CD8 Tumor-infiltrating Lymphocytes Research Articles

Prognostic significance of tumor-infiltrating CD8+ and FOXP3+ lymphocytes in residual tumors and alterations in these parameters after neoadjuvant chemotherapy in triple-negative breast cancer: a retrospective multicenter study

IntroductionThe status of tumor-infiltrating lymphocytes (TILs) has been recently proposed to predict clinical outcome of patients with breast cancer. We therefore studied the prognostic significance of CD8+ TILs and FOXP3+ TILs in residual tumors after neoadjuvant chemotherapy (NAC) and the alterations in these parameters before and after NAC in patients with triple-negative breast cancer (TNBC).MethodsOne hundred thirty-one TNBC patients who received NAC at three institutions were examined. CD8+ TIL and FOXP3+ TIL in residual tumors and biopsy specimens were evaluated by double-staining immunohistochemistry. The CD8+ TIL and FOXP3+ TIL status of the residual tumors was assessed, and the rates of their changes before and after NAC were calculated.ResultsTNBC patients with high CD8+ TIL levels or a high CD8/FOXP3 ratio in residual tumors had significantly better recurrence-free survival (RFS) and breast cancer-specific survival (BCSS) than patients with low values of these parameters. In multivariate analyses, CD8+ TIL exhibited strong prognostic significance for RFS, with a hazard ratio (HR) of 3.09 (95 % confidence interval (CI) 1.537–6.614, P=0.0013). The CD8/FOXP3 ratio was also significantly correlated with RFS (HR=2.07, 95 % CI 1.029–4.436, P=0.0412). TNBC with larger residual tumor size and positive lymph node status, which are known prognostic factors, was independently associated with worse RFS (P=0.0064 and P=0.0015, respectively). High CD8+ TIL levels were a markedly powerful indicator of improved BCSS, with an HR of 3.59 (95 % CI 1.499–9.581, P=0.0036). Nodal status was also associated with BCSS (P=0.0024). TNBC with a high rate of CD8+ TIL changes was associated with significantly better RFS compared with the low group (P=0.011). Higher rates of changes in the CD8/FOXP3 ratio were significantly correlated with both better RFS and BCSS compared with lower rates (P=0.011 and P=0.023, respectively).ConclusionsThis is the first study to demonstrate that high CD8+ TIL and a high CD8/FOXP3 ratio in residual tumors and increment of these parameters following NAC and accurately predict improved prognosis in TNBC patients with non-pathological complete response following NAC. These parameters could serve as a surrogate one for adjuvant treatment in patients with residual disease in the neoadjuvant setting.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-015-0632-x) contains supplementary material, which is available to authorized users.

An HPV-E6/E7 immunotherapy plus PD-1 checkpoint inhibition results in tumor regression and reduction in PD-L1 expression.

We have investigated if immunotherapy against human papilloma virus (HPV) using a viral gene delivery platform to immunize against HPV 16 genes E6 and E7 (Ad5 [E1-, E2b-]-E6/E7) combined with programmed death-ligand 1 (PD-1) blockade could increase therapeutic effect as compared to the vaccine alone. Ad5 [E1-, E2b-]-E6/E7 as a single agent induced HPV-E6/E7 cell-mediated immunity. Immunotherapy using Ad5 [E1-, E2b-]-E6/E7 resulted in clearance of small tumors and an overall survival benefit in mice with larger established tumors. When immunotherapy was combined with immune checkpoint blockade, an increased level of anti-tumor activity against large tumors was observed. Analysis of the tumor microenvironment in Ad5 [E1-, E2b-]-E6/E7 treated mice revealed elevated CD8(+) tumor infiltrating lymphocytes (TILs); however, we observed induction of suppressive mechanisms such as programmed death-ligand 1 (PD-L1) expression on tumor cells and an increase in PD-1(+) TILs. When Ad5 [E1-, E2b-]-E6/E7 immunotherapy was combined with anti-PD-1 antibody, we observed CD8(+) TILs at the same level but a reduction in tumor PD-L1 expression on tumor cells and reduced PD-1(+) TILs providing a mechanism by which combination therapy favors a tumor clearance state and a rationale for pairing antigen-specific vaccines with checkpoint inhibitors in future clinical trials.

Correlation between Density of CD8+ T-cell Infiltrate in Microsatellite Unstable Colorectal Cancers and Frameshift Mutations: A Rationale for Personalized Immunotherapy.

