Transcriptomic signatures of tumors undergoing T cell attack.

BACKGROUND: Studying tumor cell-T cell interactions in the tumor microenvironment (TME) can elucidate tumor immune escape mechanisms and help predict responses to cancer immunotherapy. METHODS: We selected 14 pairs of highly tumor-reactive tumor-infiltrating lymphocytes (TILs) and autologous short-term cultured cell lines, covering four distinct tumor types, and co-cultured TILs and tumors at sub-lethal ratios in vitro to mimic the interactions occurring in the TME. We extracted gene signatures associated with a tumor-directed T cell attack based on transcriptomic data of tumor cells. RESULTS: An autologous T cell attack induced pronounced transcriptomic changes in the attacked tumor cells, partially independent of IFN-γ signaling. Transcriptomic changes were mostly independent of the tumor histological type and allowed identifying common gene expression changes, including a shared gene set of 55 transcripts influenced by T cell recognition (Tumors undergoing T cell attack, or TuTack, focused gene set). TuTack scores, calculated from tumor biopsies, predicted the clinical outcome after anti-PD-1/anti-PD-L1 therapy in multiple tumor histologies. Notably, the TuTack scores did not correlate to the tumor mutational burden, indicating that these two biomarkers measure distinct biological phenomena. CONCLUSIONS: The TuTack scores measure the effects on tumor cells of an anti-tumor immune response and represent a comprehensive method to identify immunologically responsive tumors. Our findings suggest that TuTack may allow patient selection in immunotherapy clinical trials and warrant its application in multimodal biomarker strategies.

The effects of targeted immune-regulatory strategies on tumor-specific T-cell responses in vitro.

BACKGROUND: Immune-related adverse events (IrAEs) are auto-immune reactions associated with immune checkpoint inhibitor-based therapy (ICI). Steroids are currently the first-line option for irAE management; however, recent studies have raised concerns regarding their potential impairment of tumor-specific immune responses. In this study, we investigated the in vitro effects of commonly used irAE treatment drugs on the anti-tumor activity of tumor-infiltrating lymphocytes (TILs). METHODS: Impairment of anti-tumor immune responses by four drugs (antibodies: vedolizumab and tocilizumab; small molecules: mycophenolate mofetil and tacrolimus) reported to be effective in treating irAEs was tested at clinically relevant doses in vitro and compared to a standard moderate dose of corticosteroids (small molecules) or infliximab (antibodies). TIL responses against autologous tumor cell lines, in the presence or absence of irAE drugs, were determined by flow cytometry (short-term tumor-specific T-cell activation) or xCELLigence (T-cell-mediated tumor killing). RESULTS: None of the tested antibodies influenced T-cell activation or T-cell-mediated tumor killing. Low-dose mycophenolate and tacrolimus did not influence T-cell activation, whereas higher doses of tacrolimus (> 1 ng/ml) impaired T-cell activation comparably to dexamethasone. All tested small molecules impaired T-cell-mediated tumor killing, with high-dose tacrolimus reducing killing at levels comparable to dexamethasone-mediated inhibition. In addition, mycophenolate and tacrolimus alone also demonstrated anti-proliferative effects on tumor cells. CONCLUSIONS: These data support clinical testing of targeted immune-regulatory strategies in the initial phase of irAE management, as a potential replacement for corticosteroids.

Common phenotypic dynamics of tumor-infiltrating lymphocytes across different histologies upon checkpoint inhibition: impact on clinical outcome.

