TCR Repertoire Research Articles

Cyclophosphamide enhances the antitumor potency of GITR engagement by increasing oligoclonal cytotoxic T cell fitness

Only a subset of cancer patients responds to checkpoint blockade inhibition in the clinic. Strategies to overcome resistance are promising areas of investigation. Targeting glucocorticoid-induced tumor necrosis factor receptor–related protein (GITR) has shown efficacy in preclinical models, but GITR engagement is ineffective in controlling advanced, poorly immunogenic tumors, such as B16 melanoma, and has not yielded benefit in clinical trials. The alkylating agent cyclophosphamide (CTX) depletes regulatory T cells (Tregs), expands tumor-specific effector T cells (Teffs) via homeostatic proliferation, and induces immunogenic cell death. GITR agonism has an inhibitory effect on Tregs and activates Teffs. We therefore hypothesized that CTX and GITR agonism would promote effective antitumor immunity. Here we show that the combination of CTX and GITR agonism controlled tumor growth in clinically relevant mouse models. Mechanistically, we show that the combination therapy caused tumor cell death, clonal expansion of highly active CD8+ T cells, and depletion of Tregs by activation-induced cell death. Control of tumor growth was associated with the presence of an expanded population of highly activated, tumor-infiltrating, oligoclonal CD8+ T cells that led to a diminished TCR repertoire. Our studies show that the combination of CTX and GITR agonism is a rational chemoimmunotherapeutic approach that warrants further clinical investigation.

Repertoire analysis of \u03b3\u03b4 T cells in the chicken enables functional annotation of the genomic region revealing highly variable pan-tissue TCR gamma V gene usage as well as identifying public and private repertoires

BackgroundDespite increasing interest in γδ T cells and their non-classical behaviour, most studies focus on animals with low numbers of circulating γδ T cells, such as mice and humans. Arguably, γδ T cell functions might be more prominent in chickens where these cells form a higher proportion of the circulatory T cell compartment. The TCR repertoire defines different subsets of γδ T cells, and such analysis is facilitated by well-annotated TCR loci. γδ T cells are considered at the cusp of innate and adaptive immunity but most functions have been identified in γδ low species. A deeper understanding of TCR repertoire biology in γδ high and γδ low animals is critical for defining the evolution of the function of γδ T cells. Repertoire dynamics will reveal populations that can be classified as innate-like or adaptive-like as well as those that straddle this definition.ResultsHere, a recent discrepancy in the structure of the chicken TCR gamma locus is resolved, demonstrating that tandem duplication events have shaped the evolution of this locus. Importantly, repertoire sequencing revealed large differences in the usage of individual TRGV genes, a pattern conserved across multiple tissues, including thymus, spleen and the gut. A single TRGV gene, TRGV3.3, with a highly diverse private CDR3 repertoire dominated every tissue in all birds. TRGV usage patterns were partly explained by the TRGV-associated recombination signal sequences. Public CDR3 clonotypes represented varying proportions of the repertoire of TCRs utilising different TRGVs, with one TRGV dominated by super-public clones present in all birds.ConclusionsThe application of repertoire analysis enabled functional annotation of the TCRG locus in a species with a high circulating γδ phenotype. This revealed variable usage of TCRGV genes across multiple tissues, a pattern quite different to that found in γδ low species (human and mouse). Defining the repertoire biology of avian γδ T cells will be key to understanding the evolution and functional diversity of these enigmatic lymphocytes in an animal that is numerically more reliant on them. Practically, this will reveal novel ways in which these cells can be exploited to improve health in medical and veterinary contexts.

Immunogenic T cell epitopes of SARS-CoV-2 are recognized by circulating memory and naïve CD8 T cells of unexposed individuals.

BackgroundRecent studies have provided evidence of T cell reactivity to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in significant numbers of non-infected individuals, which has been attributed to cross-reactive CD4 memory T cells from previous exposure to seasonal coronaviruses. Less evidence of cross-reactive memory CD8 T cells has been documented to date.MethodsWe used the NetCTLPan neural network of the Epitope Database and Analysis Resource to select a series of 27 HLA-A*02:01 epitopes derived from the proteome of SARS-CoV-2. Their binding capacity was assessed by a HLA-A*02:01 stabilization assay and by quantifying their binding to HLA-A*02:01 monomers for the generation of tetramers. Their ability to stimulate and induce expansion of SARS-CoV-2 reactive CD8 T cells was measured by flow cytometry. The TCR repertoire of COVID convalescent and healthy unexposed donors was analysed using the MIRA database.FindingsThe HLA-A*02:01 epitopes tested were able to stabilise HLA molecules and induce activation of CD8 T cells of healthy unexposed donors. Our results, based on specific tetramer binding, provide evidence supporting the presence of frequent cross-reactive CD8 T cells to SARS-CoV-2 antigens in non-exposed individuals. Interestingly, the reactive cells were distributed into naïve, memory and effector subsets.InterpretationOur data are consistent with a significant proportion of the reactive CD8 T clones belonging to the public shared repertoire, readily available in absence of previous contact with closely related coronaviruses. Furthermore, we demonstrate the immunogenic capacity of long peptides carrying T cell epitopes, which can serve to isolate virus-specific T cell receptors among the ample repertoire of healthy unexposed subjects and could have application in COVID-19 immunotherapy. Limitations of our study are that it concentrated on one MHC I allele (HLA-A*02:01) and the low numbers of samples and epitopes tested.

