Tumor Infiltrating CD8 T-cells Research Articles

The Improved Antigen Uptake and Presentation of Dendritic Cells Using Cell-Penetrating D-octaarginine-Linked PNVA-co-AA as a Novel Dendritic Cell-Based Vaccine.

Cancer immunotherapy using antigen-pulsed dendritic cells can induce strong cellular immune responses by priming cytotoxic T lymphocytes. In this study, we pulsed tumor cell lysates with VP-R8, a cell-penetrating D-octaarginine-linked co-polymer of N-vinylacetamide and acrylic acid (PNVA-co-AA), into the DC2.4 murine dendritic cell line to improve antigen uptake and then determined the anti-tumor effect in tumor-bearing mice. DC2.4 cells were pulsed with the cell lysate of EL4, a murine lymphoma cell line, and VP-R8 to generate the DC2.4 vaccine. For the in vivo study, DC2.4 cells pulsed with EL4 lysate and VP-R8 were subcutaneously injected into the inguinal lymph node to investigate the anti-tumor effect against EL4 and EL4-specific T cell immune responses. VP-R8 significantly improved antigen uptake into DC2.4 compared to conventional keyhole limpet hemocyanin (p < 0.05). The expression of MHC class I, MHC class II, and CD86 in DC2.4 cells significantly increased after pulsing tumor lysates with VP-R8 compared to other treatments (p < 0.05). The intra-lymph node injection of DC2.4 pulsed with both VP-R8 and EL4 lysate significantly decreased tumor growth compared to DC2.4 pulsed with KLH and lysates (p < 0.05) and induced tumor-infiltrating CD8T cells. The DC2.4 vaccine also remarkably increased the population of IFN-gamma-producing T cells and CTL activity against EL4 cells. In conclusion, we demonstrated that VP-R8 markedly enhances the efficiency of dendritic cell-based vaccines in priming robust anti-tumor immunity, suggesting its potential as a beneficial additive for dendritic cell-based immunotherapy.

Radiomics Based Assessment of Tumor Infiltrating CD8 T-Cells Predicts Induction Systemic Therapy Response in HPV+ Oropharyngeal Cancer: Exploratory Analysis of Prospective Trials

The adaptive immune system is increasingly recognized to be important for tumor control in HPV+ oropharyngeal cancer (HPV+OPC), and pre-clinical models suggest tumor specific T-cell priming occurs in draining lymph nodes. De-escalation strategies incorporating T-cell infiltration present a novel prognostic biomarker. The purpose of this study was to examine the utility of a validated radiomics model of CD8 T-cell infiltration within the framework of 2 prospective HPV+OPC response-adaptive de-escalation trials. We hypothesized that nodal radiomics scores (RS) would be correlated with response to induction systemic therapy. Patients enrolled on 2 prospective phase II response adapted de-escalation trials for low- and high-risk HPV+OPC were included in the analysis. Patients received induction chemotherapy and chemoimmunotherapy in trials 1 and 2, respectively. Primary tumors and nodes were retrospectively delineated on diagnostic CT scans before and after induction therapy to ascertain volumetric tumor burden & response. RS were generated on pre-induction scans for both primary tumors and nodes. RS and tumor response were dichotomized using a 50% threshold (high vs low, responder vs non-responder, respectively). Linear regression was used to evaluate the correlation between % volume change in tumor burden and RS. Fisher's exact test was used to evaluate concordance between RS and tumor response. A total of 87 patients (N = 47 Trial 1, N = 40 Trial 2) were evaluable. The mean post-induction therapy reduction in primary and nodal volumetry was 80.7% and 67% respectively. Primary and nodal RS were not associated with primary (T1/T2 vs T3/T4) or nodal stage (N1-N2b vs N2c-N, p > 0.10 both). Both primary and nodal RS were correlated with % volume change, r = 0.24 (p = 0.034) and r = 0.37 (p = 0.002), respectively. The mean difference in % volume change in primary and node, using the RS, was 11.13% (p = 0.015) and 17. 69% (p = 0.004). There was no association between primary tumor RS and total lesion responder status (p = 0.312). However, there was an association between high vs low nodal RS and total lesion responder status (p = 0.005). This is the first report of a validated radiomics score of CD8-T cell infiltration in HPV+OPC to predict response to systemic therapy. While radiomics scores in both the primary and nodes were associated with percent volume response, this association was stronger in nodes. Higher nodal radiomics scores were associated with improved volume reduction in total lesion burden. This effect however was not observed in the primary tumor. CD8-T cell infiltration in nodes, but not in the primary tumor, was associated with overall tumor response after systemic induction therapy in HPV+ oropharyngeal cancer.