BACKGROUND: Immune checkpoint inhibitors (ICIs) have revolutionized the cancer therapeutic landscape and our perception of interactions between the immune system and tumor cells. Despite remarkable progress, disease relapse and primary resistance are not uncommon. Understanding the biological processes that tumor-infiltrating lymphocytes (TILs) undergo during ICI, how this affects the tumor microenvironment (TME) and, ultimately, clinical outcome is, therefore, necessary to further improve treatment efficacy. AIM: In the current study, we sought to characterize TILs from patients with metastatic solid tumors undergoing ICI correlating flowcytometric findings with clinical outcome. METHODS: In total, 20 patients with 10 different metastatic solid tumors treated with ICIs targeting programmed-cell death-1 (PD-1)/PD-L1 axis were included in this study. The phenotype of T cells deriving from biopsies obtained prior to treatment initiation and on-treatment was investigaded. Analyses were focused on T cells' degree of differentiation and activity and how they correlate with transcriptomic changes in the TME. RESULTS: Data indicate that patients benefitting from ICIs accumulate CD8+central memory T cells. TILs developed an effector-like phenotype over time, which was also associated with a cytolytic gene signature. In terms of modulation of T-cell responses, we observed that high expression of checkpoint molecules pre-treatment (i.e., PD-1, lymphocyte activation gene-3 [LAG-3], B and T-lymphocyte attenuator [BTLA] and T-cell immunoglobulin and mucin domain containing-3 [TIM-3]) was associated with similar gene signature and correlated to treatment benefit. Increasing expression of LAG-3 and BTLA in the CD8 compartment and their co-expression with PD-1 during treatment were, however, a common feature for patients who failed to respond to ICIs. CONCLUSIONS: Besides identifying immune profiles suggestive of response to ICI, our results provide a more nuanced picture regarding expression of checkpoint molecules that goes beyond T-cell anergy.

Differential effects of corticosteroids and anti-TNF on tumor-specific immune responses: implications for the management of irAEs.

Up to 60% of patients treated with cancer immunotherapy develop severe or life threatening immune-related adverse events (irAEs). Immunosuppression with high dose corticosteroids, or tumor necrosis factor (TNF) antagonists in refractory cases, is the mainstay of treatment for irAEs. It is currently unknown what impact corticosteroids and anti-TNF have on the activity of antitumor T cells. In our study, the influence of clinically relevant doses of dexamethasone (corresponding to an oral dose of 10-125 mg prednisolone) and infliximab (anti-TNF) on the activation and killing ability of tumor-infiltrating lymphocytes (TILs) was tested in vitro. Overall, dexamethasone at low or intermediate/high doses impaired the activation (-46 and -62%, respectively) and tumor-killing ability (-48 and -53%, respectively) of tumor-specific TILs. In contrast, a standard clinical dose of infliximab only had a minor effect on T cell activation (-20%) and tumor killing (-10%). A 72-hr resting period after withdrawal of dexamethasone was sufficient to rescue the in vitro activity of TILs, while a short withdrawal did not result in a full rescue. In conclusion, clinically relevant doses of infliximab only had a minor influence on the activity of tumor-specific TILs in vitro, whereas even low doses of corticosteroids markedly impaired the antitumor activity of TILs. However, the activity of TILs could be restored after withdrawal of steroids. These data indirectly support steroid-sparing strategies and early initiation of anti-TNF therapy for the treatment of irAEs in immuno-oncology.

Cancer immunotherapy in patients with brain metastases.

The exclusion of "real-world" patients from registration clinical trials of cancer immunotherapy represents a significant emerging issue. For instance, a large fraction of cancer patients develops brain metastases during the course of the disease, but results from large prospective clinical trials investigating this considerable proportion of the cancer patient population are currently lacking. To provide a useful tool for the clinician in a "real-world" setting, we have reviewed the available literature regarding the safety and efficacy of immune check-point inhibitors in patients with cancer metastatic to the brain. Overall, these data provide encouraging evidence that these therapeutic agents can induce intracranial objective responses, particularly in patients with asymptomatic and previously untreated brain metastases. Larger prospective studies are needed to confirm these initial results.

Nivolumab Reaches Brain Lesions in Patients with Recurrent Glioblastoma and Induces T-cell Activity and Upregulation of Checkpoint Pathways.

