Melanoma Tumor-infiltrating Lymphocytes Research Articles

Abstract 7536: CD4+T cell exhaustion in the tumor microenvironment and antitumor immunity

Abstract Background: Immune checkpoint inhibitors (ICIs) exert clinical efficacy against various types of cancer through reinvigoration of exhausted CD8+ T cells that attack cancer cells directly in the tumor microenvironment (TME). However, compared to CD8+ T cell exhaustion, the role of CD4+ T cell exhaustion in antitumor immunity remains unclear. Methods and Results: From single-cell sequencing for tumor-infiltrating lymphocytes (TILs) from patients with melanoma and mouse models, we found that CXCL13, which is highly expressed in tumor-infiltrating exhausted CD8+ T cells, induces PD-1+CXCR5+CD4+ follicular helper T (TFH)-like cell infiltration, which could contribute to antitumor immunity. Furthermore, a part of the TFH-like cells in the TME exhibits cytotoxicity and directly attacks MHC-II-expressing tumors. RNA velocity and latent time analysis in single-cell sequencing for melanoma TILs showed that the differentiation of TFH-like cells from the naive CD4+ T cells were different from that of other CD4+ T cells. In addition, the TFH-like cytotoxic CD4+ T cells were at the late stage of differentiation and some non-cytotoxic TFH-like cells might differentiate into TFH-like cytotoxic CD4+ T cells. We additionally found considerable overlapped clonotypes between the cytotoxic and non-cytotoxic clusters. TFH-like cytotoxic CD4+ T cells have high LAG-3/BLIMP1 and low TCF1 expression without self-renewal ability, whereas non-cytotoxic TFH-like cells express low LAG-3/BLIMP1 and high TCF1 with self-renewal ability, closely resembling the relationship between terminally differentiated and stem-like progenitor exhaustion in CD8+ T cells, respectively. Conclusion: Our findings provide novel insights into TFH-like CD4+ T cell exhaustion with helper progenitor and cytotoxic differentiated functions, which mediates antitumor immunity orchestrally with CD8+ T cells. Citation Format: Joji Nagasaki, Wenhao Zhou, Shusuke Kawashima, Takamasa Ishino, Katsushige Kawase, Youki Ueda, Kazuo Yamashita, Tomofumi Watanabe, Takashi Inozume, Yosuke Togashi. CD4+T cell exhaustion in the tumor microenvironment and antitumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7536.

Tracking of Leukemia and Immune Single Cell Phenotypes during Ipilimumab-Based Treatment By Long-Read Sequencing