Tumors produce glucocorticoids by metabolite recycling, not synthesis, and activate Tregs to promote growth.

Glucocorticoids are steroid hormones with potent immunosuppressive properties. Their primary source is the adrenals, where they are generated via de novo synthesis from cholesterol. In addition, many tissues have a recycling pathway in which glucocorticoids are regenerated from inactive metabolites by the enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1, encoded by Hsd11b1). Here, we find that multiple tumor types express Hsd11b1 and produce active glucocorticoids. Genetic ablation of Hsd11b1 in such cells had no effect on in vitro growth, but reduced in vivo tumor progression, which corresponded with increased frequencies of CD8+ tumor-infiltrating lymphocytes (TILs) expressing activation markers and producing effector cytokines. Tumor-derived glucocorticoids were found to promote signatures of Treg activation and suppress signatures of conventional T cell activation in tumor-infiltrating Tregs. Indeed, CD8+ T cell activation was restored and tumor growth reduced in mice with Treg-specific glucocorticoid receptor deficiency. Importantly, pharmacologic inhibition of 11β-HSD1 reduced tumor growth to the same degree as gene knockout and rendered immunotherapy-resistant tumors susceptible to PD-1 blockade. Given that HSD11B1 expression is upregulated in many human tumors and that inhibition of 11β-HSD1 is well tolerated in clinical studies, these data suggest that targeting 11β-HSD1 may be a beneficial adjunct in cancer therapy.

Metabolic dysfunction and DNA damage of exhausted tumor-infiltrating CD8 T-cells in hepatocellular carcinoma

Metabolic dysfunction and DNA damage of exhausted tumor-infiltrating CD8 T-cells in hepatocellular carcinoma

Abstract 5863: Blockade of tumor glucocorticoid production inhibits Treg function and tumor growth

Abstract Glucocorticoids (GCs) are steroid hormones with potent immunosuppressive properties. GCs are primary produced from cholesterol in the adrenals via the de novo synthetic pathway. In some tissues, however, there is also a recycling pathway in which the enzyme 11β-HSD1 generates GCs from the inactive metabolites dehydrocorticosterone (rodent) or cortisone (human). Here we find that multiple tumor types produce GCs via 11β-HSD1 in vitro and in vivo. We used CRISPR/Cas9 technology to knock out Hsd11b1 in GC-producing tumor cell lines and found that lack of 11β-HSD1 in B16 melanoma, Panc02 pancreatic adenocarcinoma, and MC38 colon carcinoma lines reduced their growth in vivo. Reduced tumor growth corresponded with increased expression of the activation marker CD44 and production of the effector cytokines IFNγ and TNFα by tumor-infiltrating CD8+ T cells (TILs). Rather than directly suppressing effector cell function, recent findings have suggested that GCs instead act primarily by activating Treg cells, and that these Treg are responsible for inhibition of effector cell function. Consistent with this, we found that tumor-derived GCs upregulate Treg expression of miRNA-342, which downregulates the mTOR component Rictor and activates Treg activity. A role of tumor-derived GCs in enhancing Treg function was demonstrated by inoculating tumor cells into mice with Treg-specific glucocorticoid receptor deficiency (GRFoxp3Cre mice). Notably, growth of tumors was reduced in GRFoxp3Cre mice, along with increased CD8+ TIL activation. Importantly, pharmacologic inhibition of 11β-HSD1 reduced WT tumor growth to the same degree as knockout of 11β-HSD1, and rendered immunotherapy-resistant tumors susceptible to inhibition with anti-PD-1 antibodies. Given that 11β-HSD1 expression is upregulated in many human tumors, these data suggest that its inhibition, which has been accomplished in a number of phase 3 clinical trials for other indications and is well-tolerated, may be beneficial in their treatment. Citation Format: Shizuka Otsuka, Matthew D. Taves, Kaitlynn M. Donahue, Jonathan D. Ashwell. Blockade of tumor glucocorticoid production inhibits Treg function and tumor growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5863.

Unbalanced Glutamine Partitioning between CD8T Cells and Cancer Cells Accompanied by Immune Cell Dysfunction in Hepatocellular Carcinoma.