Glioblastoma (GBM) is an aggressive brain tumor with poor prognosis. Although immunotherapy is being explored as a potential treatment option for patients with GBM, it is unclear whether systemic immunotherapy can reach and modify the tumor microenvironment in the brain. We evaluated immune characteristics in patients receiving the anti-PD-1 immune checkpoint inhibitor nivolumab 1 week prior to surgery, compared with control patients receiving salvage resection without prior nivolumab treatment. We observed saturating levels of nivolumab bound to intratumorally and tissue-resident T cells in the brain, implicating saturating levels of nivolumab reaching brain tumors. Following nivolumab treatment, significant changes in T-cell activation and proliferation were observed in the tumor-resident T-cell population, and peripheral T cells upregulated chemokine receptors related to brain homing. A strong nivolumab-driven upregulation in compensatory checkpoint inhibition molecules, i.e., TIGIT, LAG-3, TIM-3, and CTLA-4, was observed, potentially counteracting the treatment effect. Finally, tumor-reactive tumor-infiltrating lymphocytes (TIL) were found in a subset of nivolumab-treated patients with prolonged survival, and neoantigen-reactive T cells were identified in both TILs and blood. This indicates a systemic response toward GBM in a subset of patients, which was further boosted by nivolumab, with T-cell responses toward tumor-derived neoantigens. Our study demonstrates that nivolumab does reach the GBM tumor lesion and enhances antitumor T-cell responses both intratumorally and systemically. However, various anti-inflammatory mechanisms mitigate the clinical efficacy of the anti-PD-1 treatment.

Dose escalation study of a personalized peptide-based neoantigen vaccine (EVX-01) in patients with metastatic melanoma.

BACKGROUND: Neoantigens can serve as targets for T cell-mediated antitumor immunity via personalized neopeptide vaccines. Interim data from our clinical study NCT03715985 showed that the personalized peptide-based neoantigen vaccine EVX-01, formulated in the liposomal adjuvant, CAF09b, was safe and able to elicit EVX-01-specific T cell responses in patients with metastatic melanoma. Here, we present results from the dose-escalation part of the study, evaluating the feasibility, safety, efficacy, and immunogenicity of EVX-01 in addition to anti-PD-1 therapy. METHODS: Patients with metastatic melanoma on anti-PD-1 therapy were treated in three cohorts with increasing vaccine dosages (twofold and fourfold). Tumor-derived neoantigens were selected by the AI platform PIONEER and used in personalized therapeutic cancer peptide vaccines EVX-01. Vaccines were administered at 2-week intervals for a total of three intraperitoneal and three intramuscular injections. The study's primary endpoint was safety and tolerability. Additional endpoints were immunological responses, survival, and objective response rates. RESULTS: Compared with the base dose level previously reported, no new vaccine-related serious adverse events were observed during dose escalation of EVX-01 in combination with an anti-PD-1 agent given according to local guidelines. Two patients at the third dose level (fourfold dose) developed grade 3 toxicity, most likely related to pembrolizumab. Overall, 8 out of the 12 patients had objective clinical responses (6 partial response (PR) and 2 CR), with all 4 patients at the highest dose level having a CR (1 CR, 3 PR). EVX-01 induced peptide-specific CD4+ and/or CD8+T cell responses in all treated patients, with CD4+T cells as the dominating responses. The magnitude of immune responses measured by IFN-γ ELISpot assay correlated with individual peptide doses. A significant correlation between the PIONEER quality score and induced T cell immunogenicity was detected, while better CRs correlated with both the number of immunogenic EVX-01 peptides and the PIONEER quality score. CONCLUSION: Immunization with EVX-01-CAF09b in addition to anti-PD-1 therapy was shown to be safe and well tolerated and elicit vaccine neoantigen-specific CD4+and CD8+ T cell responses at all dose levels. In addition, objective tumor responses were observed in 67% of patients. The results encourage further assessment of the antitumor efficacy of EVX-01 in combination with anti-PD-1 therapy.

Uncoupling CD4+ TIL-Mediated Tumor Killing from JAK-Signaling in Melanoma.