Activity of ipilimumab in bone marrow-involved AML has been modest compared to extramedullary AML relapse or solid tumors like melanoma. Single cell transcriptomics (scRNA-seq) could provide deeper understanding of these differences, yet leukemia and immune cell phenotypes defined by somatic mutations or differential isoform expression often cannot be resolved with native short-read scRNA-seq data due to insufficient read coverage. To overcome this limitation, we devised a workflow ( nanoranger) that leverages improved accuracy of long-read sequencing (Oxford Nanopore, ONT) and enables targeted read-out of molecular features including single-nucleotide variants, gene fusions, isoforms, or T cell receptor (TCR) sequences from intermediate scRNA-seq cDNA libraries. To determine the performance of nanoranger, we undertook a series of assessments. First, we established its genotyping accuracy based on the ability to recover highly polymorphic TCR sequences. TCRs obtained with Illumina versus ONT from the same VD(J) amplicon library were highly concordant ( r>0.8) in the number of reads and cells per CDR3 (5,653 [ONT] vs. 5,767 [Illumina]). Second, we tested isoform profiling by targeted sequencing of PTPRC which encodes the T cell differentiation markers CD45RA (exon 4 present) and CD45RO (exon 4 absent). Not only did targeted ONT sequencing increase the single cell coverage of PTPRC from 10 to >2,600 cells, but PTPRC exon 4 expression also correlated with CD45RA surface marker expression by CITE-seq ( r=0.45). Third, we benchmarked somatic mutation detection of a homozygous TP53 R248Gmutation in the AML cell line Kasumi-1. Our ONT-based approach increased genotyped cells from 0% (via native scRNA-seq) to 22% ( nanoranger); in >98.8% of these cells, TP53 R248G was detectable. Finally, we searched for optimal nanoranger targets by assessing whole-transcriptome coverage of 5‘ single cell cDNA and found these to be loci at the 5‘ end of highly expressed transcripts shorter than 4 kB. Altogether, nanoranger reliably recovers molecular features from scRNA-seq libraries with genotyping efficiency dictated by inherent characteristics of the underlying cDNA. To better understand determinants of response and resistance of CTLA-4 blockade in AML, we applied nanoranger to bone marrow samples collected in the context of a phase I trial investigating combined decitabine and ipilimumab (NCT02890329). We genotyped 11 genes recurrently mutated in AML across 15,258 single cell profiles from 13 samples (5 responders at screening and best response, 3 non-responders at screening). At screening, somatic mutations were detectable in myeloid progenitor, monocytic and dendritic cells but also in erythroid and megakaryocytic progenitor populations, consistent with detected copy number changes. Further, we clarified AML subclones with integrated analysis of somatic mutations and chromosomal aberrations. In AML1019, we found two separate clones defined by presence or absence of RUNX1 R320* and amp(8p) or amp(21p). In AML8007, we tracked differential therapeutic responses. At screening, 3 somatic mutations ( DNMT3A V296M, TP53 C176S, TP53 R282W) and 3 chromosomal aberrations ( amp(1p), del(3p) and del(5q)) were detected. At remission, somatic mutations and del(5q) persisted, but amp(1q) and del(3p) became undetectable except in LMPP-like cells. In all 5 responders, somatic mutations remained detectable in all cellular compartments, revealing that the cytoreductive effect of decitabine/ipilimumab was not accompanied by leukemic clone eradication. The ability of nanoranger to perform isoform profiling enabled us to resolve immune phenotypes. Following CTLA-4 blockade, the PTPRC isoform CD45RA was downregulated in CD4 + and CD8 + T cells, consistent with induction of T cell differentiation by ipilimumab. As CTLA-4 exists in two isoforms, we tracked alternative splicing of exon 3 and observed lower expression of the membranous isoform in T cells from AML bone marrow compared to circulating or tumor-infiltrating lymphocytes in melanoma, pointing to context-specific isoform expression of CTLA-4. In sum, genotyping with nanoranger substantially augmented scRNA-seq analyses of ipilimumab-based therapy in bone marrow AML. This exemplifies the great potential that long-read sequencing-based single cell studies hold to better define response and resistance mechanisms of novel immunotherapies.

Artificial intelligence biomarkers for digital oncology: a case study of tumor-infiltrating lymphocytes in melanoma

Artificial intelligence biomarkers for digital oncology: a case study of tumor-infiltrating lymphocytes in melanoma

TILs Outperform Ipilimumab in Melanoma.

The first phase III study of tumor-infiltrating lymphocytes in melanoma shows that the cells are more effective than ipilimumab. Compared with the CTLA4 inhibitor, tumor-infiltrating lymphocytes yielded a higher objective response rate and longer progression-free and overall survival.