Glutamine metabolism is critical both for the proliferation of cancer cells and the activation of CD8T cells to kill cancer cells. We aim to explore the relationship between the glutamine metabolism of CD8T cells and cancer cells and tumor immunity in the tumor microenvironment. In a TCGA cohort, we found that patients with high scores of glutamine-metabolism-related genes showed poor prognoses, and that a high score of glutamine-metabolism-related genes was an independent risk factor for HCC patients. In single-cell RNA-seq data, we found that, in some patients, the glutamine metabolism gene scores of tumor cells were significantly higher than those of CD8T cells, while decreased ratios of CD8-Tef-GZMA and suppressed tumor-killing activity of CD8-Tef-APOC2 were observed. A further genetic dynamics pseudotime analysis suggested that immune remodeling of these two subpopulations was accompanied by metabolic reprogramming. CD8-Tef-APOC2 in the dominant group tended to metabolize exogenous lipids, while the metabolic program of CD8-Tef-GZMA in the nondominant group was characterized by amino acid and endogenous lipid synthesis. In addition, we found that the glutamine metabolism inhibitor JHU083 promoted the proliferation of CD8T cells and improved the efficacy of PD-1 blockers. We proposed a new tool to quantify the glutamine partitioning between tumor cells and CD8T cells, through which the unique immune microenvironment could be identified at the transcriptome level. Furthermore, the simultaneous destruction of the glutamine metabolism in tumor cells and CD8T cells facilitated the enrichment of tumor-infiltrating CD8T cells and enhanced the efficacy of immunotherapy.

Resveratrol activates CD8+ T cells through IL-18 bystander activation in lung adenocarcinoma.

Resveratrol, a natural product, has demonstrated anti-tumor effects in various kinds of tumor types, including colon, breast, and pancreatic cancers. Most research has focused on the inhibitory effects of resveratrol on tumor cells themselves rather than resveratrol's effects on tumor immunology. In this study, we found that resveratrol inhibited the growth of lung adenocarcinoma in a subcutaneous tumor model by using the β-cyclodextrin-resveratrol inclusion complex. After resveratrol treatment, the proportion of M2-like tumor-associated macrophages (TAMs) was reduced and tumor-infiltrating CD8T cells showed significantly increased activation. The results of co-culture and antibody neutralization experiments suggested that macrophage-derived IL-18 may be a key cytokine in the resveratrol anti-tumor effect of CD8T cell activation. The results of this study demonstrate a novel view of the mechanisms of resveratrol tumor suppression. This natural product could reprogram TAMs and CD8T effector cells for tumor treatment.

Tissue-resident memory and circulating T cells are early responders to pre-surgical cancer immunotherapy

Neoadjuvant immune checkpoint blockade has shown promising clinical activity. Here, we characterized early kinetics in tumor-infiltrating and circulating immune cells in oral cancer patients treated with neoadjuvant anti-PD-1 or anti-PD-1/CTLA-4 in a clinical trial (NCT02919683). Tumor-infiltrating CD8 Tcells that clonally expanded during immunotherapy expressed elevated tissue-resident memory and cytotoxicity programs, which were already active prior to therapy, supporting the capacity for rapid response. Systematic target discovery revealed that treatment-expanded tumor Tcell clones in responding patients recognized several self-antigens, including the cancer-specific antigen MAGEA1. Treatment also induced a systemic immune response characterized by expansion of activated Tcells enriched for tumor-infiltrating Tcell clonotypes, including both pre-existing and emergent clonotypes undetectable prior to therapy. The frequency of activated blood CD8 Tcells, notably pre-treatment PD-1-positive KLRG1-negative Tcells, was strongly associated with intra-tumoral pathological response. These results demonstrate how neoadjuvant checkpoint blockade induces local and systemic tumor immunity.

A Radiomic Surrogate to Predict Tumor-Infiltrating CD8 T-Cells in Soft Tissue Sarcoma

A Radiomic Surrogate to Predict Tumor-Infiltrating CD8 T-Cells in Soft Tissue Sarcoma

Transcriptional Activity and Stability of CD39+CD103+CD8+ T Cells in Human High-Grade Endometrial Cancer.

Tumor-infiltrating CD8+ T cells (TIL) are of the utmost importance in anti-tumor immunity. CD103 defines tumor-resident memory T cells (TRM cells) associated with improved survival and response to immune checkpoint blockade (ICB) across human tumors. Co-expression of CD39 and CD103 marks tumor-specific TRM with enhanced cytolytic potential, suggesting that CD39+CD103+ TRM could be a suitable biomarker for immunotherapy. However, little is known about the transcriptional activity of TRM cells in situ. We analyzed CD39+CD103+ TRM cells sorted from human high-grade endometrial cancers (n = 3) using mRNA sequencing. Cells remained untreated or were incubated with PMA/ionomycin (activation), actinomycin D (a platinum-like chemotherapeutic that inhibits transcription), or a combination of the two. Resting CD39+CD103+ TRM cells were transcriptionally active and expressed a characteristic TRM signature. Activated CD39+CD103+ TRM cells differentially expressed PLEK, TWNK, and FOS, and cytokine genes IFNG, TNF, IL2, CSF2 (GM-CSF), and IL21. Findings were confirmed using qPCR and cytokine production was validated by flow cytometry of cytotoxic TIL. We studied transcript stability and found that PMA-responsive genes and mitochondrial genes were particularly stable. In conclusion, CD39+CD103+ TRM cells are transcriptionally active TRM cells with a polyfunctional, reactivation-responsive repertoire. Secondly, we hypothesize that differential regulation of transcript stability potentiates rapid responses upon TRM reactivation in tumors.