PURPOSE: Impaired MHCI-presentation and insensitivity to immune effector molecules are common features of immune checkpoint blockade (ICB)-resistant tumors and can be, respectively, associated with loss of ß2 microglobulin (B2M) or impaired IFNγ signaling. Patients with ICB-resistant tumors can respond to alternative immunotherapies, such as infusion of autologous tumor-infiltrating lymphocytes (TIL). CD4+ T cells can exert cytotoxic functions against tumor cells; however, it is unclear whether CD4+ T-cell responses can be exploited to improve the clinical outcomes of patients affected by ICB-resistant tumors. EXPERIMENTAL DESIGN: Here, we exploited CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 gene editing to reproduce immune-resistant tumor phenotypes via gene knockout (KO). To determine the role of cytotoxic CD4+ TILs in ICB-resistant tumors, we investigated CD4+ TIL-mediated cytotoxicity in matched pairs of TILs and autologous melanoma cell lines, used as a model of patient-specific immune-tumor interaction. Around 40% of melanomas constitutively express MHC Class II molecules; hence, melanomas with or without natural constitutive MHC Class II expression (MHCIIconst+ or MHCIIconst-) were used. RESULTS: CD4+ TIL-mediated cytotoxicity was not affected by B2M loss but was dependent on the expression of CIITA. MHCIIconst+ melanomas were killed by tumor-specific CD4+ TILs even in the absence of IFNγ-mediated MHCII upregulation, whereas IFNγ was necessary for CD4+ TIL-mediated cytotoxicity against MHCIIconst- melanomas. Notably, although tumor-specific CD4+ TILs did not kill JAK1KO MHCIIconst- melanomas even after IFNγ stimulation, sensitivity to CD4+ TIL-mediated cytotoxicity was maintained by JAK1KO MHCIIconst+ melanomas. CONCLUSIONS: In conclusion, our data indicate that exploiting tumor-specific cytotoxic CD4+ TILs could help overcome resistance to ICB mediated by IFNγ-signaling loss in MHCIIconst+ melanomas. See related commentary by Betof Warner and Luke, p. 3829.

Immunologic Characterization and T cell Receptor Repertoires of Expanded Tumor-infiltrating Lymphocytes in Patients with Renal Cell Carcinoma.

The successful use of expanded tumor-infiltrating lymphocytes (TIL) in adoptive TIL therapies has been reported, but the effects of the TIL expansion, immunophenotype, function, and T cell receptor (TCR) repertoire of the infused products relative to the tumor microenvironment (TME) are not well understood. In this study, we analyzed the tumor samples (n = 58) from treatment-naïve patients with renal cell carcinoma (RCC), "pre-rapidly expanded" TILs (pre-REP TIL, n = 15) and "rapidly expanded" TILs (REP TIL, n = 25) according to a clinical-grade TIL production protocol, with single-cell RNA (scRNA)+TCRαß-seq (TCRαß sequencing), TCRß-sequencing (TCRß-seq), and flow cytometry. REP TILs encompassed a greater abundance of CD4+ than CD8+ T cells, with increased LAG-3 and low PD-1 expressions in both CD4+ and CD8+ T cell compartments compared with the pre-REP TIL and tumor T cells. The REP protocol preferentially expanded small clones of the CD4+ phenotype (CD4, IL7R, KLRB1) in the TME, indicating that the largest exhausted T cell clones in the tumor do not expand during the expansion protocol. In addition, by generating a catalog of RCC-associated TCR motifs from >1,000 scRNA+TCRαß-seq and TCRß-seq RCC, healthy and other cancer sample cohorts, we quantified the RCC-associated TCRs from the expansion protocol. Unlike the low-remaining amount of anti-viral TCRs throughout the expansion, the quantity of the RCC-associated TCRs was high in the tumors and pre-REP TILs but decreased in the REP TILs. Our results provide an in-depth understanding of the origin, phenotype, and TCR specificity of RCC TIL products, paving the way for a more rationalized production of TILs. Significance: TILs are a heterogenous group of immune cells that recognize and attack the tumor, thus are utilized in various clinical trials. In our study, we explored the TILs in patients with kidney cancer by expanding the TILs using a clinical-grade protocol, as well as observed their characteristics and ability to recognize the tumor using in-depth experimental and computational tools.

Phase 2 study of ipilimumab, nivolumab, and tocilizumab combined with stereotactic body radiotherapy in patients with refractory pancreatic cancer (TRIPLE-R).