Limited TCR repertoire and ENTPD1 dysregulation mark late-stage COVID-19

SummaryT cell exhaustion and dysfunction are hallmarks of severe COVID-19. To gain insights into the pathways underlying these alterations, we performed a comprehensive transcriptome analysis of peripheral-blood-mononuclear-cells (PBMCs), spleen, lung, kidney, liver, and heart obtained at autopsy from COVID-19 patients and matched controls, using the nCounter CAR-T-Characterization panel. We found substantial gene alterations in COVID-19-impacted organs, especially the lung where altered TCR repertoires are noted. Reduced TCR repertoires are also observed in PBMCs of severe COVID-19 patients. ENTPD1/CD39, an ectoenzyme defining exhausted T-cells, is upregulated in the lung, liver, spleen, and PBMCs of severe COVID-19 patients where expression positively correlates with markers of vasculopathy. Heightened ENTPD1/CD39 is paralleled by elevations in STAT-3 and HIF-1α transcription factors; and by markedly reduced CD39-antisense-RNA, a long-noncoding-RNA negatively regulating ENTPD1/CD39 at the post-transcriptional level. Limited TCR repertoire and aberrant regulation of ENTPD1/CD39 could have permissive roles in COVID-19 progression and indicate potential therapeutic targets to reverse disease.

Dynamics of TCR repertoire and T cell function in COVID-19 convalescent individuals

SARS-CoV-2 outbreak has been declared by World Health Organization as a worldwide pandemic. However, there are many unknowns about the antigen-specific T-cell-mediated immune responses to SARS-CoV-2 infection. Here, we present both single-cell TCR-seq and RNA-seq to analyze the dynamics of TCR repertoire and immune metabolic functions of blood T cells collected from recently discharged COVID-19 patients. We found that while the diversity of TCR repertoire was increased in discharged patients, it returned to basal level ~1 week after becoming virus-free. The dynamics of T cell repertoire correlated with a profound shift of gene signatures from antiviral response to metabolism adaptation. We also demonstrated that the top expanded T cell clones (~10% of total T cells) display the key anti-viral features in CD8+ T cells, confirming a critical role of antigen-specific T cells in fighting against SARS-CoV-2. Our work provides a basis for further analysis of adaptive immunity in COVID-19 patients, and also has implications in developing a T-cell-based vaccine for SARS-CoV-2.

Monitoring malignant T-cell clones by direct TCR expression assay in patients with leukemic cutaneous T-cell lymphoma during extracorporeal photopheresis.

Accurate assessment of malignant T-cell clones in patients with leukemic cutaneous T-cell lymphoma (L-CTCL) is crucial for diagnosis, treatment, and monitoring disease. Although multiple approaches to quantitate malignant T-cell clones have been reported, a cost-effective assay with broad coverage is not available. We report a NanoString-nCounter-Technology-based direct TCR expression assay (DTEA) that was previously developed to quantify both TCR-Vα and TCR-Vβ usages after adoptive immunotherapy. This study was performed to test the effectiveness of DTEA in assessing malignant T-cell clones in L-CTCL patients. Total RNAs extracted from peripheral blood mononuclear cells of patients before starting extracorporeal photopheresis (ECP) (n=15) and during therapy at 3months and 6 months (n=12) were used for DTEA, with customized probes for 45 TCR-Vα and 46 TCR-Vβ family members. At baseline, DTEA detected TCR-Vβ clones in all 15 patients (100%) compared to flow cytometry that detected TCR-Vβ clones in 9 of 13 patients (69.2%). In addition to predominant TCR-Vβ clones, DTEA also detected additional TCR-Vβ clones in 8 of 15 patients (53.3%). Furthermore, DTEA simultaneously identified clonal TCR-Vα usages, which allowed us to pair TCR-Vα and TCRVβ usages by malignant T-cells and identify diversified clonotypes. Changes in the relative frequencies of clonal TCR-Vβ and TCRVα usages over therapy were consistent with patients' clinical responses. Our results indicate that DTEA can effectively assess malignant T-cell clones by detecting clonal TCR-Vα and TCR-Vβ usages. By providing a global view of TCR repertoires, DTEA may also help us understand the origin(s) of malignant T-cells and pathogenesis of CTCL.