In vitro and in vivo characterization of MDNA11: A long-acting “beta-only” IL-2 superkine in syngeneic mice tumor models and nonhuman primates.

3036 Background: Use of IL-2 (Proleukin) remains limited due to its short half-life, toxicity, and its ability to preferentially activate Tregs resulting in unwanted immune suppression. Approaches to reduce binding to CD25 (IL2α), such as pegylation techniques, also results in reduced affinity to CD122 (IL2β). To bypass these limitations, we engineered MDNA11, an IL-2 Superkine containing core mutations to diminish binding to CD25 while increasing affinity to CD122. To increase half-life, MDNA11 was fused to an albumin scaffold, which is known to allow accumulation at the tumor site. Methods: MDNA11 was evaluated using in vitro and in vivo studies that included: IL-2 signaling in human PBMCs, Biacore binding analyses, PK studies in mice, and efficacy studies in syngeneic tumor models with or without immune checkpoint inhibitors (ICIs). In addition, dose-range finding studies in cynomolgus monkeys (NHP) were performed to characterize the safety and PK/PD profiles of MDNA11. Results: MDNA11 displayed enhanced STAT5 signaling in human NK and naïve CD8 T-cells with diminished Treg activity. In mice, the terminal half-life of MDNA11 was 24-fold longer than IL-2. As a result, MDNA11 triggered effective tumor growth control, as monotherapy or in combination with ICI, in multiple tumor models in spite of q1wk dosing for two weeks. MDNA11 administration to mice with pre-established CT26 colon cancer resulted in tumor-free animals and induced strong memory response and protection against subsequent re-challenges. MDNA11 also inhibited the growth of B16F10 melanomas, which translated into a durable increase in tumor infiltrating CD8 T-cells. When tested in NHP, MDNA11 led to increased circulating CD8 T-cells lasting for almost 14 days with limited effects on Tregs and eosinophils (the latter being a source of IL-5 causing vascular leak syndrome). High doses resulted in mild side effects that were transient and reversible even following repeated dosing. Conclusions: The long-acting MDNA11 Superkine has superior potency over IL-2 at activating naïve CD8 T-cells and NK cells, while exhibiting diminished Treg activation. This molecule potently inhibited tumor growth and induced durable regression and long-term memory response. Studies in NHP showed prolonged proliferation of immune effector cells lasting almost two weeks post-MDNA11 administration. The sum of these data underscores the potency of MDNA11 to trigger the host’s immune response to control or eradicate established tumors.

T-cells modified with CD70 as an alternative cellular vaccine for antitumor immunity

Abstract Successful tumor eradication primarily depends on generation and maintenance of a large population of tumor-reactive CD8 T-cells. Dendritic cells (DCs) have applied to clinics as potent antitumor therapeutic agents. However, high cost and difficulty in obtaining sufficient amounts for clinical use are the crucial drawbacks of DC-based vaccines. Here, antigenic peptide-loaded T-cells transfected with retrovirus encoding costimulatory ligands CD70, CD80, OX40L, or 4-1BBL were assessed for antigen-specific CD8 T-cell responses, and evaluated antitumor effects along with immunization of a mixture of synthetic peptides, poly-IC and anti-CD40 antibodies (TriVax). T-cells expressing CD70 (CD70-T) exhibited similar level of stimulatory functionality and therapeutic efficacy as DCs. Moreover, CD70-T prime followed by TriVax booster heterologous vaccination elicited therapeutic antitumor effect against B16 melanoma where mediated by CD8 T-cells but not CD4 T-cells or NK cells. The combination with PD-L1 blockade led to potent therapeutic efficacy which exhibited increased tumor infiltrating CD8 T-cells. CD70-T pulsed with multi-antigenic peptide generated multiple antigen-specific polyvalent CD8 T-cells that were capable of inhibiting tumor growth effectively. Moreover, CD70-T vaccination resulted in higher expansion and migration of adoptively transferred T-cells into tumor sites, and elicits enhanced therapeutic effects with peptide-based booster immunization. These results imply that T-cells endowed with CD70 enable the design of effective vaccination strategies against solid cancer, which may overcome current limitations of DC-based vaccines.