Immunophenotypic characterization of lymphocytic infiltration in canine melanocytic tumors / Caracterização imunofenotípica da infiltração linfocítica em tumores melanocíticos caninos

Melanocytic tumors correspond to approximately for 4 to 7% of tumors in dogs and up to 7% of malignant tumours. Melanomas generally exhibit aggressive biological behavior. In humans, abundant tumor-infiltrating lymphocytes (TIL) can be considered a good prognostic factor. Research on TIL in canine melanomas is scarce, and to date, there are no studies to verify its association with established prognostic factors. Our study aimed to evaluate the lymphocyte population in canine melanocytic tumors using immunohistochemical markers, and to relate this to pre-established clinicopathological prognostic variables and cell proliferation index (Ki-67). An exploratory cross-sectional study was conducted with 21 cases in 19 dogs, of which 71.5% were melanotic melanomas, 19% were melanocytomas and 9.5% were amelanotic melanomas. Ki-67 expression was elevated in amelanotic melanomas, indicating aggressive tumor behavior. We detected lymphocytes T and B through CD3 and CD20 markers, respectively, and found that 86.7% of the melanotic melanomas were positive for CD3, whereas 73.3% were negative for CD20. TIL showed a strong association with malignant tumors, as well as a correlation with other pre-established prognostic factors, such as necrosis, ulceration, and nuclear atypia. TIL need to be further investigated to verify its inclusion as a prognostic factor for canine melanomas.

Abstract LB212: Allogeneic, IL-2-independent tumor-infiltrating lymphocytes expressing membrane-bound IL-15 (cytoTIL15࣪) eradicate tumors in a melanoma PDX model through recognition of shared tumor antigens

Abstract Standard tumor-infiltrating lymphocyte (TIL) therapy requires IL-2 administration to support TIL expansion and survival, but this cytokine is associated with T cell exhaustion and can result in severe toxicities that limit patient eligibility (1). To this end, we genetically engineered TIL to express membrane-bound IL-15 (mbIL15) under the control of Obsidian’s cytoDRIVE® technology (cytoTIL15࣪), which allows regulation of protein expression via a drug-responsive domain upon acetazolamide (ACZ) administration. IL-15 is a preferred cytokine over IL-2 to mediate TIL activation and expansion, because it does not result in CD8 T cell exhaustion or stimulate regulatory CD4 T cells, and enhances development of a memory T-cell phenotype. We have previously demonstrated IL-2-independent, 3-6-fold increased cytoTIL15 persistence in an antigen-independent setting relative to unengineered TIL therapy with IL-2 (uTIL) (2). Due to the challenge of generating autologous tumor/TIL-matched pairs and most importantly, to assess cytoTIL15 cell’s functional impact on anti-tumor growth across multiple donors, we developed an allogeneic patient-derived xenograft (PDX) model. To establish the model, different melanoma tumor digests were co-incubated in vitro with select HLA-A*02-matched, allogeneic melanoma TIL donors to assess their reactivity. Tumors were screened for expression of shared antigens, such as gp100 and MART1, and TIL donor TCRs were screened with tetramers. Once established, serially passaged tumor fragments were grown, measured, and randomized into groups to receive intravenous transfer of TIL (n=8/cohort). Mice receiving uTIL were treated with four saturating doses of recombinant IL-2, and mice receiving cytoTIL15 cells received either vehicle or oral 200 mg/kg ACZ daily for the entire study, without any IL-2. Three of four cytoTIL15 cell preparations from different donors dosed with ACZ achieved significant tumor growth inhibition compared to uTIL. Four mice developed complete responses as early as 17 days post cytoTIL15 cell transfer. The level of anti-tumor response was associated with increased frequency of MART1-reactive cytoTIL15 cells. On day 20 after TIL transfer, tumors and secondary lymphoid organs were collected (n=4/cohort). Tumors treated with cytoTIL15 cells + ACZ showed an 8-10-fold increased TIL infiltration compared to uTIL or cytoTIL15 cells + vehicle. Moreover, enhanced cytoTIL15 cell infiltration and anti-tumor activity was associated with increases in pro-inflammatory cytokines (e.g., IFNγ). Taken together, these data clearly demonstrate the superiority of cytoTIL15 cells over uTIL for controlling or eradicating melanoma tumor outgrowth and the utility of an allogeneic PDX model for comparative evaluation of tumor-antigen specific TIL reactivity.