BACKGROUND: Interleukin-6 blockade and radiation combined with immunotherapy may modulate the tumour microenvironment to overcome immune resistance. We assessed the efficacy of ipilimumab, nivolumab, and tocilizumab combined with stereotactic body radiotherapy (SBRT) in patients with refractory pancreatic cancer (PC). METHODS: Patients with PC who had progressive disease (PD) or intolerance to gemcitabine- or fluorouracil-containing regimens were enrolled in Part A of the two-part, single-centre, phase 2 study (NCT04258150). SBRT with 15 Gy was administered on day one of the first cycle. Ipilimumab was administered (1 mg/kg every 6 weeks) for a maximum of two infusions. Nivolumab (6 mg/kg) and tocilizumab (8 mg/kg) were given every four weeks until the PD or unacceptable toxicity, or for up to one year. The primary end-point was the objective response rate, with a threshold of 15%. RESULTS: Twenty-six patients were enrolled and treated between April 17, 2020, and January 25, 2021. The median follow-up time at the time of data cutoff (February 7, 2022) was 4.9 months (interquartile range 2.1-7.7). No responses were observed. Five patients (19%; 95% confidence intervals [CI], 7-39) achieved a stable disease. The median progression-free survival was 1.6 months (95% CI 1.4-1.7), and the median overall survival was 5.3 months (95% CI 2.3-8.0). Overall, 19 (73%) experienced adverse events related to the treatment including two (8%) with grade 3 or higher events. CONCLUSION: The combination of ipilimumab, nivolumab, tocilizumab, and SBRT in patients with PC did not meet the prespecified criteria for expansion for full accrual.

TCR-engaging scaffolds selectively expand antigen-specific T-cells with a favorable phenotype for adoptive cell therapy.

BACKGROUND: Adoptive cell therapy (ACT) has shown promising results for the treatment of cancer and viral infections. Successful ACT relies on ex vivo expansion of large numbers of desired T-cells with strong cytotoxic capacity and in vivo persistence, which constitutes the greatest challenge to current ACT strategies. Here, in this study, we present a novel technology for ex vivo expansion of antigen-specific T-cells; artificial antigen-presenting scaffolds (Ag-scaffolds) consisting of a dextran-polysaccharide backbone, decorated with combinations of peptide-Major Histocompatibility Complex (pMHC), cytokines and co-stimulatory molecules, enabling coordinated stimulation of antigen-specific T-cells. METHODS: The capacity of Ag-scaffolds to expand antigen-specific T-cells was explored in ex vivo cultures with peripheral blood mononuclear cells from healthy donors and patients with metastatic melanoma. The resulting T-cell products were assessed for phenotypic and functional characteristics. RESULTS: We identified an optimal Ag-scaffold for expansion of T-cells for ACT, carrying pMHC and interleukin-2 (IL-2) and IL-21, with which we efficiently expanded both virus-specific and tumor-specific CD8+ T cells from peripheral blood of healthy donors and patients, respectively. The resulting T-cell products were characterized by a high frequency of antigen-specific cells with high self-renewal capacity, low exhaustion, a multifunctional cytokine profile upon antigen-challenge and superior tumor killing capacity. This demonstrates that the coordinated stimuli provided by an optimized stoichiometry of TCR engaging (pMHC) and stimulatory (cytokine) moieties is essential to obtain desired T-cell characteristics. To generate an 'off-the-shelf' multitargeting Ag-scaffold product of relevance to patients with metastatic melanoma, we identified the 30 most frequently recognized shared HLA-A0201-restricted melanoma epitopes in a cohort of 87 patients. By combining these in an Ag-scaffold product, we were able to expand tumor-specific T-cells from 60-70% of patients with melanoma, yielding a multitargeted T-cell product with up to 25% specific and phenotypically and functionally improved T cells. CONCLUSIONS: Taken together, the Ag-scaffold represents a promising new technology for selective expansion of antigen-specific CD8+ T cells directly from blood, yielding a highly specific and functionally enhanced T-cell product for ACT.

Coexisting Alterations of MHC Class I Antigen Presentation and IFNγ Signaling Mediate Acquired Resistance of Melanoma to Post-PD-1 Immunotherapy.