Abstract 4979: Ofranergene Obadenovec (VB-111), an anti-cancer gene therapy, induces immunologic responses in solid tumors transforming cold tumors to hot tumors

Abstract Introduction: Ofranergene Obadenovec (VB-111) is a non-replicating adenovirus 5 (Ad-5, El-deleted) carrying a proapoptotic human Fas-chimera transgene that targets angiogenic blood vessels and leads to vascular disruption. Improved responses seen among patients experiencing post treatment fever suggest that VB-111’s mode of action involves induction of a tumor directed immune response. The following experiments were conducted to assess if VB-111 may alter the immunogenic profile of the tumor. Methods: Biopsies were obtained from 3 patients with recurrent platinum-resistant ovarian cancer treated with intravenous VB-111 1x1013 viral particles (VPs) every 2 months in combination with weekly paclitaxel. H&amp;E and Immunohistochemistry (IHC) were performed for CD8 and CD4 intratumoral T-cells, and results were compared to pre-treatment specimens and to untreated controls. In parallel, in the Lewis Lung Carcinoma (LLC) model, mice were randomly treated with intravenous saline or VB-111 at doses of 1x109 or 1x1011 VPs. Upon sacrifice lungs were harvested and weighted. Tumor burden was assessed, and Immunohistochemistry with anti-CD8 antibody for the presence of infiltrating cytotoxic T-cells was performed. Results: Specimens from Ovarian Cancer taken before treatment with VB-111 showed no or minimal T-cell infiltration. One month after VB-111 treatment, metastatic lesions demonstrated increase in CD8 (up to 74 CD8+ cells/HPF) and CD4 tumor infiltrating T-cells. At 4.5 months following first drug administration (post 3rd dose) a liver lesion showed necrotic and fibrotic tissue with no viable tumor, lymphocytic aggregate, intensive staining for CD-8 and CD-4 T-cells and pigmented macrophages. In mice induced with LLC tumor and treated with saline, large tumor masses were observed in the lungs, and the calculated average tumor burden was 0.883g. Administration of VB-111 at 1x109 and 1x1011 VPs reduced tumor burden by 42% and 72% respectively. Concurrently, the number of tumor infiltrating CD8 T-cells was increased in correlation with VB-111 treatment dose. Conclusion: Pre clinical and clinical data suggest that VB-111 induces an Immunotherapeutic effect manifested locally with tumor infiltration with CD-8 T-cells, and evidence of tumor necrosis. The viral vector may promote transformation of the tumor microenvironment from immunologically "cold" to "hot", enhancing immune cell recognition and immune activation. Citation Format: Ronnie Shapira-Frommer, Tamar Rachmilewitz Minei, Iris Barshack, Itzhak Mendel, Niva Yakov, Yael C. Cohen, Eyal Breitbart, Dror Harats, Richard T. Penson. Ofranergene Obadenovec (VB-111), an anti-cancer gene therapy, induces immunologic responses in solid tumors transforming cold tumors to hot tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4979.

Abstract B075: Modeling response and resistance to immune checkpoint blockade in syngeneic mouse glioma

