Analysis Of Tumor-infiltrating Lymphocytes Research Articles

Abstract 3119: Treatment prediction by subset analysis of tumor infiltrating lymphocytes (TILs) in combination therapy with trastuzumab, pertuzumab, and docetaxel for advanced HER2-positive breast cancer

Abstract Background: The trastuzumab, pertuzumab, and docetaxel (TPD) regimen is strongly recommended as a treatment option for first-line therapy for advanced human epidermal growth factor receptor (HER) 2-positive breast cancer. The immune microenvironment in cancer is involved in many antitumor treatment effects, and tumor-infiltrating lymphocytes (TILs) is being established as a biomarker for therapeutic effect and prognosis. Recently, subset analysis of TILs is also under way. We have previously reported the clinical validity and benefits of evaluating TILs for neoadjuvant chemotherapy (NAC). Chemotherapy with a TPD regimen is garnering attention for its clinical outcomes and impact on cancer microenvironments. In this study, we evaluated the impact of chemotherapy with a TPD regimen, on immune micro environments in HER2-positive breast cancer using immune related proteins as indicators. Methods: The subjects consisted of 30 patients who received the TPD regimen. The expression levels of estrogen receptor (ER), progesterone receptor (PgR), Ki67, CD8, forkhead box protein (FOXP) 3, programmed death (PD) 1, and programmed death ligand (PD-L) 1 were evaluated in biopsy specimens, by immunostaining. We also examined the ratio of CD8 and FOXP3 (CFR). Results: The objective response rate (ORR) in the high CFR group was higher than in the low CFR group (p=0.013). The CD8 positive, high CFR and PD-L1 negative group had significantly longer PFS than the CD8 negative, low CFR and PDL1 positive group (p=0.045, log-rank) (p=0.007, log-rank) (p=0.040, log-rank), respectively. The high CFR group had significantly better OS than the low CFR group (p=0.034, log-rank). Receiver operating characteristic (ROC) analyses showed that, for advanced HER2-positive breast cancer patients, the CFR results [area under the curve (AUC): 0.708] were better than those for the other factors (AUC: CD8=0.681, FOXP3=0.639, PD1=0.528, PD-L1=0.681). Conclusions: This study shows with the TPD regimen, a high CFR leads to a high ORR and long PFS in HER2-positive breast cancer. CFR, therefore, may be one of the important prognostic factors for this disease. Citation Format: Koji Takada, Shinichiro Kashiwagi, Yuka Asano, Goto Wataru, Tamami Morisaki, Satoru Noda, Tsutomu Takashima, Naoyoshi Onoda, Kosei Hirakawa, Masaichi Ohira. Treatment prediction by subset analysis of tumor infiltrating lymphocytes (TILs) in combination therapy with trastuzumab, pertuzumab, and docetaxel for advanced HER2-positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3119.

Abstract LB-306: Oncolytic vaccinia virus expressing immune checkpoint blockade antibody as cancer immunotherapeutics

Abstract Oncolytic viruses can be engineered to become multifunctional cancer immunotherapeutic agents, used either as monotherapy or in combination with immune checkpoint blockade for antitumor effects. Intratumoral (IT) delivery of oncolytic viruses expressing immunomodulatory agents can alter the tumor immunosuppressive microenvironment and facilitate the proliferation and activation of antitumor effector and memory T cells. Although anti-cytotoxic T lymphocyte protein 4 (CTLA-4) antibody has been approved for the treatment of advanced melanoma, its toxicity profile and modest efficacy as a single agent have limited its use in the clinic. Poxviruses are large cytoplasmic DNA viruses and vaccinia virus is a prototypic poxvirus that has been investigated intensively as an oncolytic virus. Its 200-kb genome size allows large insertions of multiple foreign genes. Vaccinia virus has been used in humans extensively in the past during smallpox vaccination. In this study, we engineered a vaccinia (Western Reserve)-based oncolytic virus that expresses anti-muCTLA-4 antibody and human FMS-like tyrosine kinase 3 ligand (Flt3L) for the treatment of murine implantable melanoma via intratumoral injection. We used a mutant vaccinia virus E3LΔ83N as the parental virus. This virus has a deletion of the Z-DNA-binding domain of E3, a key virulence factor, which results in 1000-fold attenuation of the virus. Through homologous recombination at the thymidine kinase (TK) locus of vaccinia E3LΔ83N virus, we successfully generated an attenuated recombinant virus (E3LΔ83N-TK--hFlt3L-anti-muCTLA-4) with a deletion of TK and an insertion of a cassette that allows the expression of both hFlt3L and anti-muCTLA-4 antibody under the vaccinia synthetic early and late promoter. This virus replicates in murine and human tumor cell lines and expresses desired anti-muCTLA-4 antibody and hFlt3L in murine and human melanoma cell lines. IT delivery of this recombinant vaccinia virus is more efficacious compared with E3LΔ83N-TK- (used either alone or in combination of systemic delivery of anti-CTLA-4 antibody), in eradicating or delaying the growth of both injected tumors and non-injected distant tumors, as well as in prolonging the survival of mice in a murine bilateral B16-F10 melanoma tumor implantation model. Furthermore, immunological analyses of tumor-infiltrating lymphocytes, as well as antitumor T cells in spleens, showed highest numbers of activated Granzyme B+ CD4+ and CD8+ T cells in the non-injected distant tumors and highest numbers of antitumor CD8+ T cells in the spleens of mice treated with IT delivery of E3LΔ83N-TK--hFlt3L-anti-muCTLA-4 compared with those treated with E3LΔ83N-TK--hFlt3L or E3LΔ83N-TK-. No toxicities related to IT E3LΔ83N-TK--hFlt3L-anti-muCTLA-4 have been observed. Taken together, our results demonstrate that IT delivery of oncolytic vaccinia virus expressing anti-CTLA-4 and hFlt3L is a safe and effective strategy to enhance antitumor immunity. Citation Format: Weiyi Wang, Peihong Dai, Ning Yang, Stewart Shuman, Wei Yan, Taha Merghoub, Jedd D. Wolchok, Liang Deng. Oncolytic vaccinia virus expressing immune checkpoint blockade antibody as cancer immunotherapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-306.