Generation of Pure Highly Functional Human Anti-Tumor Specific Cytotoxic T Lymphocytes With Stem Cell-Like Memory Features for Melanoma Immunotherapy.

Adoptive immunotherapy based on the transfer of anti-tumor cytotoxic T lymphocytes (CTLs) is a promising strategy to cure cancers. However, rapid expansion of numerous highly functional CTLs with long-lived features remains a challenge. Here, we constructed NIH/3T3 mouse fibroblast-based artificial antigen presenting cells (AAPCs) and precisely evaluated their ability to circumvent this difficulty. These AAPCs stably express the essential molecules involved in CTL activation in the HLA-A*0201 context and an immunogenic HLA-A*0201 restricted analogue peptide derived from MART-1, an auto-antigen overexpressed in melanoma. Using these AAPCs and pentamer-based magnetic bead-sorting, we defined, in a preclinical setting, the optimal conditions to expand pure MART-1-specific CTLs. Numerous highly purified MART-1-specific CTLs were rapidly obtained from healthy donors and melanoma patients. Both TCR repertoire and CDR3 sequence analyses revealed that MART-1-specific CTL responses were similar to those reported in the literature and obtained with autologous or allogeneic presenting cells. These MART-1-specific CTLs were highly cytotoxic against HLA-A*0201+ MART-1+ tumor cells. Moreover, they harbored a suitable phenotype for immunotherapy, with effector memory, central memory and, most importantly, stem cell-like memory T cell features. Notably, the cells harboring stem cell-like memory phenotype features were capable of self-renewal and of differentiation into potent effector anti-tumor T cells. These “off-the-shelf” AAPCs represent a unique tool to rapidly and easily expand large numbers of long-lived highly functional pure specific CTLs with stem cell-like memory T cell properties, for the development of efficient adoptive immunotherapy strategies against cancers.

Adoptive immunotherapy with transient anti-CD4 treatment enhances anti-tumor response by increasing IL-18R\u03b1hi CD8+ T cells

Adoptive T cell therapy (ACT) requires lymphodepletion preconditioning to eliminate immune-suppressive elements and enable efficient engraftment of adoptively transferred tumor-reactive T cells. As anti-CD4 monoclonal antibody depletes CD4+ immune-suppressive cells, the combination of anti-CD4 treatment and ACT has synergistic potential in cancer therapy. Here, we demonstrate a post-ACT conditioning regimen that involves transient anti-CD4 treatment (CD4post). Using murine melanoma, the combined effect of cyclophosphamide preconditioning (CTXpre), CD4post, and ex vivo primed tumor-reactive CD8+ T-cell infusion is presented. CTXpre/CD4post increases tumor suppression and host survival by accelerating the proliferation and differentiation of ex vivo primed CD8+ T cells and endogenous CD8+ T cells. Endogenous CD8+ T cells enhance effector profile and tumor-reactivity, indicating skewing of the TCR repertoire. Notably, enrichment of polyfunctional IL-18Rαhi CD8+ T cell subset is the key event in CTXpre/CD4post-induced tumor suppression. Mechanistically, the anti-tumor effect of IL-18Rαhi subset is mediated by IL-18 signaling and TCR–MHC I interaction. This study highlights the clinical relevance of CD4post in ACT and provides insights regarding the immunological nature of anti-CD4 treatment, which enhances anti-tumor response of CD8+ T cells.

αβ T cells replacing dermal and epidermal γδ T cells in Tcrd-/- mice express an MHC-independent TCR repertoire.

The epidermis of mouse skin is usually populated by dendritic epidermal T cells (γδDETC) expressing an invariant Vγ5Vδ1+ TCR. In Tcrd-/- mice, skin-resident γδDETC are replaced by αβDETC carrying polyclonal αβ TCRs. Although they exhibit a dendritic morphology, αβDETC were reported to be less functional than genuine γδDETC, likely because their TCR is unable to interact with the original TCR ligands of γδDETC. However, the TCR repertoire of those replacement DETC in Tcrd-/- mice might provide clues for understanding the development and selection of canonical γδDETC. Here, we compare the phenotype and TCR repertoires of wild-type and Tcrd-/- mouse skin T cells. Our data reveal that αβDETC are CD4/CD8 double negative and express an MHC-independent TCR repertoire. Furthermore, we identify a second MHC-independent population of CD103hi CD4/ CD8 double-negative αβ T cells in the dermis of Tcrd-/- mice.