Abstract Immune checkpoint inhibitors are now implemented into the standard therapy of an increasing number of tumor entities and elicit remarkable durable therapy responses. However, gliomas seem resistant to checkpoint inhibition as recent evidence from a randomized clinical trial did not show a therapeutic benefit of PD-1 blockade in an unselected population of patients with recurrent glioblastoma. The blood-brain barrier per se does not seem to be a hurdle in transducing an effective peripheral immune response into tumors as evidenced by responses seen in selected glioma patients and patients with brain metastases treated with checkpoint inhibitors. This project investigates the mechanisms of response and resistance to checkpoint blockade targeting CTLA-4 and PD-1 in an experimental syngeneic Gl261 glioma model, where we found a clear and unanticipated dichotomy between responders and non-responders. We demonstrate that response to PD-1 and CTLA-4 blockade is driven by increased numbers and effector function of cytotoxic tumor-infiltrating T-cells as well as an enhanced TCRβ repertoire clonality of tumor infiltrating CD8 T-cells. Surprisingly, little overlap of the TCRβ repertoire between responder CD8 TILs was detected with only one shared TCRβ sequence motif, suggestive of a common tumor-antigen driving the expansion of reactive clones in responding mice. Moreover, we identified putative tumor neoepitopes that were predominantly abundant in non-responding tumors and thus might have undergone effective targeting by tumor-reactive T-cell in responding tumors. Resistance to PD-1 and CTLA-4 blockade was associated with increased frequencies of intratumoral macrophages (TAMs) expressing high levels of immunosuppressive markers such as PD-L1, CD38 and CD73. TAMs of nonresponding mice induced enhanced suppression of CD4 T-cell proliferation which was partially restored by PD-L1 blockade. Strikingly, additional PD-L1 blockade enhanced response rates to PD-1 and CTLA-4 blockade in Gl261-bearing mice, potentially by inhibiting the ligation of PD-L1 on TAMs to its alternative interaction partner CD80 on TILs. Collectively, we suggest a syngeneic mouse model for assessing mechanisms of response and resistance to checkpoint blockade in gliomas demonstrating a surprising heterogeneity of the TCRβ repertoire of tumor-infiltrating CD8 T-cells despite strict syngeneicity. We also provide evidence for a suppressive TAM subset associated with resistance to immune checkpoint inhibition in glioma, providing a rationale for combinatorial therapy strategies to overcome resistance to checkpoint blockade. Citation Format: Katrin Deumelandt, Jens Blobner, Jana K. Sonner, Mirco Friedrich, Edward Green, Michael O. Breckwoldt, Manuel Fischer, Jochen Meyer, Felix Sahm, Daniel Schrimpf, Andreas von Deimling, Michael Platten. Modeling response and resistance to immune checkpoint blockade in syngeneic mouse glioma [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B075.

Abstract A039: FAM49B–specific regulatory T-cells recognize and target cancer cells in the context of Qa-1

Abstract The outcome of cancer patients has recently been improved by utilizing novel immunotherapies that inhibit the regulatory immune system. Regulatory T-cells (Treg) contribute not only to suppression of excessive immune responses in the steady state but also to reduction of antitumor immune responses in cancer patients. We have shown that down-regulation of the Helios transcription factor undermines CD4 Treg stability inducing these cells to convert into effector cells that enhance the antitumor immune response. Helios is also the canonical transcription factor of CD8 Treg and essential for CD8 Treg function. CD8 Treg are Qa-1 (HLA-E in human)–restricted CD8 T-cells that mainly regulate Qa-1+ immune cells. The peptide FAM49B(p190-198) is a Qa-1 binding peptide derived from ubiquitously expressed FAM49B. Although FAM49B(p190-198) is regularly degenerated in the endoplasmic reticulum normal cells, we found that FAM49B(p190-198) is stably presented in some tumor cells. Here, we investigate the potential contribution of FAM49B(p190-198)/Qa-1 specific CD8 Treg to cancer.FAM49B(p190-198)/Qa-1 specific CD8 Treg were detected by FAM49B(p190-198)/Qa-1 tetramer after immunization with FAM49B(p190-198) peptide–loaded dendritic cells in CD8 Treg (i.e., CD44+CD122+Ly49+ CD8 T-cells). T-cell receptor cDNAs were amplified from the sorted single cells and inserted into retrovirus expression vectors for transduction in 58α–β– hybridoma cells and bone marrow cells. EL4-Qa-1 KO cells and EL4-FAM49B KO cells were generated using CRISPR/Cas9 technology. These tools allowed validation of the specificity of the TCRs and their Qa-1–restriction. For in vivo studies, TCR retrogenic mice were developed by transferring TCR-transduced Rag2–/– bone marrow hematopoietic stem cells into lethally irradiated CD45.1 mice. Retrogenic T-cells were prepared by CD8 enrichment of retrogenic mouse splenocytes and in vitro culture for 3 days in the presence of IL15c. For in vivo studies, we use an adoptive transfer model of retrogenic T-cells for EL4 tumor bearing mice. Rag2–/–γC–/– mice were inoculated with 0.5 x 10^6 EL4 WT-cells and 0.5 x 10^6 EL4 Qa-1 KO cells on each lateral flank, then intravenously injected with retrogenic T-cells (on day 13 and 17). FAM49B(p190-198)/Qa-1 specific CD8 T-cells preferentially use TCR Vα3.2 (TRAV9N-3) and Vβ5.1/5.2 (TRBV12-1/12-2) chains independent of their expression of Ly49+. We tested 17 pairs obtained from Ly49+ cells using the in vitro system and found that 11 pairs bound strongly to Fam49b(p190-198)/Qa-1 tetramer while 8 out of the 11 pairs exhibited antigen-specific activation. Interestingly, one TCR pair (No.8) responded to WT EL4 cells without antigen peptides, which was abolished in coculture wells with Qa-1 KO EL4 cells and FAM49B KO EL4 cells. This TCR hybridoma was also activated by stimulated WT B6 splenocytes but not by Qa-1 KO splenocytes suggesting its regulatory potential. Adoptive transfer of TCR No.8-expressing retrogenic T-cells delayed EL4 WT tumor growth but not EL4 Qa-1 KO tumor growth when inoculated in the same hosts. Tumor-infiltrating CD8 T-cells displayed a more activated phenotype in WT tumors compared to KO tumors, indicating that retrogenic T-cells recognize and target EL4 cells in a Qa-1-dependent manner. Citation Format: Hidetoshi Nakagawa, Hye-Jung Kim, Harvey Cantor. FAM49B–specific regulatory T-cells recognize and target cancer cells in the context of Qa-1 [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A039.