Responses to immunotherapy can be very durable but acquired resistance leading to tumor progression often occurs. We investigated a patient with melanoma resistant to anti-programmed death 1 (anti-PD-1) who participated in the CA224-020 clinical trial (NCT01968109) and had further progression after an initial objective response to anti-PD-1 plus anti-lymphocyte activation gene 3. We found consecutive acquisition of beta-2 microglobulin (B2M) loss and impaired Janus kinase 1 (JAK1) signaling that coexisted in progressing tumor cells. Functional analyses revealed a pan T-cell immune escape phenotype, where distinct alterations mediated independent immune resistance to tumor killing by autologous CD8+ tumor-infiltrating lymphocytes (TIL; B2M loss) and CD4+ TILs (impaired JAK1 signaling). These findings shed light on the complexity of acquired resistance to immunotherapy in the post anti-PD-1 setting, indicating that coexisting altered pathways can lead to pan T-cell immune escape.

Personalized therapy with peptide-based neoantigen vaccine (EVX-01) including a novel adjuvant, CAF®09b, in patients with metastatic melanoma.

The majority of neoantigens arise from unique mutations that are not shared between individual patients, making neoantigen-directed immunotherapy a fully personalized treatment approach. Novel technical advances in next-generation sequencing of tumor samples and artificial intelligence (AI) allow fast and systematic prediction of tumor neoantigens. This study investigates feasibility, safety, immunity, and anti-tumor potential of the personalized peptide-based neoantigen vaccine, EVX-01, including the novel CD8+ T-cell inducing adjuvant, CAF®09b, in patients with metastatic melanoma (NTC03715985). The AI platform PIONEERTM was used for identification of tumor-derived neoantigens to be included in a peptide-based personalized therapeutic cancer vaccine. EVX-01 immunotherapy consisted of 6 administrations with 5-10 PIONEERTM-predicted neoantigens as synthetic peptides combined with the novel liposome-based Cationic Adjuvant Formulation 09b (CAF®09b) to strengthen T-cell responses. EVX-01 was combined with immune checkpoint inhibitors to augment the activity of EVX-01-induced immune responses. The primary endpoint was safety, exploratory endpoints included feasibility, immunologic and objective responses. This interim analysis reports the results from the first dose-level cohort of five patients. We documented a short vaccine manufacturing time of 48-55 days which enabled the initiation of EVX-01 treatment within 60 days from baseline biopsy. No severe adverse events were observed. EVX-01 elicited long-lasting EVX-01-specific T-cell responses in all patients. Competitive manufacturing time was demonstrated. EVX-01 was shown to be safe and able to elicit immune responses targeting tumor neoantigens with encouraging early indications of a clinical and meaningful antitumor efficacy, warranting further study.

Neoantigen-reactive CD8+ T cells affect clinical outcome of adoptive cell therapy with tumor-infiltrating lymphocytes in melanoma.

BACKGROUNDNeoantigen-driven recognition and T cell-mediated killing contribute to tumor clearance following adoptive cell therapy (ACT) with tumor-infiltrating lymphocytes (TILs). Yet how diversity, frequency, and persistence of expanded neoepitope-specific CD8+ T cells derived from TIL infusion products affect patient outcome is not fully determined.METHODSUsing barcoded pMHC multimers, we provide a comprehensive mapping of CD8+ T cells recognizing neoepitopes in TIL infusion products and blood samples from 26 metastatic melanoma patients who received ACT.RESULTSWe identified 106 neoepitopes within TIL infusion products corresponding to 1.8% of all predicted neoepitopes. We observed neoepitope-specific recognition to be virtually devoid in TIL infusion products given to patients with progressive disease outcome. Moreover, we found that the frequency of neoepitope-specific CD8+ T cells in TIL infusion products correlated with increased survival and that neoepitope-specific CD8+ T cells shared with the infusion product in posttreatment blood samples were unique to responders of TIL-ACT. Finally, we found that a transcriptional signature for lymphocyte activity within the tumor microenvironment was associated with a higher frequency of neoepitope-specific CD8+ T cells in the infusion product.CONCLUSIONSThese data support previous case studies of neoepitope-specific CD8+ T cells in melanoma and indicate that successful TIL-ACT is associated with an expansion of neoepitope-specific CD8+ T cells.FUNDINGNEYE Foundation; European Research Council; Lundbeck Foundation Fellowship; Carlsberg Foundation.