Effector Vγ9Vδ2 T cell response to congenital Toxoplasma gondii infection.

A major γδ T cell population in human adult blood are the Vγ9Vδ2 T cells that are activated and expanded in a TCR-dependent manner by microbe-derived and endogenously derived phosphorylated prenyl metabolites (phosphoantigens). Vγ9Vδ2 T cells are also abundant in human fetal peripheral blood, but compared with their adult counterparts they have a distinct developmental origin, are hyporesponsive toward in vitro phosphoantigen exposure, and do not possess a cytotoxic effector phenotype. In order to obtain insight into the role of Vγ9Vδ2 T cells in the human fetus, we investigated their response to in utero infection with the phosphoantigen-producing parasite Toxoplasma gondii (T. gondii). Vγ9Vδ2 T cells expanded strongly when faced with congenital T. gondii infection, which was associated with differentiation toward potent cytotoxic effector cells. The Vγ9Vδ2 T cell expansion in utero resulted in a fetal footprint with public germline-encoded clonotypes in the Vγ9Vδ2 TCR repertoire 2 months after birth. Overall, our data indicate that the human fetus, from early gestation onward, possesses public Vγ9Vδ2 T cells that acquire effector functions following parasite infections.

Expansion of Unique Hepatitis C Virus-Specific Public CD8+ T Cell Clonotypes during Acute Infection and Reinfection.

Hepatitis C virus (HCV) infection resolves spontaneously in ∼25% of acutely infected humans where viral clearance is mediated primarily by virus-specific CD8+ T cells. Previous cross-sectional analysis of the CD8+ TCR repertoire targeting two immunodominant HCV epitopes reported widespread use of public TCRs shared by different subjects, irrespective of infection outcome. However, little is known about the evolution of the public TCR repertoire during acute HCV and whether cross-reactivity to other Ags can influence infectious outcome. In this article, we analyzed the CD8+ TCR repertoire specific to the immunodominant and cross-reactive HLA-A2-restricted nonstructural 3-1073 epitope during acute HCV in humans progressing to either spontaneous resolution or chronic infection and at ∼1 y after viral clearance. TCR repertoire diversity was comparable among all groups with preferential usage of the TCR-β V04 and V06 gene families. We identified a set of 13 public clonotypes in HCV-infected humans independent of infection outcome. Six public clonotypes used the V04 gene family. Several public clonotypes were long-lived in resolvers and expanded on reinfection. By mining publicly available data, we identified several low-frequency CDR3 sequences in the HCV-specific repertoire matching human TCRs specific for other HLA-A2-restricted epitopes from melanoma, CMV, influenza A, EBV, and yellow fever viruses, but they were of low frequency and limited cross-reactivity. In conclusion, we identified 13 new public human CD8+ TCR clonotypes unique to HCV that expanded during acute infection and reinfection. The low frequency of cross-reactive TCRs suggests that they are not major determinants of infectious outcome.

Protective heterologous T cell immunity in COVID-19 induced by the trivalent MMR and Tdap vaccine antigens

BackgroundT cells control viral infection, promote vaccine durability, and in coronavirus disease 2019 (COVID-19) associate with mild disease. We investigated whether prior measles-mumps-rubella (MMR) or tetanus-diphtheria-pertussis (Tdap) vaccination elicits cross-reactive T cells that mitigate COVID-19.MethodsAntigen-presenting cells (APC) loaded ex vivo with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), MMR, or Tdap antigens and autologous T cells from COVID-19-convalescent participants, uninfected individuals, and COVID-19 mRNA-vaccinated donors were co-cultured. T cell activation and phenotype were detected by interferon-γ (IFN-γ) enzyme-linked immunospot (ELISpot) assays and flow cytometry. ELISAs (enzyme-linked immunosorbant assays) and validation studies identified the APC-derived cytokine(s) driving T cell activation. TCR clonotyping and single-cell RNA sequencing (scRNA-seq) identified cross-reactive T cells and their transcriptional profile. A propensity-weighted analysis of COVID-19 patients estimated the effects of MMR and Tdap vaccination on COVID-19 outcomes.FindingsHigh correlation was observed between T cell responses to SARS-CoV-2 (spike-S1 and nucleocapsid) and MMR and Tdap proteins in COVID-19-convalescent and -vaccinated individuals. The overlapping T cell population contained an effector memory T cell subset (effector memory re-expressing CD45RA on T cells [TEMRA]) implicated in protective, anti-viral immunity, and their detection required APC-derived IL-15, known to sensitize T cells to activation. Cross-reactive TCR repertoires detected in antigen-experienced T cells recognizing SARS-CoV-2, MMR, and Tdap epitopes had TEMRA features. Indices of disease severity were reduced in MMR- or Tdap-vaccinated individuals by 32%–38% and 20%–23%, respectively, among COVID-19 patients.ConclusionsTdap and MMR memory T cells reactivated by SARS-CoV-2 may provide protection against severe COVID-19.FundingThis study was supported by a National Institutes of Health (R01HL065095, R01AI152522, R01NS097719) donation from Barbara and Amos Hostetter and the Chleck Foundation.