Abstract A186: A new strategy to identify and expand tumor-reactive CD8 TILs in human solid tumors

Abstract The immune system can recognize and destroy tumor cells through T-cell mediated mechanisms. Hence, identifying tumor antigen-specific T-cells from cancer patients and expanding them in large numbers in vitro has important implications for immunotherapy diagnostics and therapeutics. Here we show that tumor-reactive T-cells are enriched in a subset of tumor-infiltrating CD8 T-cells (CD8 TILs) identified by co-expression of CD103 and CD39 both in primary and metastatic tumors. The CD103+CD39+ CD8 TILs are present at high frequencies in melanoma and mismatch repair-deficient colon cancer but at low frequencies in microsatellite stable (MSS) colon cancer and colorectal liver metastasis. This cell population displays a distinct T-cell receptor (TCR) repertoire, with T-cell clones expanded in the tumor but present at low frequencies in the periphery. Importantly, we show in a MSS colon cancer patient that a very low number of CD103+CD39+ CD8 TILs can be expanded in vitro and that those cells recognize tumor-specific neoantigens. Finally, patients with head and neck cancer whose CD8 TILs contained a higher frequency of CD103+CD39+ cells experienced a greater overall survival. Our work suggests that CD103+CD39+ CD8 TILs are key players in the patient’s antitumor response and describe an approach for detecting and expanding those cells, which will help improve adoptive TIL therapy for cancer patients. Citation Format: Thomas Duhen, Rebekka Duhen, Ryan Montler, Tarsem Moudgil, Jitske van den Bulk, Bernard A. Fox, Shu-Ching Chang, Gary Grunkemeier, Els M.E. Verdegaal, Noel F. de Miranda, Rom Leidner, Richard B. Bell, Andrew D. Weinberg. A new strategy to identify and expand tumor-reactive CD8 TILs in human solid tumors [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A186.

Abstract B008: Treatment of IDO1 and TDO2 positive tumors with a kynurenine-degrading enzyme: A highly differentiated approach from IDO1 inhibition

Abstract Despite the sustained clinical benefit demonstrated by immune checkpoint inhibitors, a majority of patients derive minimal or no appreciable benefit, indicating the urgent need to incorporate novel immunomodulatory targets and therapeutic strategies. Indoleamine 2,3-dioxygenase 1 (IDO1) and tryptophan 2,3-dioxygenase 2 (TDO2) catalyze the first and rate-limiting step in the immunosuppressive tryptophan/kynurenine pathway and are both upregulated in a number of tumor types. Although small-molecule IDO1 inhibitors are being clinically evaluated in several tumor types, so far they have not demonstrated significant clinical benefits either as a single agent or in combination with immune checkpoint inhibition. We are developing pegylated kynureninase (Kynase), a kynurenine degrading enzyme, to treat a broader population with IDO1 and/or TDO2 expressing tumors. We believe that a more robust antitumor immune response can be achieved by depleting kynurenine, produced by both IDO1 and TDO2, with Kynase, than by inhibiting only IDO1. The human Kynase (HsKYN) has been successfully engineered to exhibit vastly improved catalytic activity and stability toward kynurenine over the wild-type human enzyme. HsKYN achieved durable and near complete plasma kynurenine depletion in mice, rats and non-human primates. HsKYN demonstrated single agent efficacy in CT26, MC38 and B16-IDO syngeneic mouse models. Tumor gene expression analysis using NanoString revealed that HsKYN treatment upregulated T-cell and NK cell activation signature. More importantly, HsKYN significantly increased the tumor-infiltrating CD8 T-cells and their activation/polyfunctionality, and reduced the Treg population. As a direct comparison, the lead IDO1 inhibitor epacadostat did not impose any meaningful effects on the same immune cell populations. Furthermore, HsKYN showed beneficial combination efficacy with anti-PD-1 that was superior to combined Epacadostat / anti-PD-1. Evidence to date suggest that HsKYN is well tolerated in multiple species. Therefore, immunoprofiling, efficacy and safety results strongly support that Kynase is a more effective therapeutic approach than IDO1 inhibition. HsKYN is moving toward clinical development for treatment of cancers where IDO1 and/or TDO2 pathways play a significant immunosuppressive role. Citation Format: Silvia Coma, Jillian Cavanaugh, James Nolan, Jeremy Tchaicha, Karen McGovern, Everett Stone, John Blazeck, Candice Lamb, George Georgiou, Mark G Manfredi, Michelle Zhang. Treatment of IDO1 and TDO2 positive tumors with a kynurenine-degrading enzyme: A highly differentiated approach from IDO1 inhibition [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B008.