Colorectal cancers with microsatellite instability (MSI) represent 15% of all colorectal cancers, including Lynch syndrome as the most frequent hereditary form of this disease. Notably, MSI colorectal cancers have a higher density of tumor-infiltrating lymphocytes (TIL) than other colorectal cancers. This feature is thought to reflect the accumulation of frameshift mutations in sequences that are repeated within gene coding regions, thereby leading to the synthesis of neoantigens recognized by CD8(+) T cells. However, there has yet to be a clear link established between CD8(+) TIL density and frameshift mutations in colorectal cancer. In this study, we examined this link in 103 MSI colorectal cancers from two independent cohorts where frameshift mutations in 19 genes were analyzed and CD3(+), CD8(+), and FOXP3(+) TIL densities were quantitated. We found that CD8(+) TIL density correlated positively with the total number of frameshift mutations. TIL densities increased when frameshift mutations were present within the ASTE1, HNF1A, or TCF7L2 genes, increasing even further when at least one of these frameshift mutations was present in all tumor cells. Through in vitro assays using engineered antigen-presenting cells, we were able to stimulate peripheral cytotoxic T cells obtained from colorectal cancer patients with peptides derived from frameshift mutations found in their tumors. Taken together, our results highlight the importance of a CD8(+) T cell immune response against MSI colorectal cancer-specific neoantigens, establishing a preclinical rationale to target them as a personalized cellular immunotherapy strategy, an especially appealing goal for patients with Lynch syndrome.

Tim-3 expression represents dysfunctional tumor infiltrating T cells in renal cell carcinoma.

Renal cell carcinoma (RCC) is the most common cancer of kidney. Evidences have shown that RCC is sensitive to various immunotherapies. Tim-3 plays a role in suppressing Th1-mediated immune responses. However, no study has yet examined the effect of Tim-3 on tumor infiltrating lymphocytes (TILs) in RCC. We investigated the expression and function of Tim-3 on TIL CD4+ T cells and TIL CD8+ T cells from 30 RCC patients. Levels of Tim-3 were significantly increased on both TIL CD4+ T cells and TIL CD8+ T cells and were associated with higher stages of the cancer. Also, GATA-3 and interferon gamma (IFN-γ) were down-regulated, whereas T-bet was up-regulated in TIL Tim-3+ T cells, indicating that Tim-3 expression defined a population of dysfunctional TIL Th1/Tc1 cells. Mechanism analyses showed that TIL Tim-3-expressing CD8+ T cells exhibited impaired Stat5 and p38 signaling pathway. Blocking the Tim-3 pathway restored cell proliferation and increased IFN-γ production in TIL CD4+ and CD8+ T cells of RCC. These results suggest that Tim-3 may be used as a novel target for increasing immune responses in RCC tumor microenvironment.

Abstract 4064: Targeting the anti-tumor function of B cells in non-small cell lung cancer patient tumors

Abstract Despite improvements in surgical techniques and combined chemotherapies, the 5-year survival rate for all stages of non-small cell lung cancer (NSCLC) is only 18%. Understanding the function of tumor infiltrating lymphocytes (TILs) in NSCLC patient tumors will contribute to the development of rationally designed treatments and improved statistics. B cells in tumors (TIL-Bs) are detected in NSCLC and their frequency correlates with improved survival, however, they are understudied in human tumors. We hypothesize that TIL-Bs help generate potent, long-term immune responses against cancer. We used un-manipulated, primary human B cells from fresh tumor, tumor-adjacent, and normal (cancer-free) lung tissue. We performed a comprehensive flow cytometric analysis of all TILs in NSCLC: CD4 and CD8 TILs, T regulatory cells, TIL-Bs, NK and NKT cells. We further examined the phenotype of TIL-Bs by analyzing markers of lymphocyte activation (anti-tumor; CD69, CD86, HLA-DR, CD27) and exhaustion (pro-tumor; CD95, CD21, PD-1, PD-L1). We developed an in vitro antigen presentation assay that analyzed if TIL-Bs present tumor-specific antigens to CD4 TILs to help stimulate an anti-tumor response in NSCLC patient tumors. The total number of B cells at the site of the tumor versus the tumor-adjacent tissue was increased compared to other immune subsets. In analyzing the markers of activation and exhaustion, we observed a spectrum of activation of TIL-Bs. Further, we observed that TIL-Bs present autologous tumor antigens to CD4 TILs in a subset of NSCLC patients. These data suggest that some patients have functional TIL-Bs and some patients have nonfunctional TIL-Bs. XAGE-1b is an immunogenic, lung cancer-specific antigen that generates CD4 and CD8 T cell responses in lung cancer patients. It was overexpressed in the tumor cells of our NSCLC patients, and we demonstrated that some TIL-Bs could present recombinant XAGE-1b antigen to CD4 TILs. The TIL-Bs that could not present tumor-specific antigens had higher expression of B cell exhaustion markers on the cell surface. Further, because PD-1 expression is increased on CD4 TILs in our NSCLC patients, we are combining blockade of the immune inhibitory PD-1:PD-L1 pathway in our antigen presentation assays. In conclusion, the anti-tumor function of TIL-Bs can be stimulated in some NSCLC patients, and TIL-Bs that cannot be stimulated have increased immune exhaustion. Ultimately, results from this study will advance the understanding of the anti-tumor function of TIL-Bs in solid tumors and will help develop a model of B cell exhaustion in human cancer. It will also help predict which TIL-B functions to target in future TIL-B-specific immunotherapies or in combination with current successful immunotherapies for NSCLC patients like blockade of the immune inhibitory PD-1:PD-L1 pathway. Citation Format: Tullia Carmela Bruno, Brandon L. Moore, Daniel J. Munson, Peggy Ebner, Jeffrey Kern, Jill E. Slansky. Targeting the anti-tumor function of B cells in non-small cell lung cancer patient tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4064. doi:10.1158/1538-7445.AM2015-4064