Abstract B32: Lymphocytes and immune checkpoint biomarker expression in sarcomas

Abstract Recent clinical trials of immune checkpoint inhibitors have raised hopes for sarcoma therapy, but they have not taken into account the variability of the immune microenvironment in their design or interpretation. Here, we present a comprehensive analysis of tumor-infiltrating lymphocytes (TILs) across a spectrum of sarcoma types, in order to better identify sarcoma populations that are more likely to respond to specific immunotherapies. Methods: From a tissue microarray database of 1918 formalin-fixed, paraffin-embedded sarcoma tissue samples spanning 24 subtypes, immunohistochemistry was performed to quantify TILs, to characterize major T-cell subsets (CD4, CD8, FOXP3), and to evaluate expression of targetable immune checkpoint ligand PDL1 (programmed death ligand 1) and its immune cell receptor PD1. PDL1 was scored by percentage of tumor cells exhibiting positive membranous staining; all other markers were quantified by counting positive-staining lymphocytes. Results: Lymphocytes were present in 93% of samples (ranging from 1-1300 TILS/mm2), of which 63% were CD8+. Dedifferentiated liposarcoma (DDLPS) showed the greatest enrichment, with a median count of 103 TILs/mm2. Other subtypes with notably high TIL counts include undifferentiated pleomorphic sarcoma (UPS; 38 TILs/mm2), myxofibrosarcoma (35 TILs/mm2), well-differentiated liposarcoma (26 TILs/mm2), osteosarcoma (21 TILs/mm2), and leiomyosarcoma (19 TILs/mm2). Without exception, translocation-associated sarcoma subtypes had very low levels of infiltrating lymphocytes (median 9 TILs/mm2). The subtype with the lowest levels of TILs was chondrosarcoma (0 TILs/mm2). PDL1 was present (≥1% tumor cell staining cutpoint) in 7% of samples, and stained ≥10% of tumor cells in 5.3% of samples. The subtypes that account for the majority of this PDL1 positivity are UPS (15/72), osteosarcoma (12/224), malignant peripheral nerve sheath tumor (9/74), myxofibrosarcoma (8/52), and DDLPS (6/60). Angiosarcoma had the highest proportion of positive cases (60%), but this was based on a small number of samples (n=5). PD1 lymphocyte staining was also low overall, with 14% of samples having at least one PD1+ TIL per mm2. The subtypes with the highest proportions of PD1+ cases were DDLPS (36/79), UPS (23/80), leiomyosarcoma (8/19), and epithelioid sarcoma (4/8). Conclusions: Lymphocyte infiltration is predominantly limited to complex karyotype sarcomas and is rarely seen in translocation-associated sarcomas. The very highest levels of infiltrates are observed in DDLPS and the very lowest in chondrosarcoma. CD8+ effector T cells are the predominant lymphocyte subset present in sarcomas. PDL1 and PD1 expression are low overall for sarcomas, but some subtypes (UPS, DDLPS) appear to employ PDL1 immune blockade in up to 20% of cases. Further studies are pending to determine the significance of these findings for immunotherapy response. Citation Format: Amanda R. Dancsok, David Thomas, Jean-Yves Blay, Robert G. Maki, Torsten O. Nielsen, Elizabeth G. Demicco. Lymphocytes and immune checkpoint biomarker expression in sarcomas [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr B32.

Absence of myeloid Klf4 reduces prostate cancer growth with pro-atherosclerotic activation of tumor myeloid cells and infiltration of CD8 T cells.

The microenvironment of prostate cancer often includes abundant tumor-associated macrophages (TAMs), with their acquisition of an M2 phenotype correlating with local aggressiveness and metastasis. Tumor-derived M-CSF contributes to TAM M2 polarization, and M-CSF receptor inhibition slows prostate cancer growth in model systems. As additional cytokines can direct TAM M2 polarization, targeting downstream transcription factors could avoid resistance. Klf4 and C/EBPβ each contribute to monocyte development, and reduced expression of macrophage Klf4 or C/EBPβ favors their adoption of a pro-inflammatory M1 state. We find that a Hi-Myc C57BL/6 prostate cancer line grows more slowly in syngeneic Klf4(f/f);Lys-Cre compared with Klf4(f/f) mice when inoculated subcutaneously, but grows equally rapidly in C/EBPβ(f/f);Lys-Cre and C/EBPβ(f/f) hosts. In the absence of myeloid Klf4, TAMs have reduced expression of surface mannose receptor and Fizz1 mRNA, both M2 markers. Global gene expression analysis further revealed activation of pro-inflammatory, pro-atherosclerotic pathways. Analysis of tumor-infiltrating lymphocytes (TILs) demonstrated markedly increased activated CD8 T cell numbers, and CD8 T cell depletion obviated the inhibitory effect of myeloid Klf4 deletion on prostate cancer growth. These findings suggest that reducing expression or activity of the Klf4 transcription factor in tumor myeloid cells may contribute to prostate cancer therapy.

Co-administration of RANKL and CTLA4 Antibodies Enhances Lymphocyte-Mediated Antitumor Immunity in Mice.

Purpose: Novel partners for established immune checkpoint inhibitors in the treatment of cancer are needed to address the problems of primary and acquired resistance. The efficacy of combination RANKL and CTLA4 blockade in antitumor immunity has been suggested by recent case reports in melanoma. Here, we provide a rationale for this combination in mouse models of cancer.Experimental Design: The efficacy and mechanism of a combination of RANKL and CTLA4 blockade was examined by tumor-infiltrating lymphocyte analysis, tumor growth, and metastasis using a variety of neutralizing antibodies and gene-targeted mice.Results: RANKL blockade improved the efficacy of anti-CTLA4 mAbs against solid tumors and experimental metastases, with regulatory T-cell (Treg)-depleting anti-CTLA4 mAbs of the mouse IgG2a isotype showing greatest combinatorial activity. The optimal combination depended on the presence of activating Fc receptors and lymphocytes (NK cells for metastatic disease and predominantly CD8+ T cells for subcutaneous tumor control), whereas anti-RANKL alone did not require FcR. The significantly higher T-cell infiltration into solid tumors post anti-RANKL and anti-CTLA4 was accompanied by increased T-cell effector function (cytokine polyfunctionality), and anti-RANKL activity occurred independently of Treg depletion. The majority of RANKL expression in tumors was on T cells whereas RANK-expressing cells were mostly tumor-associated macrophages (TAM), with some expression also observed on dendritic cells (DC) and myeloid-derived suppressor cells (MDSC).Conclusions: These results provide a rationale for the further investigation of RANKL-RANK interactions in tumor immunity and a basis for development of translational markers of interest in human clinical trials. Clin Cancer Res; 23(19); 5789-801. ©2017 AACR.