NKG2A Blockade Potentiates CD8 T Cell Immunity Induced by Cancer Vaccines

Tumor-infiltrating CD8 Tcells were found to frequently express the inhibitory receptor NKG2A, particularly in immune-reactive environments and after therapeutic cancer vaccination. High-dimensional cluster analysis demonstrated that NKG2A marks a unique immune effector subset preferentially co-expressing the tissue-resident CD103 molecule, but not immune checkpoint inhibitors. To examine whether NKG2A represented an adaptive resistance mechanism to cancer vaccination, we blocked the receptor with an antibody and knocked out its ligand Qa-1b, the conserved ortholog of HLA-E, in four mouse tumor models. The impact of therapeutic vaccines was greatly potentiated by disruption of the NKG2A/Qa-1b axis even in a PD-1 refractory mouse model. NKG2A blockade therapy operated through CD8 Tcells, but not NK cells. These findings indicate that NKG2A-blocking antibodies might improve clinical responses to therapeutic cancer vaccines.

PO-365 Dissecting the synergistic effect of chemotherapy and immunotherapy on anti-tumoral T cell functions in breast cancer

IntroductionImmunotherapies that target immune checkpoint molecules proved effective for the treatment of several solid tumours. However, the majority of breast cancer patients does not respond to this kind of approach, underlining the need of new strategies able to elicit an effective anti-tumoral immune response. The combination of conventional and immuno-based therapies represents a promising strategy to elicit such response. Our aim is to evaluate the efficacy of immunotherapy and chemotherapy combination in breast cancer, to dissect its effect on the endogenous anti-tumoral cytotoxic CD8 +T cells, and finally to assess the impact of a spontaneously developing tumour on T-cell biology.Material and methodsThe experiments are performed in K14cre;Cdh1F/F;Trp53F/F (KEP) mice, a spontaneous breast tumour model that closely resembles human invasive lobular carcinoma. Mice are treated with α-CTLA-4 and α-PD-1 antibodies, as a single treatment modality or in combination with MTD dosing Cisplatin or docetaxel. Therapy is initiated when the tumour has reached 50mm2 and continues until it reached 225mm2 or after a pre-defined time point, depending on the experiment.Results and discussionsMammary tumours in KEP mice show an altered T-cell balance compared to tumor-free mammary glands, with a reduced infiltration of CD8+ T cells and conventional CD4+ T cells, and higher percentage of T regulatory cells. Tumor-infiltrating CD8+ T cells (TILs) have an increased expression of multiple inhibitory receptors, and reduced IFNγ production, compared to peripheral CD8 +T cells of KEP mice. In vitro exposure of sorted CD8 +TILs to IL-15 or IL-2 augments their capacity to produce IFNγ, indicating that their suppressed state is reversible. Treatment of tumor-bearing KEP mice with a-CTLA-4 and a-PD-1 does not affect the tumour growth, but a synergistic therapeutic benefit is observed when α-CTLA-4 and α-PD-1 are combined with cisplatin, and not docetaxel, in a CD8+ T-cell dependent mechanism.ConclusionCD8 +TILs display some features of dysfunctional T cells. However, ex vivo cytokine stimulation can restore part of their effector functions, indicating that they are not terminally dysfunctional. Nonetheless, only the combination with chemotherapy and immunotherapy is able to elicit an efficient CD8 +T cell response, in a drug-dependent manner. We are currently dissecting the mechanisms underlying the synergistic therapy response, in order to ultimately contribute to the rational design of new immunomodulatory treatment strategies for breast cancer.

Medical image computing to assess tumor infiltrating CD8 T cells, tumor immune phenotype and response to anti-PD-1/PD-L1 immunotherapy in prospective phase 1 trials.

3016Background: The aim was to develop a radiomic estimator of tumor infiltrating CD8 T-cells, to assess its association with tumor immune phenotype, and to evaluate outcomes of cancer patients enr...