Abstract 1315: High IFNγ and perforin and low GM-CSF and sCD40L production correlate with CD8 TIL growth in breast cancer

Abstract Introduction: Adoptive cell therapy (ACT) with expanded autologous tumor-infiltrating lymphocytes (TIL) shows promise in melanoma but has not been developed for breast cancer (BC) because BC-TIL have not demonstrated consistent antitumor potency. This study examined the characteristics and potency of BC-TIL generated by using TIL culture techniques established for our ACT protocol in melanoma. Methods: Clinically annotated fresh specimens of tumor tissue were collected during surgical treatment of 8 patients with breast cancer and 26 patients with melanoma. TIL for each specimen were grown from 3-24 tumor fragments, depending on tumor size. After several weeks of cell growth, the expression of CD3, CD4 and CD8 on TIL was measured by flow cytometry. Then TIL were co-cultured with autologous tumor cells, and IFNγ expression level was measured by ELISA as an indicator of antitumor potency. Luminex assay was used to measure the expression of 18 immune mediators (GM-CSF, IFNγ, IL-1α, IL-1Rα, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL-17a, IP-10, sCD40L, MCP-1, TNFβ, sCD137, granzyme-B, and perforin) in the BC-TIL culture supernatant. Mixed effect model with random intercept was used for univariable and multivariable analysis to determine which immune mediators were associated with high CD8 cell percentage in TIL cultures. Results: TIL were successfully grown from 62 of 64 (97%) BC fragments and from 487 of 622 (78%) melanoma fragments (p = 0.004). TIL reached a sufficient number for co-culture in 16±3 days for BC-TIL and in 25±9 days for melanoma-TIL (p<0.0001). In the successful TIL cultures, 6 of 62 (9.6%) BC-TIL and 290 of 487 (60.0%) melanoma-TIL showed antitumor potency: 47.0% BC-TIL and 70.7% melanoma-TIL contained more CD8 cells than CD4 cells. The total number of expanded BC-TIL was inversely associated with MCP-1 expression (p<0.0001) and AJCC stage (p<0.0007). In univariable analysis, low expression of IL-10 and sCD40L, and high expression of perforin were associated with increased CD8 cell percentage in TIL cultures (P<0.001, P<0.03, and P<0.0001, respectively). In multivariable analysis, low GM-CSF and sCD40L, and high IFNγ and perforin were associated with high CD8 cell percentage in TIL cultures (P<0.002, P<0.008, P = 0.02, and P<0.001, respectively). Conclusions: Antitumor potency of BC-TIL was less frequently but BC fragments can grow TIL faster than melanoma fragments. The CD4:CD8 cell ratio was higher in BC-TIL than in melanoma-TIL. Blocking antibodies against IL-10, GM-CSF and sCD40L should be investigated to improve CD8 expansion from BC-TIL. In addition, because BC-TIL contained more CD4, it might be possible to promote tumor regression via mediation of a BC antigen by CD4 cells. Citation Format: Hitoe Torisu-Itakura, Yueqin Quan, Myung Shin Sim, Maggie L. DiNome, Peter A. Sieling, Delphine J. Lee, Mark B. Faries. High IFNγ and perforin and low GM-CSF and sCD40L production correlate with CD8 TIL growth in breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1315. doi:10.1158/1538-7445.AM2015-1315

Correlation of tumor-infiltrating lymphocytes with bladder cancer recurrence in patients with solitary low-grade urothelial carcinoma.