Abstract 3636: Potent antitumor activity of duvortuxizumab, a CD19 x CD3 DART® molecule, in lymphoma models

Abstract Duvortuxizumab (JNJ-64052781 or MGD011) is a CD19 x CD3 DART® protein designed to engage and redirect CD3+ T-cells to eliminate CD19+ B-cells through T-cell-mediated cytotoxicity. Duvortuxizumab displays potent killing activity in lymphoma cell lines and animal models and is currently in clinical development for the treatment of B-cell malignancies. Here we examined duvortuxizumab activity alone and in combination with standard chemotherapy regimens in preclinical lymphoma models. Duvortuxizumab plus bendamustine increased T-cell mediated cytotoxicity of CD19+ Raji and DOHH2 lymphoma cells in the presence of isolated human pan T-cells (effector:target = 10:1). Minimal inhibition of T-cell activation was observed at bendamustine concentrations up to 40 µM. A Burkitt’s lymphoma model was used to evaluate the in vivo effects of duvortuxizumab plus bendamustine. NOD scid gamma mice were subcutaneously implanted with Daudi tumor cells and inoculated with either peripheral blood mononuclear cells or purified, activated human pan T-cells. Duvortuxizumab was dosed at 0.5 mg/kg (x7, twice weekly); bendamustine was dosed once at 25 mg/kg on day 1. Dosing with bendamustine alone inhibited tumor growth by 79% (p=0.0002) whereas duvortuxizumab alone resulted in complete tumor regression with no signs of relapse for up to 90 days. Dosing with duvortuxizumab plus bendamustine resulted in complete and durable tumor regression and elimination. Analysis of tumor-infiltrating lymphocytes (TILs) 3 days after the start of therapy showed that bendamustine inhibited CD3+ T-cell infiltration and activation whereas duvortuxizumab increased both. The combination showed greater activation of CD4+ and CD8+ T-cells. At 48 days after the last duvortuxizumab dose, TILs had a large percentage of CD3+ CD45RA- CCR7- cells, indicative of effector memory T-cells. This activation was accompanied by higher expression of PD-1 on T-cells and PD-L1 on tumor cells. Similar effects were seen when duvortuxizumab was combined with cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP). In vitro, a decrease in T-cell activation was observed with duvortuxizumab/CHOP, which may have been caused by inclusion of prednisone; however, cytotoxicity against tumor cells was maintained. In a patient sample-derived model of diffuse large B-cell lymphoma, duvortuxizumab (twice weekly for 4 consecutive weeks) in combination with CHOP (given once) resulted in rapid tumor regression that was more sustained than CHOP treatment alone. In summary, duvortuxizumab has potent anti-tumor activity as a single agent and in combination with standard chemotherapy in lymphoma preclinical models. Duvortuxizumab-mediated tumor killing and T-cell activation were maintained or increased in the presence of multiple chemotherapeutics, suggesting the potential clinical utility of combining duvortuxizumab with standard therapies in the treatment of B-cell malignancies. Citation Format: Liat Izhak, Dana E. Cullen, Maha Elgawly, Leopoldo Luistro, Syd Johnson, Jaime Bald, A Kate Sasser, Sriram Balasubramanian. Potent antitumor activity of duvortuxizumab, a CD19 x CD3 DART® molecule, in lymphoma models [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 3636. doi:10.1158/1538-7445.AM2017-3636

Abstract 2619: Combination of ECP1014 and anti-PD-L1 reduces tumor growth in the CT26 murine colon carcinoma model of a cold tumor

Abstract Objective: The objective of this study was to evaluate the effect of ECP1014, a new selective COX2 inhibitor, in combination with a PDL1 inhibitor on tumor growth in a CT26 murine colon carcinoma model. Colon cancer has been described as a “cold tumor” and antiPDL1monotherapy has shown limited or no benefit. In addition, many colon cancer cell lines produce high levels of PGE2, which may in turn dampen the immune system. The hypothesis was that combining ECP1014, which we have shown potently decreases PGE2 in CT26, with a checkpoint inhibitor would increase immune response to the tumor, turning it “hot” and producing superior tumor control versus either agent alone. Methods: Balb/C mice were implanted with CT26 cells and randomly assigned to 7 treatment groups; vehicle (G1), 10mg/kg rat IgG2b control (G2), 10mg/kg antiPDL1 (G3), 10mg/kg ECP1014 (G4), 10mg/kg ECP1014+10mg/kg rat IgG2b (G5), 10mg/kg ECP1014+10mg/kg antiPDL1 (G6), 1mg/kg ECP1014+10mg/kg antiPDL1(G7). ECP1014 was given daily by oral gavage; rat IgG2b and antiPDL1 (clone 10F.9G2) were administered IP. Treatment was started on day 3 post implant and mice were followed for 16 days. Tumor measurements were taken every 2 to 3 days, and a nonparametric approach was taken to assess the pairwise differences among groups using day 16 tumor volumes. In addition, a responder analysis was used. Tumor infiltrating lymphocytes (TIL) were also assessed by flow cytometry on day 16. Results: All animals developed tumors. G4 to 7 demonstrated statistically significantly (p<0.05) slowing of tumor growth when compared to vehicle (G1). In particular, G7 produced an 87% suppression of tumor growth compared to vehicle. Three animals in G7 showed regression of tumor growth and one additional animal had no detectable growth until day 16. G6 produced 65% suppression of tumor growth compared to G1. G2 to 5 produced 30, 54, 73 and 50% suppression of tumor growth respectively when compared to G1. G7 had the highest number of responders 75% vs. 62.5% for G6 and G4, with all other groups having < 25% responders. TIL analysis from G4, G6 and G7 also displayed the highest levels of CD8+ Tcells (6.3%, 6.8%, and 6.7% respectively, versus 3.8% for antiPDL1 and 2.5% for vehicle groups). Summary: ECP1014 alone was superior to antiPDL1 in slowing tumor growth. However, the combination of ECP1014+antiPDL1showed the greatest tumor response and growth suppression and markedly increased CD8+ Tcells infiltrating into the tumor vs. single agent anti-PDL1 treatment. Citation Format: Bobby W. Sandage, John J. Talley, Eduardo J. Martinez, Maryland R. Franklin, Mary Anne Meade, Dan Saims, Matt Thayer, Scott Wise, David Draper, Wilber Leopold. Combination of ECP1014 and anti-PD-L1 reduces tumor growth in the CT26 murine colon carcinoma model of a cold tumor [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 2619. doi:10.1158/1538-7445.AM2017-2619