385 Identification and enrichment of neoantigen-reactive T cells to optimize adoptive cell transfer with tumor-infiltrating lymphocytes (TIL)

BackgroundAdoptive cell transfer (ACT) using tumor-infiltrating lymphocytes (TIL) has achieved an overall response rate of 39% in metastatic melanoma patients at Moffitt Cancer Center. In these trials, a substantial fraction of patients were non-responders by RECIST, but demonstrated a mixed response to therapy. These results suggest that the infused TIL product contained tumor-reactive T cells with therapeutic potential, which could be further optimized to improve ACT with TIL. We hypothesized that outcomes might be improved by identifying and enriching neoantigen-reactive TIL within bulk products. The purpose of this study is to define approaches to optimize ACT with TIL, by identifying, enriching, and analyzing neoantigen reactive TIL from the ACT infusion product of previously treated metastatic melanoma patients.MethodsPatient-derived cryopreserved tumor tissue, PBMC, and TIL from completed metastatic melanoma TIL trials were used for this study. Whole exome and RNA sequencing were performed on DNA and RNA extracted from tumor tissue and compared to DNA from autologous PBMC. Genetic sequencing and gene expression data were utilized to determine protein-modifying somatic mutations. Peptides were then predicted for their ability to be presented on MHC molecules, prioritized, and up to 192 custom 25-mers were synthesized per patient sample. Neoantigen peptides were loaded onto patient-derived dendritic cells (DC) and co-cultured with autologous TIL. These TIL were then sorted by FACS on their ability to upregulate 41BB and OX40 and expanded through the rapid expansion protocol (REP). Enriched TIL were subsequently screened for neoantigen reactivity by 41BB/OX40 upregulation, cytokine release, and degranulation.ResultsProtein-altering somatic mutations from metastatic melanoma tissues ranged from 49 to 1631 mutations (median = 389). On average, 16.2% of TIL were sorted for upregulation of 41BB/OX40 upon co-culture with DC pulsed with the neoantigen peptide pool (range: 2.7–31.1%). CD4+ TIL displayed a 3.75-fold upregulation of 41BB/OX40, while CD8+ TIL saw a 1.88-fold increase (n=6). This coincided with substantial production of IFNγ, TNFα, and granzyme B (n=6). Neoantigen-reactive (41BB+/OX40+) and non-reactive (41BB-/OX40-) TIL expanded to similar degrees in REP (average of 639-fold vs. 611-fold; n=6). Restimulation of enriched neoantigen-specific TIL resulted in superior pro-inflammatory functionality (granzyme B, IFNγ, and TNFα) when compared to non-reactive TIL.ConclusionsTIL from metastatic melanoma patient samples were successfully enriched for neoantigen-reactive TIL, which maintained increased reactivity against these predicted peptides upon restimulation when compared non-reactive TIL. These data support further investigation into the use of neoantigen-enriched TIL products to enhance efficacy of ACT.Trial RegistrationNCT01005745, NCT01659151, NCT01701674Ethics ApprovalNCT01005745 was approved by USF IRB approval number Ame5_107905.NCT01659151 was approved by Advarra IRB approval number 14.03.0083.NCT01701674 was approved by USF IRB approval number Ame13_Pro00009061.All participants gave informed consent before taking part.

Tumor-infiltrating lymphocytes (TILs) in feline melanocytic tumors: A preliminary investigation