The aim of the present study was to correlate tumor-infiltrating lymphocytes (TIL) with bladder cancer recurrence in patients with solitary low-grade non-muscle-invasive bladder cancer (NMIBC). We retrospectively identified from the institutional database 115 patients with solitary low-grade NMIBC after transurethral resection (TURBT) without adjuvant therapy and with complete follow-up, between 1996 and 2006. Tumor specimens were retrieved and tissue microarrays were constructed. Patients were divided in two groups: those who developed recurrent disease (n = 69) and those without recurrence (n = 46) during a follow-up period of a minimum of 5 years. Immunohistochemical staining for TIL with anti-CD3, CD4, CD8, CD20, CD56, CD68, and granzyme B (GrB) was performed. Student's t test, Mann-Whitney U test, as well as uni- and multivariate analyses were applied to compare the two patient groups. TIL were predominantly observed in cancer stroma. The number of CD3+ and CD8+ lymphocytes observed in the non-recurrent group of patients was lower than that in recurrent patients (p = 0.0001, p = 0.0002, respectively). Also, in uni- and multivariate analyses, levels of CD3+ TIL (OR = 5.4035; p = 0.0001 and OR = 5.8280; p = 0.0102) and CD8+ TIL (OR = 3.2857; p = 0.0036 and OR = 5.3257; p = 0.0092) showed prognostic value with regard to NMIBC recurrence. Our results suggest that CD3+ and CD8+ TIL are predictive of bladder cancer recurrence in patients with solitary low-grade NMIBC which might facilitate identification of patients with higher risk of recurrence. However, prospective validating studies have to confirm these results before immunohistochemical staining for CD3 and CD8 TIL can be included in the clinical workup of these patients.

Progressive loss of differentiated effector CD8 T cells infiltrating melanoma (TUM7P.1021)

Abstract CD8 tumor infiltrating lymphocytes (TIL) are critical for tumor control and their presence is a positive prognostic factor. Understanding CD8 TIL dysfunction is vital for developing cancer immunotherapies. Here, using a Granzyme B Cre ROSA YFP reporter system, we characterized the differentiation of CD8 T cells over the course of tumor growth using a murine melanoma model. At early stages of the CD8 T cell response (day 10 after tumor injection), the majority of the cells had differentiated to express Granzyme B (YFP+). However, over a period of four days, most of these effector cells were replaced by CD8+ T cells that had no evidence of effector differentiation. Concurrently, we found an increase in expression of the inhibitory receptor PD-1 in differentiated (YFP+) relative to non-differentiated (YFP-) CD8 TIL in late stage tumors. This suggests that there is both a loss of differentiated CD8 TIL and that the differentiated CD8 T cells that remain are more dysfunctional in late stage tumors. To provide insight into the cause and effect of this fluctuation in differentiation, we present data establishing the contribution of checkpoint inhibitory molecules and dendritic cell activation state in the loss of effector characteristics, and whether cytokine production and other effector activities correlate with the shift in effector differentiation status.

Targeting the anti-tumor function of B cells in non-small cell lung cancer patient tumors (TUM9P.1008)

Abstract Understanding the function of tumor infiltrating lymphocytes (TILs) in NSCLC patient tumors will contribute to the development of rationally designed treatments and an increase in the current 18% survival rate. We hypothesize that TIL-Bs help generate potent, long-term immune responses against cancer. We used un-manipulated, primary human B cells from fresh tumor and tumor adjacent lung tissue to determine the phenotype and function of TIL-Bs in NSCLC patients. The total number of B cells at the site of the tumor versus the tumor-adjacent tissue was increased compared to other immune subsets. In analyzing the markers of activation and exhaustion, we observed a spectrum of activation of TIL-Bs. Further, we observed that TIL-Bs present autologous tumor antigens to CD4 TILs in a subset of NSCLC patients. These data suggest that some patients have functional TIL-Bs and some patients have nonfunctional TIL-Bs; TIL-Bs that could not present tumor-specific antigens had higher expression of B cell exhaustion markers. Lastly, we are combining blockade of the immune inhibitory PD-1:PD-L1 pathway in our antigen presentation assays because PD-1 expression is increased on CD4 TILs in our NSCLC patient tumors. Results from this study will advance the understanding of the anti-tumor function of TIL-Bs in solid tumors and will develop a model of B cell exhaustion in human cancer. Ultimately, we will predict which TIL-B functions to target in future NSCLC immunotherapies.

Epithelial and Tumor-associated Endothelial Expression of B7-H3 in Cervical Carcinoma

B7-H3 is a transmembrane protein and a member of the B7 family of immune regulatory ligands. It exerts both inhibitory and stimulatory effects on the activation of T cells. We investigated the expression of B7-H3 in invasive squamous cell carcinoma (ISCC) of the uterine cervix by immunohistochemistry, and aimed to determine whether expression of this factor is involved in the progression of the morphologic spectrum from normal cervical epithelia to cervical intraepithelial neoplasia and cervical ISCC. In addition, we sought to examine the relation of B7-H3 to the abundance of tumor-infiltrating and tumor-associated CD8(+) lymphocytes and to the evidence of phosphohistone H3, which is a core histone protein detected during mitosis. B7-H3 immunostaining was scored with regard to quantity and intensity of positively stained cells, and was noted in membranous and cytoplasmic patterns in epithelial cells and on endothelia of stromal blood vessels. Compared with those in intraepithelial neoplasias, immunoscores were significantly increased in ISCC (P<0.0001 for epithelial and endothelial expression, respectively). High scoring was associated with International Federation of Gynecology and Obstetrics stages IB and higher. Immunoscores of epithelial and endothelial B7-H3 expression were correlated significantly (P=0.0358). Epithelial and endothelial expression of B7-H3 was inversely related with CD8(+) tumor-infiltrating lymphocyte (P<0.0001). Moderate/strong B7-H3 epithelial as well as endothelial expression was mutually increased with intermediate/strong phosphohistone H3 scores (P=0.0396 and P=0.0483, respectively). There was no statistical relation with survival; however, no patient with negative scoring died of her tumor. Our results indicate that B7-H3 expression in cervical ISCC may play an important role in overcoming CD8(+) T-cell immunoregulation to acquire aggressive growth.