Rapid Identification of the Tumor-Specific Reactive TIL Repertoire via Combined Detection of CD137, TNF, and IFNγ, Following Recognition of Autologous Tumor-Antigens.

Detecting the entire repertoire of tumor-specific reactive tumor-infiltrating lymphocytes (TILs) is essential for investigating their immunological functions in the tumor microenvironment. Current in vitro assays identifying tumor-specific functional activation measure the upregulation of surface molecules, de novo production of antitumor cytokines, or mobilization of cytotoxic granules following recognition of tumor-antigens, yet there is no widely adopted standard method. Here we established an enhanced, yet simple, method for identifying simultaneously CD8+ and CD4+ tumor-specific reactive TILs in vitro, using a combination of widely known and available flow cytometry assays. By combining the detection of intracellular CD137 and de novo production of TNF and IFNγ after recognition of naturally-presented tumor antigens, we demonstrate that a larger fraction of tumor-specific and reactive CD8+ TILs can be detected in vitro compared to commonly used assays. This assay revealed multiple polyfunctionality-based clusters of both CD4+ and CD8+ tumor-specific reactive TILs. In situ, the combined detection of TNFRSF9, TNF, and IFNG identified most of the tumor-specific reactive TIL repertoire. In conclusion, we describe a straightforward method for efficient identification of the tumor-specific reactive TIL repertoire in vitro, which can be rapidly adopted in most cancer immunology laboratories.

Tumor-Infiltrating T Cells From Clear Cell Renal Cell Carcinoma Patients Recognize Neoepitopes Derived From Point and Frameshift Mutations.

Mutation-derived neoantigens are important targets for T cell-mediated reactivity toward tumors and, due to their unique tumor expression, an attractive target for immunotherapy. Neoepitope-specific T cells have been detected across a number of solid cancers with high mutational burden tumors, but neoepitopes have been mostly selected from single nucleotide variations (SNVs), and little focus has been given to neoepitopes derived from in-frame and frameshift indels, which might be equally important and potentially highly immunogenic. Clear cell renal cell carcinomas (ccRCCs) are medium-range mutational burden tumors with a high pan-cancer proportion of frameshift mutations. In this study, the mutational landscape of tumors from six RCC patients was analyzed by whole-exome sequencing (WES) of DNA from tumor fragments (TFs), autologous tumor cell lines (TCLs), and tumor-infiltrating lymphocytes (TILs, germline reference). Neopeptides were predicted using MuPeXI, and patient-specific peptide-MHC (pMHC) libraries were created for all neopeptides with a rank score < 2 for binding to the patient's HLAs. T cell recognition toward neoepitopes in TILs was evaluated using the high-throughput technology of DNA barcode-labeled pMHC multimers. The patient-specific libraries consisted of, on average, 258 putative neopeptides (range, 103-397, n = 6). In four patients, WES was performed on two different sources (TF and TCL), whereas in two patients, WES was performed only on TF. Most of the peptides were predicted from both sources. However, a fraction was predicted from one source only. Among the total predicted neopeptides, 16% were derived from frameshift indels. T cell recognition of 52 neoepitopes was detected across all patients (range, 4-18, n = 6) and spanning two to five HLA restrictions per patient. On average, 21% of the recognized neoepitopes were derived from frameshift indels (range, 0-43%, n = 6). Thus, frameshift indels are equally represented in the pool of immunogenic neoepitopes as SNV-derived neoepitopes. This suggests the importance of a broad neopeptide prediction strategy covering multiple sources of tumor material, and including different genetic alterations. This study, for the first time, describes the T cell recognition of frameshift-derived neoepitopes in RCC and determines their immunogenic profile.

