PD-L1 in TILs was only observed in PABC (9/21, 42.9%); none of the controls had strong PD-L1, p=0.01. The high expression of PD-L1 in PABC TILs was independent of tumor grade, hormone receptor and HER2 status, and other histologic features including lymph node metastasis. Expression of PD-1 in TILs was similar in both PABC and controls (mean CS 6.81 and 5.36, respectively). Immunoreactivity in the tumor cells themselves was rare with only two PABC and four control cases expressing PD-1 and PD-L1. Conclusion: 1. TILs in PABC have significantly higher PD-L1 expression. 2. Strong expression of PD-L1 in TILs was only observed in PABC. 3. High PD-L1 expression in TILs was independent of the tumor characteristics in this series. 4. PD-1 is expressed similarly in TILs in both PABC and controls. 5. Rare cases may have PD-1 and PD-L1 expression in the tumor cells themselves. The results of our study showing significant expression of PD-L1 in PABC TILs add to the understanding of the role of the microenvironment in breast cancer progression. These complex interactions between tumors cells and the local immune system may predict response to therapy and investigation into the role of immune based therapies is under way in these aggressive breast carcinomas that affect young women. Citation Format: Blanco Jr LZ, Pincus JL, Siziopikou KP. PD-L1 is highly expressed in tumor infiltrating lymphocytes in pregnancy associated breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr PD6-07.

PUB115 Clinical Significance of PD-L1 Status and the Expression of PD-1 in Tumor-Infiltrating Lymphocytes (TILs) in Lung Cancer

PD-L1 status in lung cancer may be a predictive biomarker for the use of PD1/PD-L1 inhibitors. Nonetheless, it is still unclear which immunohistochemistry platform for PD-L1 assessment should be implemented for optimal prognostic/predictive information. We investigated whether the PD-L1 status in early-stage lung cancer is associated with specific clinical features or survival, as well as the PD1 expression in TILs. IHC was performed on FFPE surgical tumors, arrayed on tissue microarrays with duplicate cores. PD-L1 status was assessed with two ABs: Ventana (SP124) (PD-L1V) and Cell Signaling (E1L3N) (PD-L1CS). PD1 expression in TILs was determined with the AB Ventana (NAT105). All patients (n=482) had early stage T1/T2-N0 and underwent curative surgery between 1987 and 2002. PD-L1 membranous staining was scored as positive if >5% of tumor cells. PD1 was considered as positive if >10% in TILs. Tabled 1PD-L1(V)PD-L1(CS)PD1All cases (including carcinoids)7%18%50%Histology Sq Non-Sqp=0.0043 12% 4%p<0.0001 30% 12%56% 49%pStage IA IB5% 10%p=0.017 15% 24%51% 53%Grading G1 G2 G3p=0.004 1% 6% 12%p=0.0002 7% 18% 27%p=0.001 41% 45% 61% Open table in a new tab Patient’s median age (IQR) 68 years (59-73), gender male/female 53%/47%, smoking status never/former/current/missing 14%/26%/49%/11%, histology squamous-cell carcinoma (Sq)/Non-squamous (Non-Sq)-NSCLC/carcinoid 37%/55%/7%, p-stage IA/IB 52%/48%. A table shows the association between biomarker status (PD-L1(V), PD-L1(CS) and PD1 )and tumor features (including Pearson’s Chi-Square p-values <0.05). All carcinoids were negative for PD-L1, except for one. The degree of agreement between the two PD-L1 Abs was moderate (Kappa=0.47). Median OS was 76 months. No statistically significant association between PD-L1 status and OS was observed. PD1 expression in TILs was associated with worse survival in Sq tumors. Median OS for cases with PD1 positive vs negative TILs was 54 vs 79 months, respectively (log-rank p=0.08; Wilcoxon p=0.02). In this study we observed a higher expression of PD-L1 using the CS antibody with a moderate agreement between the two antibodies. Sq tumors showed a significantly higher expression of PD-L1 compared with non-Sq tumors. PD-1 expression in TILs was associated with worse survival in Sq tumors.

PD1 and PDL1 expression in primary central nervous system diffuse large B-cell lymphoma are frequent and expression of PD1 predicts poor survival.