Distinct T cell receptor diversity and integrative analyses of clinically defined high-grade serous ovarian cancer

<h3>Objectives:</h3> To characterize distinct T cell repertoires between clinically well-defined groups of high-grade serous ovarian cancer (HGSC) patients, we obtained pre- treatment tumor samples via a laparoscopic triage algorithm. <h3>Methods:</h3> Pre-treated frozen tumor samples (one primary and two metastatic sites) were collected from patients with HGSC based on a laparoscopic triage algorithm: 1) patients who underwent complete gross resection after primary surgery (R0, n=10), 2) patients who received neoadjuvant chemotherapy (NACT) with excellent response (NACT-ER, n=10), or 3) patients who received NACT with poor response (NACT-PR, n=10). These samples were subjected to high-throughput TCR sequencing analysis and integrative analyses, with immune assessments and high-depth whole-genome sequencing from the matched samples among the groups. <h3>Results:</h3> We identified a rearranged complementary-determining region 3 (CDR3) of the T-cell receptor β (TCRβ) gene using a total of 90 samples. Importantly, we identified higher TCR β-chain CDR3 diversity (i.e., a higher number of unique productive sequences, less clonal relatedness, and higher TCR convergence) in the R0 group than in the NACT groups. We also found enrichment of specific variable (V), diversity (D), and joining (J) genes usage in each group and significant unique mutual exclusivity and co-occurrence of V, D, and J genes in all three groups. In addition, we also found significant positive correlations between clonal relatedness and neoantigens (R=0.31, p=0.007), copy number variations (R=0.43, p<0.001), and mutation load (R=0.27, p=0.019) by integrative analyses. We also observed significant negative correlations between TCR clonal relatedness and the percentage of T cells (CD8+ and CD4+, <0.001) and B cells (CD20+) from immune profiling data. <h3>Conclusions:</h3> Our findings provide a comprehensive understanding of TCR repertoire diversity in clinically defined HGSC subgroups, and could have prognostic and therapeutic strategies in HGSC.

Single-Cell RNA Sequencing Approaches for Tracing T Cell Development

Abstract T cell development occurs in the thymus, where uncommitted progenitors are directed into a range of sublineages with distinct functions. The goal is to generate a TCR repertoire diverse enough to recognize potential pathogens while remaining tolerant of self. Decades of intensive research have characterized the transcriptional programs controlling critical differentiation checkpoints at the population level. However, greater precision regarding how and when these programs orchestrate differentiation at the single-cell level is required. Single-cell RNA sequencing approaches are now being brought to bear on this question, to track the identity of cells and analyze their gene expression programs at a resolution not previously possible. In this review, we discuss recent advances in the application of these technologies that have the potential to yield unprecedented insight to T cell development.

Notch-induced endoplasmic reticulum-associated degradation governs mouse thymocyte β-selection.

Signals from the pre-T cell receptor and Notch coordinately instruct β-selection of CD4-CD8-double negative (DN) thymocytes to generate αβ T cells in the thymus. However, how these signals ensure a high-fidelity proteome and safeguard the clonal diversification of the pre-selection TCR repertoire given the considerable translational activity imposed by β-selection is largely unknown. Here, we identify the endoplasmic reticulum (ER)-associated degradation (ERAD) machinery as a critical proteostasis checkpoint during β-selection. Expression of the SEL1L-HRD1 complex, the most conserved branch of ERAD, is directly regulated by the transcriptional activity of the Notch intracellular domain. Deletion of Sel1l impaired DN3 to DN4 thymocyte transition and severely impaired mouse αβ T cell development. Mechanistically, Sel1l deficiency induced unresolved ER stress that triggered thymocyte apoptosis through the PERK pathway. Accordingly, genetically inactivating PERK rescued T cell development from Sel1l-deficient thymocytes. In contrast, IRE1α/XBP1 pathway was induced as a compensatory adaptation to alleviate Sel1l-deficiency-induced ER stress. Dual loss of Sel1l and Xbp1 markedly exacerbated the thymic defect. Our study reveals a critical developmental signal controlled proteostasis mechanism that enforces T cell development to ensure a healthy adaptive immunity.