Abstract 3993: A different spatiotemporal distribution of TIL subtypes is associated with response to neoadjuvant chemotherapy in locally advanced breast cancer patients

Abstract Background: Tumor infiltrating lymphocytes (TILs) are described as an important immune modulator in the tumor microenvironment in cancer and were shown to be associated with breast cancer (BC) outcome. Besides the analysis of total TILs, the discrimination into TIL subtypes is essential as they exhibit pro- or anti-tumorigenic potential. The spatiotemporal TIL distribution at the invasive tumor front (ITF) and the tumor center (TC) might provide insights into tumor progression. Here we analyzed TIL infiltration in core biopsies of BC patients (pts) prior to neoadjuvant chemotherapy (NACT) and correlated these findings with clinical parameters and tumor cell spread. Material and Methods: Core biopsies from 87 pre-NACT BC pts were cut, deparaffinized and antigen retrieval and automated immunohistochemistry were performed using the following primary antibodies: CD3 (clone: SP7; 1:400, DCS Innovative Diagnostik Systeme, Hamburg, Germany), CD4 (clone: 1F6; 1:40, Zytomed Systems, Berlin, Germany), CD8 (clone: C8/144B; 1:150), CD20 (clone: L26; rtu), CD68 (clone: PG-M1; 1:500, all DAKOCytomation, Glostrup, Denmark). Binding of primary antibody was visualized using the OptiView DAB kit (Ventana Medical Systems, Tucson, USA). The densities of total TILs (H&E staining) and TIL subtypes were evaluated microscopically at the TC and ITF and classified into three categories: Low = 0-10%, Moderate = 11-30%, High ≥ 31% infiltration. TIL results were correlated with clinical parameters and disseminated tumor cells (DTCs) in the bone marrow, determined by immunocytochemistry applying the pan cytokeratin antibody A45-B/B3. Results: TILs were differentially distributed at both tumor areas. The ITF was mainly infiltrated by CD3+ T- and CD20+ B cells, while low amounts of CD68+ macrophages, CD4+ and CD8+ T cell subtypes were present. Only CD3+ T cells were present in a higher level at the TC. A high total TIL amount and a high CD3+ infiltration at the ITF was associated with poorly differentiated tumors. Pre-NACT tumors < T2 had a high CD4+ T cell infiltration at the TC, whereas in small post-NACT tumors a high infiltration of total TILs and all TIL subtypes at the ITF, except CD68+ cells, was observed. BC pts responding to NACT exhibited significantly more total TILs (p=0.02) and CD3+ T cells (p=0.02) at the TC. A high CD4+ T cell infiltration at the TC was significantly associated with the presence of DTCs post-NACT (p=0.029). CD68+ macrophages located at TC or ITF were not related to any clinical parameter. Conclusion: The differential association of TIL subtypes with clinical parameters and NACT response underlines the necessity of detailed TIL analysis for gaining insights into immune modulatory processes in the tumor microenvironment. In this regard, BC patients not responding to standard NACT might be identified for application of additional immunotherapy. Citation Format: Lisa Koenig, Fabian Dominik Mairinger, Oliver Hoffmann, Ann-Kathrin Bittner, Kurt Werner Schmid, Rainer Kimmig, Sabine Kasimir-Bauer, Agnes Bankfalvi. A different spatiotemporal distribution of TIL subtypes is associated with response to neoadjuvant chemotherapy in locally advanced breast cancer patients [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 3993. doi:10.1158/1538-7445.AM2017-3993

Abstract 3849: HuPBL humanized models as a tool to evaluate the efficacy and immune regulation of immune checkpoint inhibitors

Abstract After the success of PD-1, PD-L1 and CTLA-4 antibodies, a number of immune-checkpoint inhibitors are already in advanced stages. Meanwhile, tumor infiltrating lymphocytes (TIL) are well recognized as features of tumor progression and keys to effective cancer immunotherapy. Therefore, there is an urgent need to establish an effective pre-clinical platform for the evaluation of therapeutical outcomes in the human immune-system and the understanding of TILs. Either traditional syngeneic models or human target knockin models have their drawbacks: syngeneic models disable the development of antibodies that do not recognize mouse receptors; neither of them can achieve a good clinical prediction due to their inherent property as a murine system. To get a reconstitution of the human immune system in murine models, we have established several customized Human peripheral blood lymphocytes (hu-PBL) humanized models in NOG (NOD/Shi-scid/IL-2Rγnull) mice either systemically or focalized in tumors. Those models were validated by immune-checkpoint inhibitors for efficacy and by conducting TIL analysis with the support of sophisticated Flow Cytometry-Based Approach. Here we report that the engrafted human cells can expand and infiltrate in tumors in response to xenogenic stimulation with functional Th and Tc detected. The cell lineages are mainly CD3+ T lymphocytes (90%) and to lesser extent NK/NKT cells; the redistribution in tumors is unique not only compared to splenic and peripheral blood T cells, but also varies among different cancer cell lines when the same donor was applied. In addition, cell surface expression of immune checkpoint targets showed unique redistributions. We further investigated the correlation between the efficacy and the immune-regulation in these models: Models with more dense infiltration at the early phase usually grew more slowly than its NON-PBMC control, which also are prone to be better responders to immunotherapy due to the higher Effector cell: Target cell ratio (E:T ratio). When compared the efficacy of 2 different hPD-1 test antibodies in A375 systemic humanized model, the one with better efficacy also achieved stronger inhibition on PD-1 expression in TILs. Treatment with the clinically used Opdivo showed not only different responses in HCC827, A375 and A431 xenograft models but also unique profiles in TILs subsets and related immune-checkpoint expression. These models can be used for the purpose of pre-clinical immune-checkpoint inhibitor and bi-specific antibody screening under human background, as well as for further investigations regarding distinct functions of TILs subsets and target expression. Citation Format: Xuzhen Tang, Xianzhi Zhai, Hui Qi, Qin Hong, Qiyao Zhang, Lei Jin, Shaoyu Yan, Zhuozhi Wang, Yong Zheng, Qingyang Gu, Norman Zhang, Jing Li, Qunsheng Ji. HuPBL humanized models as a tool to evaluate the efficacy and immune regulation of immune checkpoint inhibitors [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 3849. doi:10.1158/1538-7445.AM2017-3849

Magnitude and duration of immune checkpoint up-regulation and changes in the immune microenvironment post chemo-radiation (CRT) in esophageal cancer.