Primary central nervous system diffuse large B-cell lymphoma (PCNS-DLBCL) is a rare and aggressive type of diffuse large B-cell lymphoma (DLBCL) whit poorly understood pathogenesis. Finding biomarkers associated with patient survival may be important for understanding its physiopathology and to develop new therapeutic approaches. We investigated 32 PCNS-DLBCL from immunocompetent patients for BCL2, CMYC, LMO2, and P53 expression and for cytogenetic aberrations of BCL2, BCL6, and MYC genes, all known for their prognostic value in systemic DLBCL (s-DLBCL). We analyzed PD1 and PDL1 protein expression in both tumor infiltrating lymphocytes (TILs) and tumor cells. Finally, we searched for correlation between biological data and clinical course. The PCNS-DLBCL expressed BCL2, CMYC, LMO2, and P53 at similar frequency than s-DLBCL but without significant prognostic on survival. None cases harbored aberrations involving BCL2 and MYC gene whereas BCL6 abnormalities were present in 20.7% of cases but without value on survival. Expression of PD1 in TILs and PDL1 in tumor cells was observed at higher rates than in s-DLBCL (58% and 37%, respectively). The PD1 expression in TILs correlated with PDL1 expression in tumor cells (P=.001). Presence of PD1 positive TILs was associated with poorer overall survival (P=.011). Patients with PDL1 overexpression tended to better response to chemotherapy (P=.23). In conclusion PCNS-DLBCL pathogenesis differs from s-DLBCL without prognostic value of the phenotypic and cytogenetic parameters known for their pejorative impact in the latter. The PD1/PDL1 pathway plays a strong role in PCNS-DLBCL and represents an attractive target for this aggressive lymphoma.

OX40 and other immunoregulatory molecules are highly expressed on tumor infiltrating lymphocytes in oral, head and neck squamous cell carcinoma

OX40 is a potent co-stimulatory receptor on the surface of T lymphocytes. An OX40 agonist was recently tested in a Phase I clinical trial and was found to be well tolerated and enhanced both humoral and cellular immunity in patients with metastatic cancer. Both PD-1 and CTLA-4 blockade are showing therapeutic clinical activity in late stage cancer patients. Hence, we hypothesize that the immune suppressed tumor microenvironment that characterizes oral, head and neck squamous cell carcinoma (OHNSCC) can be overcome by strategies that favor antitumor inflammatory T cell responses and may be an ideal tumor site for immune modulation. This investigation aims to provide a better understanding of the expression of OX40, PD-1, CTLA-4 and CD103 in OHNSCC tumor infiltrating lymphocytes (TIL). Additionally we evaluated whether phenotypic differences existed in patients with HPV positive vs. negative status. Methods TIL and autologous blood were isolated from 29 patients undergoing surgery for OHNSCC. A sample of tissue was obtained from the resection specimen of the primary tumor and sent to the laboratory with autologous blood. Tumor was minced and subjected to digestive enzymes. Blood and tumor samples were separated over ficoll gradient to enrich for lymphocytes. Samples were frozen at -140°c and later thawed in batches for analysis. Proteins were then labeled with fluorescently conjugated antibodies and analyzed by flow cytometry. Results The percentage of FoxP3+CD25+CD4+ (Tregs) of CD4+ T cells is significantly higher in OHNSCC Tumor Infiltrating Lymphocytes (TIL) compared to blood (p < .0005), regardless of HPV status. OX40 expression is significantly higher in the TIL Treg population relative to blood Tregs (p < .0005), also independent of HPV status. PD1 expression is increased in the Tregs (p < .0005), conventional CD4 (p=.0066), and CD8 T cells (p < .0005), in TIL relative to blood. CTLA-4 expression is increased in TIL relative to blood in all subsets with the highest expression observed in the Treg population (.0056). CD103 expression is increased on CD8 T cells in TIL relative to blood (p < .0005). CD8s from HPV positive patients have higher percentage CD103 positive cells than CD8s from HPV negative patients (p=.0056). The highest percentages of PD-1, CTLA-4 and OX40 triple positive cells were found in the Treg population of TIL compared to blood (p < .0005). Due to increased expression of OX40, CTLA-4 and PD-1 in TIL of OHNSCC patients we hypothesize that future combination clinical trials that target these pathways will be therapeutically beneficial.