Clinical and basic implications of dynamic T cell receptor clonotyping in hematopoietic cell transplantation.

TCR repertoire diversification constitutes a foundation for successful immune reconstitution after allogeneic hematopoietic cell transplantation (allo-HCT). Deep TCR Vβ sequencing of 135 serial specimens from a cohort of 35 allo-HCT recipients/donors was performed to dissect posttransplant TCR architecture and dynamics. Paired analysis of clonotypic repertoires showed a minimal overlap with donor expansions. Rarefied and hyperexpanded clonotypic patterns were hallmarks of T cell reconstitution and influenced clinical outcomes. Donor and pretransplant TCR diversity as well as divergence of class I human leukocyte antigen genotypes were major predictors of recipient TCR repertoire recovery. Complementary determining region 3–based specificity spectrum analysis indicated a predominant expansion of pathogen- and tumor-associated clonotypes in the late post–allo-HCT phase, while autoreactive clones were more expanded in the case of graft-versus-host disease occurrence. These findings shed light on post–allo-HCT adaptive immune reconstitution processes and possibly help in tracking alloreactive responses.

Abstract 1675: Leveraging RNA sequencing for scalable tumor immune repertoire profiling

Abstract Background: Therapeutic strategies that modulate the interaction between tumors and tumor-infiltrating lymphocytes (TILs), such as checkpoint blockade, have tremendous promise but yield clinical responses in only a subset of patients. Accumulating evidence indicates that better characterizing TILs using repertoire-sequencing (rep-seq) may aid in predicting immunotherapy outcomes. However, repertoire profiling of the immune infiltrate is often implemented as a stand-alone assay or via single-cell sequencing, which can be difficult to incorporate into diagnostic platforms at scale. We developed and optimized a method to integrate immune repertoire profiling into a high-volume RNA-seq pipeline. Our approach yields accurate quantification of abundant TIL receptors without diminishing the clinical value of the remaining transcriptome. Here, we demonstrate the capabilities of our method in 500 TCR/BCR repertoires from a diverse cohort of tumor samples. Methods: Hybrid capture probes tiling T-cell and B-cell receptor (TCR and BCR) genes were used to enrich immune receptor transcripts detected by the Tempus RNA-seq workflow. Receptor sequences were aligned, assembled, and annotated against IMGT reference sequences using the TRUST4 assembly tool. Shannon Entropy was calculated to evaluate repertoire evenness. Richness (productive clonotypes) and immune infiltration predictions were correlated with a one-tailed Pearson correlation coefficient (95% CI). Results: Immune repertoires were extracted from RNA-seq of 501 tumor samples across 38 different cancer types. TCR and BCR repertoires (including small gamma/delta TCR repertoires) were robustly assembled in our cohort, with over 1.2 million total clonotypes identified. The distribution of repertoire evenness demonstrated expected trends, including dominant monoclonal expansion (&amp;gt;70-90% total receptor reads) of putative tumor cell receptors in T-cell/B-cell-driven malignancies. Repertoire richness also followed tissue-specific expectations where TIL-low cancers (e.g., glioblastoma multiforme) typically exhibited low-richness repertoires, while TIL-high cancers (e.g., non-small cell lung cancer) often yielded thousands of productive clonotypes. Repertoire richness was significantly correlated with RNA-based cell-type-specific transcriptional profiles for T-cells (r = 0.43, P&amp;lt;5e-24) and B-cells (r = 0.2, P&amp;lt;3.6e-6). Benchmarking our method on co-extracted DNA with amplicon-based repertoire sequencing confirmed accurate detection of highly abundant TIL receptors using an orthogonal approach. Conclusions: Our repertoire profiling method effectively leveraged routine RNA-seq to profile TIL receptors, ultimately allowing for the seamless integration of repertoire-based biomarker evaluation and improved immune infiltrate estimation through a high-volume RNA-seq pipeline. Citation Format: Taylor S. Harding, Brittany Mineo, Jenna Malinauskas, Jason Perera, Aly A. Khan. Leveraging RNA sequencing for scalable tumor immune repertoire profiling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1675.