4060 Background: PD-1 inhibitors in metastatic gastroesophageal cancer have demonstrated response rates of approximately 25%. Unfortunately, the majority of patients do not respond. Therefore, a rationale strategy of combining immunotherapeutic agents with CRT in earlier stage esophageal cancer may prevent metastatic disease in a greater proportion of patients. This study assessed the impact of CRT on the immune microenvironment and the expression patterns of multiple immune checkpoints to optimally design neoadjuvant clinical trials. Methods: To determine the effects of CRT on resected esophageal adenocarcinomas (EAC), we examined the immune microenvironment pre and post CRT using IHC, LCM followed by qRT-PCR, and functional analysis of tumor-infiltrating lymphocytes. Additionally, to assess the duration and dependency of radiation-induced PD-L1 sensitization, esophagojejunostomy were performed on rats to induce gastroduodenoesophageal reflux and EAC formation. First, tumor bearing animals were dosed with single fraction 13 Gy or 16 Gy radiation to determine safety, dose correlation, and PD-L1 sensitization using qRT-PCR post-radiation. Next, longitudinal PD-L1 expression levels within individual tumor bearing animals were determined using serial endoscopic biopsies at baseline, 1, 5 and 9 weeks post 16 Gy radiation. Results: The majority of cancers displayed enhanced interferon γ and activated CD8+ T lymphocytes at the tumor stroma interface. These tumors also demonstrated enhanced upregulation of PD-L1 and multiple other immune checkpoints including – TIM3, GITR, IDO1, LAG3, CD137, OX40 and KIR. The animal model results indicated PD-L1 upregulation is dose dependent and transiently elevated post radiation exposure. Conclusions: Collectively, these findings provide insights into the evolving immune landscape after CRT and have significant implications for future neoadjuvant trial designs that will combine radiotherapy with immune checkpoint inhibitors. Currently, we are conducting a neoadjuvant trial assessing Nivolumab or Nivolumab/Ipilimumab in combination with CRT in stage II/III operable esophageal cancer.

Therapeutic effect of local Interleukin-15 with radiotherapy in breast cancer.

158 Background: Interleukin (IL)-15 is a key regulator of T cell homeostasis with activity in cancer and a favorable toxicity profile compared to IL-2. IL-15 stimulates the proliferation and effector differentiation of CD8+T cells, and the proliferation and activation of natural killer (NK) cells. We observed IL-15 upregulation by gene arrays in radiotherapy (RT)-treated TSA mouse breast cancer, suggesting that it may play a role in RT-induced anti-tumor immunity. However, the upregulation was modest prompting us to test the hypothesis that administration of IL-15 may enhance in situ vaccination by RT. Methods: BALB/c mice with established poorly immunogenic TSA tumors were sham-treated, treated with tumor-targeted RT (8GyX3 days), IL-15 given peri-tumorally (2 ug/mouse/day for 10 days) starting on the first day of RT, and RT+IL-15, and monitored for tumor growth and survival. Tumor infiltrating lymphocytes (TIL) were analyzed by flow cytometry and immunostaining. In some experiments, Batf3-/-mice were used as tumor recipient. Results: IL-15 by itself was ineffective, but it significantly increased tumor control by RT (p=0.0007, RT versus RT+IL-15) leading to complete responses in 50% of the mice, most of them durable. Analysis of TILs showed significantly increased NK cells (CD45+ CD3- DX5+) in tumors treated with RT+IL-15 (p<0.0004 versus sham-treated; p<0.02 versus RT). NK cells were also more activated as indicated by expression of CD122 and CD137. Depletion of NK cells completely abrogated the therapeutic effect of the combination, while CD8 T cell depletion reduced tumor control and rate of complete regression. Interestingly, Batf3-/- mice, which lack CD103+ DCs, showed reduced response to RT+IL-15 compared to WT mice. Conclusions: Data suggest that local IL-15 with RT is an effective strategy to induce anti-tumor immunity to poorly immunogenic breast cancer. NK cells are critical mediators of the response, and may act by both killing tumor cells and promoting priming of CD8 T cells. Experiments are ongoing to determine the mechanisms of durable complete responses. <footer>Acknowledgments: IL-15 was provided by NCI BRB. Garcia-Martinez E was supported by GEICAM grant.</footer>

Abstract A10: A distinct gene module for T cell dysfunction uncoupled from T cell activation and controlled by metallothioneins

Abstract Reversing the dysfunctional (also known as “exhausted”) T cell state that arises in cancer and chronic viral infections is the focus of therapeutic interventions; however, current therapies are effective in only some patients and for only some tumor types. To gain a deeper molecular understanding of the dysfunctional T cell state, we leveraged mouse models to analyze RNA profiles of CD8+ tumor-infiltrating lymphocytes (TILs) at various functional states. In a comparative study we identified metallothioneins, regulators of zinc metabolism, as drivers of T cell dysfunction and used genetic perturbation to identify, for the first time, a distinct gene module for T cell dysfunction that is uncoupled from T cell activation and associated with a CD8+ Treg signature. An analysis of TILs at single-cell resolution revealed that our dysfunction module negatively correlates with activation signatures also at the single-cell level, indicating that our uncoupled signatures for T cell dysfunction and activation can represent cell-intrinsic states. Clustering of the single-cells revealed a previously uncharacterized subpopulation of CD8+ TILs that was strongly enriched for our novel dysfunction signature, but not for activation signatures. This subpopulation was depleted of cells from a metallothionein KO mouse model in which T cell dysfunction was not observed, implying that we have indeed identified a phenotypically dysfunctional subpopulation. Using CRISPR/Cas9 genome editing we validate that Gata-3, a transcription factor expressed specifically in our identified subpopulation, is a driver of the dysfunctional phenotype in CD8+ TILs. Our results open novel avenues for specifically targeting the dysfunctional T cell state while leaving T cell activation programs intact. Citation Format: Meromit Singer, Chao Wang, Le Cong, Nemanja D. Marjanovic, Monika S. Kowalczyk, Huiyuan Zhang, Jackson Nyman, Kaori Sakuishi, Sema Kurtulus, David Gennert, Junrong Xia, John YH Kwon, James Nevin, Rebecca H. Herbst, Itai Yanai, Orit Rozenblatt-Rosen, Vijay K. Kuchroo, Aviv Regev, Ana C. Anderson. A distinct gene module for T cell dysfunction uncoupled from T cell activation and controlled by metallothioneins. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A10.