The presence and the role of tumor-infiltrating lymphocytes (TILs) in different types of tumors, but particularly in melanoma, has become more and more investigated during the last decade, both in human and veterinary medicine. Melanocytic tumors are quite rare in cats, with diffuse iris melanoma being the most commonly diagnosed in this species.The aim of this study was to characterize the lymphocytic infiltration in feline melanocytic tumors and to analyze their association with the histological features of malignancy recognized in these tumors, as well as with the expression of the most commonly used immunohistochemical markers.Thirty-eight feline melanocytic tumors were retrospectively selected; histological and immunohistochemical characterization of the tumors (histologic criteria of malignancy; S100, Melan A, and PNL2 expression) and of TILs (presence/absence, density, distribution, and grade; CD3, CD20 expression) were performed and associations between them tested.Results showed that TILs grade increased with cellular pleomorphism (P < 0.05) and, within the group of cutaneous melanocytic tumors, also with the mitotic count (P < 0.05). On the other hand, TILs grade was inversely associated with the percentage of neoplastic cells positive for Melan A (P < 0.05) and PNL2 (P < 0.05). Both CD3+ and CD20+ lymphocytes increased significantly with TILs grade and in association with mitotic count, when stratified in low/high quantity.This preliminary study suggests that TILs in feline melanoma may be associated with histologic features of malignancy and loss of melanocytic-specific markers, such as Melan A and PNL2. Further studies, with a larger cohort and follow-up information, are recommended.

Addressing the importance of melanoma tumor-infiltrating lymphocytes in disease progression and clinicopathological characteristics.

Tumor-infiltrating lymphocytes (TILs) in primary cutaneous melanoma are considered to represent the host's antitumor immunological response; however, whether there are associations between TIL grade and histopathological characteristics and disease survival remains controversial. BRAF mutational status has been established as a routine screening method in advanced malignant melanoma, and worse prognosis rates have been demonstrated in patients harboring BRAF mutations. However, the general impact of BRAF mutational status on survival and histopathological characteristics is still debated. The aim of the present study was to compare the value of the assessment of TIL grade in stages I-II nodular and superficial spreading melanoma and BRAF mutational status, and its influence on clinicopathological characteristics. Altogether, 85 patients at stage IA-IIC who underwent melanoma surgical treatment at the Riga East University Hospital between 2012 and 2017 were retrospectively enrolled in the study. The histopathological characteristics were assessed according to the current World Health Organization and The American Joint Committee on Cancer 8th edition guidelines. The current study showed that patients with melanoma with high TIL grade had significantly better progression-free survival than patients with low TIL grade (hazard ratio, 4.9; 95% CI, 2.3-11.2; P<0.0001). BRAF mutations were observed in 52 patients (61.2%). BRAF mutational status in melanoma was associated with Clark invasion level (P=0.045), patient age (P=0.02) and TIL (P=0.04). The assessment of TIL grade in stage I-II melanoma demonstrated prognostic significance value and may help improve risk assessment in the future.

Disease-Associated Risk Variants in ANRIL Are Associated with Tumor-Infiltrating Lymphocyte Presence in Primary Melanomas in the Population-Based GEM Study.

Genome-wide association studies have reported that genetic variation at ANRIL (CDKN2B-AS1) is associated with risk of several chronic diseases including coronary artery disease, coronary artery calcification, myocardial infarction, and type 2 diabetes mellitus. ANRIL is located at the CDKN2A/B locus, which encodes multiple melanoma tumor suppressors. We investigated the association of these variants with melanoma prognostic characteristics. The Genes, Environment, and Melanoma Study enrolled 3,285 European origin participants with incident invasive primary melanoma. For each of ten disease-associated SNPs at or near ANRIL, we used linear and logistic regression modeling to estimate, respectively, the per allele mean changes in log of Breslow thickness and ORs for presence of ulceration and tumor-infiltrating lymphocytes (TIL). We also assessed effect modification by tumor NRAS/BRAF mutational status. Rs518394, rs10965215, and rs564398 passed false discovery and were each associated (P ≤ 0.005) with TILs, although only rs564398 was independently associated (P = 0.0005) with TILs. Stratified by NRAS/BRAF mutational status, rs564398*A was significantly positively associated with TILs among NRAS/BRAF mutant, but not wild-type, cases. We did not find SNP associations with Breslow thickness or ulceration. ANRIL rs564398 was associated with TIL presence in primary melanomas, and this association may be limited to NRAS/BRAF-mutant cases. Pathways related to ANRIL variants warrant exploration in relationship to TILs in melanoma, especially given the impact of TILs on immunotherapy and survival.