Manipulating the tumor microenvironment ex vivo for enhanced expansion of tumor-infiltrating lymphocytes for adoptive cell therapy.

Cultured tumor fragments from melanoma metastases have been used for years as a source of tumor-infiltrating lymphocytes (TIL) for adoptive cell therapy (ACT). The expansion of tumor-reactive CD8(+) T cells with interleukin-2 (IL2) in these early cultures is critical in generating clinically active TIL infusion products, with a population of activated 4-1BB CD8(+) T cells recently found to constitute the majority of tumor-specific T cells. We used an agonistic anti-4-1BB antibody added during the initial tumor fragment cultures to provide in situ 4-1BB costimulation. We found that addition of an agonistic anti-4-1BB antibody could activate 4-1BB signaling within early cultured tumor fragments and accelerated the rate of memory CD8(+) TIL outgrowth that were highly enriched for melanoma antigen specificity. This was associated with NFκB activation and the induction of T-cell survival and memory genes, as well as enhanced IL2 responsiveness, in the CD8(+) T cells in the fragments and emerging from the fragments. Early provision of 4-1BB costimulation also affected the dendritic cells (DC) by activating NFκB in DC and promoting their maturation inside the tumor fragments. Blocking HLA class I prevented the enhanced outgrowth of CD8(+) T cells with anti-4-1BB, suggesting that an ongoing HLA class I-mediated antigen presentation in early tumor fragment cultures plays a role in mediating tumor-specific CD8(+) TIL outgrowth. Our results highlight a previously unrecognized concept in TIL ACT that the tumor microenvironment can be dynamically regulated in the initial tumor fragment cultures to regulate the types of T cells expanded and their functional characteristics.

Immune-mediated antitumor effect by type 2 diabetes drug, metformin

Metformin, a prescribed drug for type 2 diabetes, has been reported to have anti-cancer effects; however, the underlying mechanism is poorly understood. Here we show that this mechanism may be immune-mediated. Metformin enabled normal but not T-cell-deficient SCID mice to reject solid tumors. In addition, it increased the number of CD8(+) tumor-infiltrating lymphocytes (TILs) and protected them from apoptosis and exhaustion characterized by decreased production of IL-2, TNFα, and IFNγ. CD8(+) TILs capable of producing multiple cytokines were mainly PD-1(-)Tim-3(+), an effector memory subset responsible for tumor rejection. Combined use of metformin and cancer vaccine improved CD8(+) TIL multifunctionality. The adoptive transfer of antigen-specific CD8(+) T cells treated with metformin concentrations as low as 10 μM showed efficient migration into tumors while maintaining multifunctionality in a manner sensitive to the AMP-activated protein kinase (AMPK) inhibitor compound C. Therefore, a direct effect of metformin on CD8(+) T cells is critical for protection against the inevitable functional exhaustion in the tumor microenvironment.

Comprehensive analysis of predictive biomarkers for patients with curative colorectal cancer patients and high risk of recurrence.

607 Background: Preoperative serum systemic inflammatory response (SIR) has been reported in patients with various cancers including colorectal cancer (CRC) as predictive biomarker of early recurrence. Molecular phenotypes of CRC including microsatellite instability (MSI) status and tumor infiltrating lymphocytes (TILs) have also known to be associated with recurrence in curative CRC patients. We comprehensively evaluated SIR status, MSI status and TILs in curative CRC patients and investigated which can be promising as high risk markers of recurrence in these patients. Methods: This retrospective study enrolled 157 CRC patients with stage I-III undergoing curative operation for whom preoperative neutrophil/lymphocyte ratio (NLR), platelet/lymphocyte ratio (PLR) and CRP were available as indicators of SIR status. For analysis of molecular phenotypes, we evaluated TILs by counting the number of intra-tumor Foxp3 and CD8 positive T cells by IHC analysis. Next, MSI status was determined in using 5 mononucleotide repeat microsatellite markers. Finally, we investigated the impact of SIR status, TILs and MSI status on the recurrence of these patients. Results: This study included a total of 90 males and 67 females, with an average age of 66.9 years. Twenty-nine patients (18.4%) developed recurrence. Kaplan-Meier analysis using SIR indicators revealed that patients with high CRP, NLR and PLR levels were significantly poorer disease free survival (DFS) than those with low levels. Low infiltrating CD8-positive T cells were significantly predictive factors for poor DFS, but there was no correlation between MSI status and recurrence, In univariate analysis, low infiltrating CD8-positive T cells, high CEA, CRP, NLR and PLR levels in serum were significantly predictive factors for poor DFS, respectively. Multivariate analysis showed that low infiltrating CD8-positive T cells and high serum CRP levels were independent predictive markers for recurrence in curative CRC patients. Conclusions: We demonstrated that evaluation of both CRP levels in preoperative serum and tumor infiltrating CD8 lymphocytes in CRC tissues are useful to predict the curative CRC patients with early relapse.