Abstract S1-09: Evaluation of tumor-infiltrating lymphocytes (TILs) as predictive and prognostic biomarker in different subtypes of breast cancer treated with neoadjuvant therapy - A metaanalysis of 3771 patients

Abstract Background: Tumor-infiltrating lymphocytes (TILs) have been shown to be predictive for response to neoadjuvant therapy, in particular in triple-negative and HER2 positive breast cancer, suggesting that subtypes of breast cancer are immunogenic. The role of TILs in luminal breast cancer as well as the impact on prognosis in the different subtypes is less clear. In this study we evaluated TILs in a total of 3771 breast carcinomas from 6 prospective neoadjuvant clinical trials and evaluated their relevance for pCR, DFS and OS in different molecular subtypes. Methods: A total of 3771 tumors from the clinical studies GeparDuo, GeparTrio, GeparQuattro, GeparQuinto, GeparSixto, GeparSepto were evaluated for stromal TILs by standardized methodology. Data on pCR were available for all tumors, DFS and OS was available for 2560 tumors. Logistic regressions, Cox regressions and Kaplan-Meier analyses were performed. In addition, a combined analysis of pCR, survival and TILs in different subtypes was performed. Results: In the complete cohort of 3771 tumors, increased TILs (>=60%) were observed in 19% of tumors, these tumors with >=60% TILs had a pCR rate of 44% (p<0.0005). Increased stromal TILs (>=60%) were observed in 30% of TNBC (n=906), in 19% of HER2+ tumors (n=1379) and in 13% of HR+/HER2- tumors (n=1366). In all three subtypes, increased TILs were significantly associated with increased pCR rates (p<0.0005). In univariate logistic regression analysis of stromal TILs as a continuous variable, a 10% increase in TILs increased the probability to achieve a pCR by 16% in TNBC (OR 1.16), 13% in HER2+ (OR 1.13) and 33% in HR+/HER2- (OR 1.31,p<0.0005 for all three groups). Similar results were observed in multivariate logistic regression (p<0.0005 for all three groups). In univariate Cox regression, increased TILs were associated with improved DFS survival in TNBC (HR 0.93 per 10% TILs, p=0.01) and HER2+ BC (HR 0.93 per 10% TILs, p=0.02). In luminal (HR+/HER-) tumors there was no effect of TILs observed on DFS (p=0.46). In the luminal group increased TILs were associated with reduced OS (HR 1.10 per 10% increase in TILs, p=0.01), and increased TILs (>=60%) were associated with worse survival in Kaplan-Meier analysis in luminal tumors (DFS: p=0.04, OS: p<0.0005). A detailed analysis of subgroups of luminal BC as well as the link to pCR will be presented. Conclusion: Our results suggest that tumor-infiltrating lymphocytes are a strong predictive marker for response to neoadjuvant chemotherapy in all molecular subtypes, and this predictive effect is translated into a survival benefit in HER2+ BC and TNBC. In contrast, a survival benefit is not observed in luminal BC, suggesting a different biology of the immunological infiltrate in this subtype. Citation Format: Denkert C, von Minckwitz G, Darb-Esfahani S, Ingold Heppner B, Klauschen F, Furlanetto J, Pfitzner B, Huober J, Schmitt W, Blohmer J-U, Kümmel S, Engels K, Lederer B, Schneeweiss A, Hartmann A, Jakisch C, Untch M, Hanusch C, Weber K, Loibl S. Evaluation of tumor-infiltrating lymphocytes (TILs) as predictive and prognostic biomarker in different subtypes of breast cancer treated with neoadjuvant therapy - A metaanalysis of 3771 patients [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 S1-09.

TILs in Head and Neck Cancer: Ready for Clinical Implementation and Why (Not)?

The assessment of tumor infiltrating lymphocytes (TILs) has recently emerged as a prognostic biomarker in several solid tumors. Quantification and subtyping of TILs reflects the immune response in the tumor microenvironment, contributing to either tumoral immune attack or escape and thereby affecting outcome. Despite the growing evidence of its value as prognosticator, TILs analysis has not yet found its way to daily clinical practice. The aim of this review is to evaluate whether the current knowledge on TILs in head and neck cancer justifies its clinical implementation. Therefore, we summarize the data on TILs in squamous cell cancer of the head and neck with focus on the most important subsets (T lymphocytes and more specifically CD8+ cytotoxic T cells and FoxP3+ regulatory T cells) and site-specific characteristics such as Human Papilloma Virus infection. In addition, we discuss methodological problems and pitfalls that can account for discordant findings and that may hamper inclusion of TILs assessment in routine practice of pathologists and oncologists.

Multispectral imaging for highly accurate analysis of tumour-infiltrating lymphocytes in primary melanoma.

The quality and quantity of the infiltration of immune cells into tumour tissues have substantial impacts on patients' clinical outcomes, and are associated with response to immunotherapy. Therefore, the precise analysis of tumour-infiltrating lymphocytes (TILs) is becoming an important additional pathological biomarker. Analysis of TILs is usually performed semiquantitatively by pathologists on haematoxylin and eosin-stained or immunostained tissue sections. However, automated quantification outperforms semiquantitative approaches, and is becoming the standard. Owing to the presence of melanin pigment, this approach is seriously hampered in melanoma, because the spectrum of melanin lies close to that of commonly used immunohistochemical stains. Aim of this study is to overcome the technical issues due to the presence of melanin for an automated and accurate quantification of TILs in melanoma. Here, we successfully applied a novel multispectral imaging (MSI) technique to enumerate T cells in human primary melanomas. This microscopy technique combines imaging with spectroscopy to obtain both quantitative expression data and the tissue distributions of different cellular markers. We demonstrate that MSI allows complete and accurate analysis of TILs, successfully avoiding the blurring of images by melanin pigments, in whole tissue slide primary melanoma lesions, which could otherwise not be accurately detected by conventional digital image methodologies. Our study highlights the potential of MSI for accurate assessment of immune cell infiltrates, including those in notoriously difficult tissues, such as pigmented melanomas. Quantification of tumour infiltration by different immune cell types is crucial in the search for new biomarkers to predict patient responses to immunotherapies. Our findings show that this innovative microscopy technique is an important extension of the armamentarium of pathologists.

A high number of stromal tumor-infiltrating lymphocytes is a favorable independent prognostic factor in M0 (stages I-III) esophageal squamous cell carcinoma.