Qualitative Analysis of Tumor-Infiltrating Lymphocytes across Human Tumor Types Reveals a Higher Proportion of Bystander CD8+ T Cells in Non-Melanoma Cancers Compared to Melanoma.

Background: Human intratumoral T cell infiltrates can be defined by quantitative or qualitative features, such as their ability to recognize autologous tumor antigens. In this study, we reproduced the tumor-T cell interactions of individual patients to determine and compared the qualitative characteristics of intratumoral T cell infiltrates across multiple tumor types. Methods: We employed 187 pairs of unselected tumor-infiltrating lymphocytes (TILs) and autologous tumor cells from patients with melanoma, renal-, ovarian-cancer or sarcoma, and single-cell RNA sequencing data from a pooled cohort of 93 patients with melanoma or epithelial cancers. Measures of TIL quality including the proportion of tumor-reactive CD8+ and CD4+ TILs, and TIL response polyfunctionality were determined. Results: Tumor-specific CD8+ and CD4+ TIL responses were detected in over half of the patients in vitro, and greater CD8+ TIL responses were observed in melanoma, regardless of previous anti-PD-1 treatment, compared to renal cancer, ovarian cancer and sarcoma. The proportion of tumor-reactive CD4+ TILs was on average lower and the differences less pronounced across tumor types. Overall, the proportion of tumor-reactive TILs in vitro was remarkably low, implying a high fraction of TILs to be bystanders, and highly variable within the same tumor type. In situ analyses, based on eight single-cell RNA-sequencing datasets encompassing melanoma and five epithelial cancers types, corroborated the results obtained in vitro. Strikingly, no strong correlation between the proportion of CD8+ and CD4+ tumor-reactive TILs was detected, suggesting the accumulation of these responses in the tumor microenvironment to follow non-overlapping biological pathways. Additionally, no strong correlation between TIL responses and tumor mutational burden (TMB) in melanoma was observed, indicating that TMB was not a major driving force of response. No substantial differences in polyfunctionality across tumor types were observed. Conclusions: These analyses shed light on the functional features defining the quality of TIL infiltrates in cancer. A significant proportion of TILs across tumor types, especially non-melanoma, are bystander T cells. These results highlight the need to develop strategies focused on the tumor-reactive TIL subpopulation.

Acquired resistance to cancer immunotherapy.

In recent times, advances in cancer immunotherapy have yielded impressive, durable clinical responses in patients with varied subtypes of cancer. However, a significant proportion of patients who initially demonstrate encouraging tumor regression develop resistance and progress over time. The identification of novel therapeutic approaches to overcome resistance may result in significantly improved clinical outcomes and remains an area of high scientific priority. This review aims to summarize the current knowledge regarding the role of both tumor-intrinsic and tumor-extrinsic factors in the development of resistance to cancer immunotherapy and to discuss current and possible future therapeutic strategies targeting these mechanisms.

MERTK Acts as a Costimulatory Receptor on Human CD8+ T Cells.

The TAM family of receptor tyrosine kinases (TYRO3, AXL, and MERTK) is known to be expressed on antigen-presenting cells and function as oncogenic drivers and as inhibitors of inflammatory responses. Both human and mouse CD8+ T cells are thought to be negative for TAM receptor expression. In this study, we show that T-cell receptor (TCR)-activated human primary CD8+ T cells expressed MERTK and the ligand PROS1 from day 2 postactivation. PROS1-mediated MERTK signaling served as a late costimulatory signal, increasing proliferation and secretion of effector and memory-associated cytokines. Knockdown and inhibition studies confirmed that this costimulatory effect was mediated through MERTK. Transcriptomic and metabolic analyses of PROS1-blocked CD8+ T cells demonstrated a role of the PROS1-MERTK axis in differentiation of memory CD8+ T cells. Finally, using tumor-infiltrating lymphocytes (TIL) from melanoma patients, we show that MERTK signaling on T cells improved TIL expansion and TIL-mediated autologous cancer cell killing. We conclude that MERTK serves as a late costimulatory signal for CD8+ T cells. Identification of this costimulatory function of MERTK on human CD8+ T cells suggests caution in the development of MERTK inhibitors for hematologic or solid cancer treatment.