Tumor-Infiltrating Lymphocytes in Patients with Melanoma: Which Is Its Prognostic Value?

Oncological impact of tumor-infiltrating lymphocytes (TILs) in melanoma remains controversial. We aimed to determine the significance of TILs on melanoma-specific survival (MSS), recurrence-free survival (RFS), and sentinel lymph node status (SLN). A retrospective analysis of patients undergoing melanoma resection during the period 2009-2019 was performed. Using the Melanoma Institute Australia grading system for TILs, the cohort was divided into two groups: group 1 (G1), patients with TILs grades 1, 2, or 3 and Group 2 (G2), patients with TILs grade 0. From a total of 386 melanoma resections, 151 (39%) were included in G1 and 39 (10%) in G2. Among the 151 patients who underwent SLN biopsy, the positivity rate according to the TILs grades 0, 1, 2, and 3 was 32%, 18%, 14%, and 0%, respectively, p = 0.02. With an average follow-up of 48months, the 5-year MSS (G1: 86% vs G2: 75%, p = 0.002) and the 5-year RFS (G1: 81% vs G2: 60%, p = 0.004) were significantly higher in G1 than G2. Tumor-infiltrating lymphocytes in melanoma are associated with the SLN status and with a better MSS and RFS.

TIL in Melanoma-Similar Approaches, Different Results, Unanswered Questions.

Prior treatment with anti-PD-1 and/or BRAF-targeted therapies was associated with limited activity of tumor infiltrating lymphocytes (TIL) in metastatic melanoma. Recent insights into mechanisms of action for TIL and anti-PD-1 provide the foundation for understanding differences in outcomes in different trials or populations, and a roadmap for developing improved products. See related article by Seitter et al., p. 5289.

523 ANRIL inherited variants associated with primary melanoma TIL grade

Genome-wide association studies have reported an association between genetic variants in or near the ANRIL (CDKN2B-AS1) gene and age-related diseases. Despite these variants’ proximity to CDKN2A, which encodes melanoma tumor suppressor genes, their associations with melanoma prognostic factors are unknown. Here we investigated the associations of these variants with primary melanoma Breslow thickness, ulceration, and tumor-infiltrating lymphocyte (TIL) grade. Our analysis included 3,285 European-origin participants in the Genes, Environment and Melanoma (GEM) Study with incident invasive primary melanoma.

Epigenetic reprogramming of tumor cell–intrinsic STING function sculpts antigenicity and T cell recognition of melanoma

Lack or loss of tumor antigenicity represents one of the key mechanisms of immune escape and resistance to T cell-based immunotherapies. Evidence suggests that activation of stimulator of interferon genes (STING) signaling in tumor cells can augment their antigenicity by triggering a type I IFN-mediated sequence of autocrine and paracrine events. Although suppression of this pathway in melanoma and other tumor types has been consistently reported, the mechanistic basis remains unclear. In this study, we asked whether this suppression is, in part, epigenetically regulated and whether it is indeed a driver of melanoma resistance to T cell-based immunotherapies. Using genome-wide DNA methylation profiling, we show that promoter hypermethylation of cGAS and STING genes mediates their coordinated transcriptional silencing and contributes to the widespread impairment of the STING signaling function in clinically-relevant human melanomas and melanoma cell lines. This suppression is reversible through pharmacologic inhibition of DNA methylation, which can reinstate functional STING signaling in at least half of the examined cell lines. Using a series of T cell recognition assays with HLA-matched human melanoma tumor-infiltrating lymphocytes (TIL), we further show that demethylation-mediated restoration of STING signaling in STING-defective melanoma cell lines can improve their antigenicity through the up-regulation of MHC class I molecules and thereby enhance their recognition and killing by cytotoxic T cells. These findings not only elucidate the contribution of epigenetic processes and specifically DNA methylation in melanoma-intrinsic STING signaling impairment but also highlight their functional significance in mediating tumor-immune evasion and resistance to T cell-based immunotherapies.