Esophageal cancer is a highly invasive tumor with a poor prognosis. Lymphocytes play an important role in systemic immune responses, but their role in cancers varies depending on the specific tumor microenvironment. The aim of this study was to provide evidence for tumor-infiltrating lymphocytes (TILs) as a prognostic biomarker in esophageal squamous cell carcinoma. TIL analysis was retrospectively performed on full-face hematoxylin and eosin-stained sections from 127 patients. A majority (92.6%) of tumors had at least 10% stromal TILs (sTILs) (range, 10%-90%), and 84.3% of cancers had at least 10% intraepithelial TILs (iTILs) (range, 10%-40%). Multivariate analysis showed progressively better overall survival (P<0.001, hazard ratio=0.968, 95% confidence interval 0.955-0.981) and disease-free survival (P=0.005, hazard ratio=0.982, 95% confidence interval 0.970-0.995) in patients with higher sTILs. Marginal increases in overall survival and disease-free survival were found in the higher iTILs cohort versus the lower iTILs cohort, but the difference was not significant. In conclusion, in addition to tumor stage increasing stromal lymphocytic infiltration is an independent prognostic factor for esophageal squamous cell carcinoma treated by radical resection.

Abstract B108: The HDAC inhibitor HBI-8000 enhances immunotherapy with either PD-1 or PD-L1 blockade in the MC38 model of colon cancer

Abstract Immunomodulatory antibodies targeting the immune checkpoint receptors CTLA-4, PD-1 and its ligand PD-L1, have resulted in significantly improved clinical benefits over the standard of care in many cancer types. While these immune checkpoint inhibitors have successfully exploited the adaptive immune system to produce remarkable clinical responses in various cancers, a significant number of patient's tumors are either innately resistant or develop resistance, and the disease eventually progresses. For this reason, rational drug combinations with checkpoint inhibitors and other immunomodulating agents are imperative to improve the rate of durable responses and patient survival. Histone deacetylase inhibitors (HDACi) have been successful as monotherapy for hematologic malignancies, but their clinical effectiveness against solid tumors has been limited to date. The long-held belief that HDACi are immunosuppressive has been challenged recently by data demonstrating the ability of HDACi to enhance anti-tumor immunity through effects on tumor immunogenicity, regulatory T cells, myeloid-derived suppressor cells (MDSCs), as well as effector cell functions. HBI-8000 (chidamide) is a novel, orally bioavailable Class I (HDAC 1,2,3) and Class II (HDAC 10) selective HDACi with a demonstrated positive effects on antitumor immunity, enhancing the activity of CTL and NK cells, as well as having direct activity against tumor cells through reversal of apoptosis resistance. We report here that HBI-8000 enhances the activity of both PD-1 and PD-L1 blocking antibody (Ab) in the MC38 syngeneic mouse model of colon cancer. Combinations of HBI-8000 with PD-1 or PD-L1 Ab resulted in statistically significant decreases in tumor progression and increases in survival compared to single agent therapy. Analysis of tumor infiltrating lymphocytes (TIL) revealed a significant increase in the percentage of intratumoral CD8+ T cells in tumors treated with both HBI-8000/PD-1 Ab and HBI-8000/PD-L1 Ab combinations. The TIL analysis also revealed a trend towards lower percentages of Tregs and granulocytic MDSCs. To evaluate the potential role of this combination as a 2nd line treatment option, i.e., in subjects failing 1st line checkpoint antibody therapy, we developed a model of immune checkpoint antibody failure, where tumor-bearing mice were initially treated (1st line) with either PD-1 or PD-L1 blocking Ab. Mice which had either slow disease progression or stable disease followed by progression, were randomized into one of six 2nd line treatment groups, including combinations of anti PD-1 or PD-L1 blocking Ab with HBI-8000. Fifty percent of mice failing 1st line PD-1 Ab treatment experienced a complete response, and were rescued by treatment with HBI-8000 combined with PD-L1 Ab. Altogether our data suggest that the addition of the HDACi HBI-8000 can improve the anti-tumor response to either PD-1 or PD-L1 blocking antibodies, increase the percentage of intratumoral immune effector cells and potentially counter innate resistance to checkpoint inhibitors. Our data also suggests that combination therapy with HBI-8000 and a checkpoint inhibitor can rescue tumor-bearing mice that have failed 1st line therapy with different checkpoint inhibitor. These results provide a strong rationale for the evaluation of therapeutic regimens utilizing PD-L1 or PD-1 blocking antibodies in combination with HBI-8000. Citation Format: Reid P. Bissonnette, Alain Rolland, Bob Goodeneow, Mireille Gillings. The HDAC inhibitor HBI-8000 enhances immunotherapy with either PD-1 or PD-L1 blockade in the MC38 model of colon cancer [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B108.

Abstract B109: Epigenetic reprogramming of T-cells from metastatic melanoma patients enhances central memory and decreases Th2/Treg phenotypes

Abstract Immunotherapy strategies for the treatment of melanoma have achieved impressive clinical outcomes over the past decade. Response rates to checkpoint blockade by PD-1 and CTLA-4 antibodies range from 15-40%, while in adoptive cell therapy using tumor infiltrating lymphocytes (TILs), anti-tumor response is observed in approximately 50%. However, the need to improve immunotherapies is evident as the majority of patients are unresponsive to treatment. Dysfunctional T-cells are thought to contribute to failed responses to checkpoint inhibition. As such we sought to investigate the ability of drugs targeting the epigenetic regulatory machinery as a means to alter T-cell function(s) and improve the anti-melanoma response. Here we demonstrate that the HDAC6 selective inhibitor ACY1215 disrupts mTORC signaling pathways in T-cells obtained from melanoma patients. Phosphorylation of mTOR, RAPTOR and the downstream molecules AKT, SGK1, PKCa and S6K were reduced on CD4 and CD8 T-cells after ACY1215 in vitro treatment (p&amp;lt;0.05). The levels of the Th2 cytokines IL-4, IL-6, IL-10 generated by ACY1215-treated T-cells (p&amp;lt;0.05) were also decreased. Similar results were achieved with an SGK1 inhibitor, in agreement with published data demonstrating SGK1 as a regulator of Th2 polarization. Since the mTOR/RAPTOR complex is known to be involved in determining T regulatory (Treg) function, the effects of ACY1215 on Tregs were evaluated. Treatment in vitro with ACY1215 decreased phosphorylated mTOR and RAPTOR in Tregs, and reduced the levels of FOXP3. In a functional suppression assay, ACY1215-treated Tregs displayed a reduced ability to impair proliferation of effector T-cells (Teff) compared to control (DMSO: 10% vs ACY1215: 25% Teff proliferation, p&amp;lt;0.05). To explore whether HDAC inhibition during expansion of tumor infiltrating lymphocytes (TIL) for adoptive transfer would improve their quality and anti-tumor reactivity, TIL isolated from melanoma surgical biopsies were cultured in vitro with IL-2 and ACY1215. Treatment with ACY1215 led to an accumulation of central memory CD4 and CD8 TILs (p&amp;lt;0.01 and p&amp;lt;0.05, respectively), which was maintained even after rapid expansion with anti-CD3 and anti-CD28 stimulation in vitro. Similarly, ACY1215 treatment of T-cells derived from peripheral blood of melanoma patients and healthy donors also displayed an increased central memory phenotype, characterized by expression of CD45RO, CD62L and CCR7 (p&amp;lt;0.05). Inhibition of AKT has been shown to increase T-cells with memory characteristics, and the use of an AKT inhibitor also resulted in accumulation of central memory T-cells. Confirming the observed phenotypic changes, microarray analysis of ACY1215-treated TILs revealed up-regulation of genes associated with a T-cell central memory and inflammatory response (e.g. SELL, LEF1, TNFRSF9) and downregulation of genes associated with Treg function (e.g. LGMN, CXCL8). Collectively these data suggest that reprogramming T-cells with epigenetic modulators may improve melanoma immunotherapy by reducing Treg suppression and production of immunosuppressive cytokines, while favoring generation of central memory T-cells. Citation Format: Andressa L. Sodre, David M. Woods, Amod Sarnaik, Brian C. Betts, Jeffrey S. Weber. Epigenetic reprogramming of T-cells from metastatic melanoma patients enhances central memory and decreases Th2/Treg phenotypes [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B109.