Association between Chemotherapy-Response Assays and Subsets of Tumor-Infiltrating Lymphocytes in Gastric Cancer: A Pilot Study.

PurposeThe purpose of this pilot study was to evaluate the association between adenosine triphosphate-based chemotherapy response assays (ATP-CRAs) and subsets of tumor infiltrating lymphocytes (TILs) in gastric cancer.Materials and MethodsIn total, 15 gastric cancer tissue samples were obtained from gastrectomies performed between February 2007 and January 2011. Chemotherapy response assays were performed on tumor cells from these samples using 11 chemotherapeutic agents, including etoposide, doxorubicin, epirubicin, mitomycin, 5-fluorouracil (5-FU), oxaliplatin, irinotecan, docetaxel, paclitaxel, methotrexate, and cisplatin. TILs in the tissue samples were evaluated using antibodies specific for CD3, CD4, CD8, Foxp3, and Granzyme B.ResultsThe highest cancer cell death rates were induced by etoposide (44.8%), 5-FU (43.1%), and mitomycin (39.9%). Samples from 10 patients who were treated with 5-FU were divided into 5-FU-sensitive and -insensitive groups according to median cell death rate. No difference was observed in survival between the two groups (P=0.216). Only two patients were treated with a chemotherapeutic agent determined by an ATP-CRA and there was no significant difference in overall survival compared with that of patients treated with their physician's choice of chemotherapeutic agent (P=0.105). However, a high number of CD3 TILs was a favorable prognostic factor (P=0.008). Pearson's correlation analyses showed no association between cancer cell death rates in response to chemotherapeutic agents and subsets of TILs.ConclusionsCancer cell death rates in response to specific chemotherapeutic agents were not significantly associated with the distribution of TIL subsets.

Generation of Potent Tumor-Specific CTLs from High-Dose IL-2 Activated naïve CD8 T Cells with Overcoming the Tumor-Derived Immune Suppression

(Background) Adoptive T cell therapy using tumor-infiltrating lymphocytes (TILs) is a promising treatment for cancer patients unresponsive to conventional therapies. However, clonal expansion of T cells easily faced with T cell exhaustion or terminal differentiation which related to suppression of the tumor-killing function in tumor microenvironment. Naïve CD8+T cells have an astounding capacity to react to antigens by massive expansion and differentiation into cytotoxic effector cells. However, it is the main key factor that how can increase the limited number of tumor-specific naïve CD8 T cells due to the negative selection during T cell thymic development.(Methods) In human, naïve, memory T cells and TILs were activated with CD3/CD28 dynabeads and culture on CD2-coated plate to generate various effector T cell populations (Teff N, Teff M and activated TILs). Those different effectors were analyzed the expression of surface exhaustion phenotypes, intracellular transcription factors (T-bet/Eomes) and granzyme/perforin secretions using FACS and western blot assay. Telomere length of three kinds of effector cells was analyzed. High-dose IL-2 (1ug/mL) activated naïve CD8 T cells were stimulated with tumor antigen-loaded dendritic cells (DCs) and then, ELISPOT/cytokine ELISA assay was performed to evaluate tumor-specific (TA) CTL function. In murine model, we also checked the functional difference of each effector cells as like the same methods. In addition, tumor challenging test using EG7-EL4 cell line (OVAp expression) was performed in C57BL/6 mice which adoptively transferred with OT-I thy1.1 Teff N, Teff M and activated TILs.(Results) In vitro expansion of all human naïve, memory and TIL CD8+ T cells was induced successfully. After 3-5 days of expansion, effectors from different progenitors were assessed for the several activation markers CD44, OX40 and CD27 and were considered as the CD62LlowCD44highOX40highCD27high populations. Population frequency of Teff N was significantly higher than Teff M (p < 0.05) or activated TILs (p < 0.005). Telomere length, which correlates with replicative capacity, was greatest in TeffN, shorter in TeffM and shortest in aTILs. When compared the T cell exhaustion phenotypes in three different effector cells, TeffN showed the very low expression of inhibitory markers including PD-1, CTLA-4, and KLRG-1.aTILs expressed most high exhaustion phenotypes with shorter telomere length. Moreover, the secretion of cytotoxic granules such as granzyme B and perforin gradually increased in all effector cells but, among the fully effector cells status, TeffN possess the highest expression level compared to TeffM and aTILs wheras similar level of IFN-r. To further confirm expression profile of T-box transcription factors, the expression of both T-bet and Eomes in TeffN increased at relatively early time point (D+3) and sustained high expression levels during effector status (D+5 and D+8). High expression of T-bet in TeffN promoted the full effector generation and Eomes expression linked to formation of long-term tumor-specific memory population. In CTL function, high-dose IL2-activated naïve T cells were successfully increased and generated the tumor-specific CTLs co-cultured with TA DCs, which inducing the outstanding CTL function compared to those from memory CD8 T cells or aTILs.(Discussion) High-dose IL-2 could increase the tumor-specific naïve CD8 T cells without resulting in clonal exhaustion and could generate the potent tumor-specific CTLs with overcoming the tumor-derived immune suppression compared with CD8+ TILs or secondary effectors from memory T cells. DisclosuresNo relevant conflicts of interest to declare.