PET Imaging of TIGIT Expression on Tumor-Infiltrating Lymphocytes.

Therapeutic checkpoint inhibitors on tumor-infiltrating lymphocytes (TIL) are being increasingly utilized in the clinic. The T-cell immunoreceptor with Ig and ITIM domains (TIGIT) is an inhibitory receptor expressed on T and natural killer cells. The TIGIT signaling pathway is an alternative target for checkpoint blockade to current PD-1/CTLA-4 strategies. Elevated TIGIT expression in the tumor microenvironment correlates with better therapeutic responses to anti-TIGIT therapies in preclinical models. Therefore, quantifying TIGIT expression in tumors is necessary for determining whether a patient may respond to anti-TIGIT therapy. PET imaging of TIGIT expression on TILs can therefore aid diagnosis and in monitoring therapeutic responses. Antibody-based TIGIT imaging radiotracers were developed with the PET radionuclides copper-64 (64Cu) and zirconium-89 (89Zr). In vitro characterization of the imaging probes was followed by in vivo evaluation in both xenografts and syngeneic tumor models in mouse. Two anti-TIGIT probes were developed and exhibited immunoreactivity of >72%, serum stability of >95%, and specificity for TIGIT with both mouse TIGIT-expressing HeLa cells and ex vivo-activated primary splenocytes. In vivo, the 89Zr-labeled probe demonstrated superior contrast than the 64Cu probe due to 89Zr's longer half-life matching the TIGIT antibody's pharmacokinetics. The 89Zr probe was used to quantify TIGIT expression on TILs in B16 melanoma in immunocompetent mice and confirmed by ex vivo flow cytometry. This study develops and validates novel TIGIT-specific 64Cu and 89Zr PET probes for quantifying TIGIT expression on TILs for diagnosis of patient selection for anti-TIGIT therapies.

Immunotyping and Quantification of Melanoma Tumor-Infiltrating Lymphocytes.

The density of tumour-infiltrating lymphocytes (TILs) in melanoma is correlated with improved clinical prognosis; however, standardized TIL immunotyping and quantification protocols are lacking. Herein, we provide a review of the technologies being utilized for the immunotyping and quantification of melanoma TILs.

Multiplex Immunohistochemistry Analysis of Melanoma Tumor-Infiltrating Lymphocytes.

Tumor-infiltrating lymphocytes (TILs) are an important prognostic indicator in melanoma and play a key role in the patient's response to immune checkpoint blockade. However, until recently, it was not possible to combine multi-parameter markers to define the TILs and their histological context. Multiplex immunohistochemistry (mIHC) is a new technology which addresses this issue and enables simultaneous detection of melanoma and multiple immune subsets in formalin fixed paraffin embedded tissue. Following antigen retrieval, melanoma tissue sections are stained by OPAL on an autostainer, including serial rounds of epitope labelling with monoclonal antibodies followed by tyramide signal amplification (TSA). The stained tissue sections are then imaged on the Vectra instrument, and digital images are processed by analysis software (inForm and HALO) to derive tissue segmentation and immune subset densities within the tumor and tumor stroma. Spatial relationships between immune cells and tumor cells are then analyzed using a novel R algorithm. Taken together, multiplex IHC describes the histological context of the immune system in melanoma. The data is objective and allows for characterization of individual melanomas as T cell inflamed (hot), immune excluded, or no immune cells (cold).

Single-Cell Gene Expression, Clonality, and Feature Barcoding of Melanoma Tumor-Infiltrating Lymphocytes.