Quantitative analysis of tumor-infiltrating lymphocytes in meningiomas of the brain

Tumor-infiltrating lymphocytes (TIL) are an important component of the microenvironment in brain tumors and particularly in meningiomas. It is considered that immune cell infiltrates in meningiomas of the brain are composed of different populations of immune cells that perform a wide range of functions.Objective: To investigate the quantitative composition and distribution of CD4, CD8, CD20 tumor-infiltrating lymphocytes (TIL) in the tumor microenvironment of the brain meningiomas.Material and methods. Using immunohistochemistry 60 cases of meningiomas were analyzed.Results. The lowest quantity of CD4+ and CD8+ TIL was established in angiomatous subtypes and the median amount of CD4+ and CD8+ TIL in meningothelial subtypes was the highest, it was equal to 9 (1; 26) and 16 (8; 47) cells respectively. It was found that the number of CD4+ TIL was significantly higher in meningothelial variants than the number of CD4+ TIL in angiomatous (p = 0,035) and fibroblastic cases (p = 0,040). The number of CD8+ TIL in meningothelial subtypes was statistically significantly higher than in angiomatous (p = 0,010) and transitional (p = 0,003). We revealed that in grade I meningiomas number of CD8+ TIL significantly exceeded the number of CD4+ TIL (p = 0,003). The CD8+/CD4+ TIL ratio was 1,66. The median number of CD4+ TIL in anaplastic meningiomas was 9 (3; 20) cells, and CD8+ TIL – 14 (9; 80) cells. Although the number of CD8+ TIL exceeded the number of CD4+ TIL, but the results were not statistically significant (p = 0,164). The CD8+/CD4+ TIL ratio was 2,99. Median quantity of CD20+ TIL was increased in the following row: fibroblastic – 1 (0; 6) cells, transitional – 5,5 (0; 10) cells, meningothelial – 14,5 (0; 39) cells and anaplastic meningiomas – 24 (2,5; 46) cells.Conclusions. In benign meningiomas quantity of CD8+ TIL significantly exceeded the number of CD4+ TIL, that can play an important role in limiting the rate of growth of these tumors. There was no statistically significant difference in the number of CD4+, CD8+ and CD20+ TIL between grade I and grade III meningiomas. Interaction of immune cells in the tumor microenvironment is complex and largely depends on the phenotype of TIL, its establishment will be able to uncover the mechanisms of progression in anaplastic meningiomas.

Peritumoral IL-15 Potentiates Radiation-Induced Anti-Tumor Immunity

Radiotherapy (RT) can induce T cell-mediated anti-tumor immune responses by multiple mechanisms but is often unable to overcome immunosuppression in the tumor microenvironment. The common gamma-chain cytokines interleukin (IL)-2 and IL-15 promote the proliferation of activated T cells and, therefore, are prime agents for immunotherapy strategies aimed at sustaining anti-tumor T cell responses. The benefits of high dose IL-2, however, are undermined by serious toxicity and by regulatory T cell (Treg) stimulation. In contrast, IL-15 is well-tolerated and lacks Treg stimulatory activity, making it an attractive candidate for testing in combination with RT. Here we tested the hypothesis that IL-15 strengthens the pro-immunogenic effect of local RT to potentiate a durable anti-tumor immune response. The poorly immunogenic mouse TSA breast cancer cells were implanted s.c. in syngeneic BALB/c mice and randomly assigned to one of 4 treatment groups when tumors reached 5mm average diameters: control, RT, IL-15 or RT+IL-15. RT was delivered locally in 8 Gy fractions on days 13, 14 and 15. IL-15 (2 μg/mouse) was administered s.c. peritumorally daily for 10 days starting on day 12. Mice were followed for tumor growth. A parallel experiment was done to characterize tumor-infiltrating lymphocytes (TILs) at the end of treatment (day 22). Low dose IL-15 given peritumorally as a monotherapy induced marginal tumor growth control and had no effect on survival (median survival = 45 days compared to 76 days for control). Local RT significantly delayed tumor growth (p < 0.05 compared to control) and improved survival (median= 76 days, p < 0.05). However, highest survival advantage was seen in mice given IL-15+RT (median=102 days, p < 0.05 compared to all groups) with 1 of 6 mice showing complete tumor rejection and development of anamnestic response against tumor re-challenge. Analysis of TILs showed marked infiltration of CD8+ T cells expressing activation marker CD137 (35.3% in RT+IL-15 vs 5.9% in control, p < 0.05) while the increase was modest with either monotherapy (18.8% in RT, 20.7% in IL-15, p < 0.05 compared to control). In addition, we found a significant increase in the ratio of effector CD4+ T cells to Tregs (2.5 in RT+IL-15 versus 0.78 in control, p < 0.05) whereas monotherapy had no effect (1.14 in RT, 0.96 in IL-15). Overall these results support the rational combination of low dose intratumoral IL-15 with local RT to re-awaken immunity against poorly immunogenic tumors. We are currently elucidating the mechanisms involved in preclinical models in preparation for future testing in patients.