Abstract 1677: Characterization of the pharmacodynamic activity of AGEN1181, an Fc-enhanced CTLA-4 antibody, alone and in combination with the PD-1 antibody balstilimab

Abstract AGEN1181 is a novel, Fc-engineered monoclonal antibody that targets cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), with enhanced FcyR-dependent functionality designed to improve T cell priming and activation, deplete intratumoral regulatory T cells (Treg), and improve memory T cell response. AGEN1181 is currently undergoing Phase I clinical evaluation, both as a single agent and in combination with the anti-PD-1 antibody balstilimab, in patients with advanced solid tumors (NCT03860272). Encouraging signals of clinical benefit have emerged as part of this ongoing study, including complete and durable responses in patients that would traditionally be considered unresponsive to conventional CTLA-4/PD-1 therapy. Here we report on pharmacodynamic analyses associated with AGEN1181 exposure in patients on study in this trial. As monotherapy, AGEN1181 was administered on a once every 3 or once every 6 week cycle (doses escalated from 0.1 - 4 mg/kg). Alternatively, AGEN1181 was dosed once every six weeks in combination with balstilimab (3 mg/kg once every 2 weeks). Tumor mutational burden (TMB) and gene expression were assessed from pre-treatment and on-treatment biopsies by whole exome sequencing (WES) and RNASeq, respectively. Multiplex immunohistochemistry was used to assess Treg depletion and CD8 T-cell activation in response to AGEN1181. TCR repertoire was analyzed in PBMCs collected on-treatment with AGEN1181. FcγRIIIA genotyping was performed by real-time PCR. Immunophenotyping of peripheral blood mononuclear cells collected pre- and post-treatment were analyzed by flow cytometry. Consistent with its proposed mechanism of action, dose-dependent pharmacodynamic effects have been observed in patients while on-treatment with AGEN1181. Peripheral CD4+Ki67+, CD4+ICOS+ and CD4+HLA-DR+ T cells increased over baseline in response to monotherapy. This effect was further enhanced in subjects treated with AGEN1181 when given in combination with balstilimab. Responders expressed either the low affinity FcγRIIIA allele or were heterozygous - suggesting that AGEN1181 has the potential to expand clinical activity to patients with low affinity Fc binding, in contrast to conventional CTLA-4 inhibitors. Additionally, we show that AGEN1181 induced selective Treg depletion within the tumor microenvironment with concomitant increases in CD8 T cell infiltration. TCRSeq analysis demonstrated increased clonality and decreased downsampled diversity, providing further evidence that the Fc-enhanced activity of AGEN1181 can promote improved T cell priming and Treg depletion. In summary, the pharmacodynamic analyses presented here are consistent with the Fc-enhanced design of AGEN1181 and its potential to broaden the therapeutic reach of CTLA-4-based immunotherapy. Citation Format: Irina Shapiro, Min Lim, Simarjot Pabla, Steven J. O'Day, Anthony El-Khoueiry, Chethan Ramamurthy, Andrea J. Bullock, Michael S. Gordon, Jennifer Buell, Dhan Chand, Anna Wijatyk. Characterization of the pharmacodynamic activity of AGEN1181, an Fc-enhanced CTLA-4 antibody, alone and in combination with the PD-1 antibody balstilimab [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1677.

Abstract 1653: A single-cell atlas of the effect of chemotherapeutics over intratumoral immune cells reveals that combining an alkylating agent and a vinca alkaloid can activate antigen presenting cells and increase tcf1+ stem-like CD8+ T-cells, thus improving anti-PD-1 efficacy in triple negative breast cancer and lymphoma