We describe here a protocol to measure gene expression, T cell receptor (TCR) sequence, and protein expression by single T cells extracted from melanoma, using 10× Chromium technology. This method includes freezing and thawing of the melanoma infiltrating lymphocytes, staining of cells with fluorescent and barcode-conjugated antibodies, sorting of T cells, and loading the cells on the 10× Chromium Controller. After sequencing, analysis includes quality control, genetic demultiplexing to resolve genetically different samples, and T cell clonality and clustering analysis. Single cell RNA sequencing paints the complete portrait of individual T cells, including their clonality and phenotype, and it reconstructs a complete picture of the T cell infiltrate in a tumor that is represented as cell clustering similar to a pointillism painting.

Using Mass Cytometry to Analyze the Tumor-Infiltrating Lymphocytes in Human Melanoma.

Here we describe the application of mass cytometry to analyze tumor-infiltrating lymphocytes in human melanoma. Mass cytometry is the coupling of flow cytometry and mass spectrometry, which allows for the simultaneous measurement of 40+ cell parameters on a per cell basis. Heavy metal-labeled antibodies can bind to proteins (CD markers, transcription factors, cytokines) on the cell surface and in the cytoplasm/nucleus. As labeled cells pass through the CyTOF, the instrument detects the heavy metals. Combining these signals allows description of melanoma tumor-infiltrating lymphocytes at a greater depth than alternative phenotyping strategies and enables detailed analyses of a variety of cellular parameters, including immune cell lineage, activation status, and functional polarization.

Abstract IA02: Next targets for immune checkpoint blockade in melanoma

Abstract Multiple mechanisms of melanoma-induced immune escape contribute to the failure of T-cell responses to control tumor growth in humans. Many inhibitory pathways play a critical role in impeding T-cell responses to tumor antigens (TAs), including PD-1, the T-cell immunoglobulin and mucin-domain containing-protein 3 (Tim-3), and the T-cell immunoreceptor with Ig and ITIM domains (TIGIT). These inhibitory receptors (IRs) are upregulated by TA-specific CD8+T cells in the tumor microenvironment (TME) while their respective ligands are expressed by antigen-presenting cells (APCs) and tumor cells. Circulating TA-specific CD8+ T cells and CD8+ tumor-infiltrating lymphocytes (TILs) that coexpress PD-1, TIGIT and Tim-3 exhibit different levels of T-cell dysfunction. Dysfunctional/exhausted CD8+ TILs are not totally inert and can exhibit cytolytic functions but are likely kept in check by multiple inhibitory pathways that can be targeted by immune checkpoint blockade. TIGIT is an attractive target for the next-generation immune checkpoint blockade for several reasons. First, it is highly expressed by the majority of CD8+ TILs together with PD-1. Second, the TIGIT ligands, CD155/PVR, and CD112, are highly expressed in the TME by melanoma cells and APCs. Third, dual PD-1/TIGIT blockade augments the expansion and function of human TA-specific CD8+ T cells in vitro and promotes tumor rejection in animal models. Fourth, TIGIT also acts in Tregs and NK cells to regulate antitumor immune responses. CD8+ TILs in melanoma and other solid tumors downregulate the costimulatory molecule DNAM-1/CD226, which competes with TIGIT for binding to the same ligands CD155 and CD112. Therefore, in addition to the TIGIT-mediated T-cell intrinsic inhibitory effects, the downregulation of CD226 expression by CD8+TILs in the TME also contributes to impede T-cell responses to melanoma. Collectively, our findings provide the rationale for dual PD-1/TIGIT blockade together with therapeutic strategies to counteract CD226 downregulation in the TME to improve the clinical benefits of single PD-1 blockade in patients with melanoma and other solid tumors. Citation Format: Joe-Marc Chauvin, Mignane Ka, Ornella Pagliano, Julien Foucade, Carmine Menna, Diwakar Davar, John Kirkwood, Vijay Kuchroo, Ana Anderson, Alan Korman, Hassane Zarour. Next targets for immune checkpoint blockade in melanoma [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr IA02.