Potential synergistic anti-tumor activity between lenalidomide and sorafenib in hepatocellular carcinoma.

The immune modulatory drug lenalidomide has shown promising anti-tumor activity in a clinical trial of patients with advanced hepatocellular carcinoma (HCC). The present study explored whether lenalidomide can enhance the anti-tumor activity of sorafenib, the standard molecular targeted therapy for HCC. The anti-tumor efficacy of single-agent or combination treatment was measured by change in tumor volume and animal survival using an orthotopic liver cancer model. Distribution of T-cell subpopulations in tumor-infiltrating lymphocytes (TILs) and splenocytes derived from tumor-implanted mice was measured by flow cytometry. Depletion of relevant T-cell subpopulations or cytokines was done by co-administration of relevant antibodies with study drug treatment. Tumor cell apoptosis and tumor angiogenesis were measured by transferase deoxytidyl uridine end labeling assay and immunohistochemical study, respectively. Combination of sorafenib and lenalidomide produced significant synergistic anti-tumor efficacy in terms of tumor growth delay and animal survival. This synergistic effect was associated with a significant increase in interferon-γ expressing CD8(+) lymphocytes in TILs and a significantly higher number of granzyme- or perforin-expressing CD8(+) T cells, compared with vehicle- or single-agent treatment groups. Combination treatment significantly increased apoptotic tumor cells and vascular normalization in tumor tissue. The synergistic anti-tumor effect was abolished after CD8 depletion. Lenalidomide can enhance the anti-tumor effects of sorafenib in HCC through its immune modulatory effects, and CD8(+) TILs play an important role in the anti-tumor synergism.

Tumour‐infiltrating lymphocytes and expression of programmed death ligand 1 (PD‐L1) in melanoma brain metastases

In this study we aimed to characterize immune infiltrates and expression of programmed death 1 (PD-1) and programmed death ligand 1 (PD-L1) in a series of melanoma BM to provide a basis for experimental therapy using immune checkpoint inhibitors. We investigated expression of PD-1, PD-L1, CD3, CD8, CD45RO, forkhead box protein 3 (FoxP3), CD20 and BRAF V600E by immunohistochemistry in melanoma BM samples. Forty-three specimens [27 of which (62.8%) were BRAF V600E-positive] were available. CD3(+) tumour-infiltrating lymphocytes (TILs) were evident in 33 specimens (76.7%), CD8(+) in 39 (90.7%), CD45RO(+) in 32 (74.4%), PD-1(+) in 27 (62.8%), FoxP3(+) in 21 (48.8%) and CD20(+) TILs in 19 (44.2%). Tumour PD-L1 expression was observed in 22 specimens (51.1%), and in nine of these (40.9%) expression was observed in more than 5% of tumour cells. PD-L1 expression was associated with higher density of PD-1(+) (P < 0.001), CD3(+) (P = 0.014) and FoxP3(+) (P < 0.001) TIL infiltration. Density of CD3(+) TILs was associated with density of CD8(+) (P < 0.001), PD-1(+) (P < 0.001) and CD45RO(+) (P < 0.001) TILs. PD-L1 expression or PD-1(+) , CD3(+) , CD8(+) or CD45RO(+) TILs density did not correlate with BRAF V600E status, previous systemic therapy or survival (P > 0.05). Melanoma BM showed considerable lymphocytic infiltrates and expression of PD-L1 in the majority of investigated specimens, with high PD-L1 expression found predominantly in regions of abundant inflammation. Our data indicate that clinical studies should investigate the value of checkpoint inhibitors in patients with melanoma BMs.