Abstract Checkpoint inhibitors (CIs) instigate anticancer immunity, albeit only in a fraction of patients. Triple negative breast cancer (TNBC) is among the most aggressive and lethal BC types, and currently available therapies have an unsatisfactory clinical impact. The association of CIs with chemotherapy had encouraging results in randomized TNBC trials, but so far there has been no clear evidence of what should be considered the best combinatorial therapy. In B-cell non-Hodgkin's lympoma (NHL), CIs' clinical activity has been so far unsatisfactory. We designed the current study to define what could be considered the most efficient combinatorial regimen of chemotherapy plus CI in TNBC and NHL. We investigated different options including platinum (P), doxorubicin (D), taxol (T), vinorelbine (V), and cyclophosphamide ( C), i.e. the most effective chemotherapy drugs for these diseases. Capecitabine was not considered as found not to be effective in combination with CIs. As TNBC patients have a large intratumoral immune cell heterogeneity, we investigated two immune competent TNBC models: 4T1 with a predominant lymphoid intratumoral infiltrate and EMT6 with a predominant myeloid infiltrate. A C57BL/6 Eμ-myc transgenic NHL model, with a predominant lymphatic dissemination pattern, was also studied. We found that 1) Intermittent, medium-dosage C (C140), was more effective than other combinatorial regimens including the CI anti-PD1 plus low-dose C, or regular-dose T, D, or P. The association of V and anti-PD1 further increased the preclinical efficacy of C140; 2) both CD3+CD4+ and CD3+CD8+ T cells (at variance with other subsets of NK and myeloid cells) were needed to control local and metastatic neoplastic growth in mice treated with C140, V and anti-PD1. 3) The combinatorial therapy including C140, V and anti-PD1 was the most efficient in activating antigen presenting cells (APCs), and the investigation of TCR repertoire indicated that this therapy generated a significantly larger clonal expansion of both CD3+CD4+ and CD3+CD8+ T cells. 4) Single-cell transcriptome analysis of &amp;gt;50,000 intratumoural immune cells showed after C140, V and anti-PD1 therapy a gene signature suggestive of a change resulting from exposure to a mitogen, ligand, or an antigen for which it is specific, as well as APC-to-T-cell adhesion. This transcriptional program also increased intratumoural tcf1+ stem-like CD8+ T-cells and altered the balance between terminally and progenitor exhausted T-cells, favoring the latter. These data support the clinical investigation of this therapy in TNBC and NHL, and we are currently investigating the related transcriptional mechanisms inducing APC activation and T cell rewiring. Citation Format: Paolo Falvo, Stefania Orecchioni, Roman Hillje, Alessandro Raveane, Giulia Mitola, Patrizia Mancuso, Chiara Camisaschi, Lucilla Luzi, PierGiuseppe Pelicci, Francesco Bertolini. A single-cell atlas of the effect of chemotherapeutics over intratumoral immune cells reveals that combining an alkylating agent and a vinca alkaloid can activate antigen presenting cells and increase tcf1+ stem-like CD8+ T-cells, thus improving anti-PD-1 efficacy in triple negative breast cancer and lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1653.

Abstract 1772: A novel approach for autologous pan-cancer cellular immunotherapy reveals dramatic expansion of ab and gd TCR T cell clonotypes indicative of an antigen-driven response

Abstract Introduction: We developed a unique autologous cellular therapeutic strategy using a proprietary engineered leukocyte stimulator cell lines (ENLIST cells) that express an array of immunomodulatory proteins to activate and expand tumor cell killing effector cells from human peripheral blood mononuclear cells (PBMCs). Our clinical aim is to use these expanded cells, called SUPLEXA therapy in cancer patients. Results of prior immunophenotyping of SUPLEXA cells by mass cytometry (CyTOF) reveals that they are comprised of NK cells, CD8+ T cells and γδ T cells that express markers indicative of cytolytic function, e.g. granzymes and perforin. Using an unbiased approach to better define the specificity of SUPLEXA, we performed TCR repertoire analysis using the iRepertoire and 10X Genomics RNA sequencing platforms. Results: SUPLEXA was prepared from healthy donor PBMCs. RNA was subsequently prepared from SUPLEXA cells and donor PBMCs to compare TCR αβ and γδ chain clonality and diversity using the iRepertoire DAM-PCR sequencing platform. SUPLEXA cells showed dramatic and significantly increased clonality among the 4 TCR chains that were analyzed as compared to normal PBMCs. We also observed a diversification of TCRαβ and TCRγδ sequences in SUPLEXA as compared to PBMCs. In addition, to the increased diversity, clonal expansion was also observed. Single-cell RNA sequencing of flow-sorted CD3+ T cells using the 10X Genomics platform with paired TCRαβ and TCRγδ PCR sequencing feature overlays revealed high frequencies of T cells with paired TCR clonotypes. We discovered that the top 5 paired TCRαβ clonotype sequences accounted for 32.7% of T cells, while CD8+ T cells from a normal donor contains only 3.0% in the top 5 clonotypes. Similar high frequency clonotypes were observed for γδ T cells by 10X single-cell RNA sequencing. Conclusions: The creation of SUPLEXA from healthy adult PBMCs results in dramatic expansion of αβ and γδ TCR T cell clonotypes. Given that SUPLEXA cells demonstrate broad, dose-dependent tumor cytolytic activity, these findings suggest that the expanded αβ and γδ T cell clonotypes may react to tumor neoantigens that are present during the activation process by ENLIST cells, which are derived from the SK-MEL-2 melanoma cell line. Accordingly, TCR reagents made using the paired αβ and γδ T cell sequences from the expanded clonotypes will be useful for future tumor neoantigen discovery. Plans for a phase 1 clinical trial are currently underway. Citation Format: Frank Borriello, Joshua W. Keegan, Brandon L. Hancock, Fei Guo, James R. Lederer. A novel approach for autologous pan-cancer cellular immunotherapy reveals dramatic expansion of ab and gd TCR T cell clonotypes indicative of an antigen-driven response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1772.