Tissue resident memory T cells are found in the primary and metastatic tumors of breast cancer patients (TUM2P.922)

Abstract Tumor tissues are composed of significant numbers of Tumor Infiltrating Lymphocytes (TILs), which may include CD8+ T cells, CD4+ T cells, and B cells. Of the T cell population, it is known that these cells are often functionally suppressed and defective in their ability to kill and proliferate. However, little is known about how T cells traffic into tumor sites. Furthermore, it is unclear how the various T cell memory subtypes play a role in attempting to eradicate tumor cells. Such knowledge is critical for the development of successful cancer immunotherapies. Here we assayed peripheral blood mononuclear cells (PBMCs), tumor tissue, and sentinel lymph nodes (SLNs) of breast cancer patients to phenotype memory T cells. We found higher proportions of EM cells among CD8 TILs as compared to SLN CD8 T cells. Conversely, the CD4 compartment in both TILs and SLNs was dominated by CM CD4 T cells. Further analysis showed that breast cancer patient CD8+ TILs in primary tumors were made up of a significant population of Tissue Resident Memory cells (TRMs) as identified by CD103 expression and high CD69 expression. We also showed that the CD8+ TILs found in a metastatic brain lesion of a breast cancer patient also contained TRMs. Finally we showed that healthy breast tissue from a patient with no clinical signs of breast cancer also contained CD8+ TRMs.

Tumour-infiltrating CD4+ and CD8+ lymphocytes as predictors of clinical outcome in glioma

Background:T lymphocyte infiltration has been detected in glioma, although its significance remains unclear. The purpose of the present study was to explore the prognostic value of CD4+ and CD8+ tumour-infiltrating lymphocytes (TILs) in glioma, and the prognostic value of infiltrating Forkhead box protein 3 (FoxP3+) regulatory T cells were also investigated.Methods:CD4+, FoxP3+ and CD8+ TILs were assessed by immunohistochemical staining of tissue microarray cores from 284 gliomas. Kaplan–Meier analysis and Cox proportional hazards models were used to examine the survival function of these TILs in 90 glioblastoma patients.Results:The number of CD8+ TILs was inversely correlated with tumour grade (P=0.025), whereas the number of CD4+ TILs was positively correlated with tumour grade (P=0.002). FoxP3+ TILs were only observed in glioblastomas, but not in low-grade astrocytomas or oligodendroglial tumours. Among patients with glioblastoma, none of CD4+ TILs, FoxP3+ TILs and CD8+ TILs alone was significantly associated with patient prognosis. However, the presence of high CD4+ and low CD8+ TIL levels was an independent predictor of poor progress-free survival (multivariate hazard ratio (HR) 1.618, 95% confidence interval (CI) 1.245–2.101, P<0.001) and poor overall survival (multivariate HR 1.508, 95% CI 1.162–1.956, P=0.002). Moreover, pseudoprogression was more often found in patients with high CD4+ TILs and high CD8+ TILs.Conclusions:The combination of CD4+ and CD8+ TILs is a predictor of clinical outcome in glioblastoma patients, and a high level of CD4+ TILs combined with low CD8+ TILs was associated with unfavourable prognosis.

Identification by digital immunohistochemistry of intratumoral changes of immune infiltrates after vaccine in the absence of modifications of PBMC immune cell subsets

Preclinical studies have demonstrated that the combination of systemic subcutaneous (s.c.) vaccination with intratumoral (i.t.) vaccination was superior in the induction of antitumor activity vs. vaccination with either route alone. A subsequent phase I study employing i.t.-s.c. vaccination was carried out in men with locally recurrent or progressive prostate cancer. rF-PSA-TRICOM (PROSTVAC) vaccine was administered intraprostatically and rV-PSA-TRICOM followed by rF-PSA-TRICOM vaccine was administered systemically. In that study no dose limiting toxicities were observed, 19/21 patients had stable or improved prostate-specific antigen (PSA) values and tumor-infiltrating lymphocytes (TILs) increased in post- vs. pre-treatment tumor biopsies, analyzed employing conventional immunohistochemistry (IHC). In the studies reported here, 31 phenotypes of peripheral blood mononuclear cells (PBMCs) were analyzed prevaccination and postvaccination as well as the functions of PBMC regulatory T cells (Tregs) and natural killer cells. A trend was observed in decreases in serum PSA with the reduction of circulating Tregs postvaccination. Digital IHC was employed prevaccination and postvaccination to measure CD4 and CD8 TILs, as well as Treg TILs by conventional IHC. Few correlations were observed with CD4, CD8 or Treg in TILs vs. PBMCs. However, patients with lower levels of CD4 TILs prevaccination showed the greatest increases in CD4 TILs postvaccine, while Treg TILs decreased postvaccine. There was also a strong correlation between decreases in serum PSA and increases in CD8 TILs postvaccine. These studies provide additional rationale for the use of i.t.-s.c. vaccinations and demonstrate a noncoordinate expression of specific immune subsets in PBMCs vs. tumor.