T-cell Clonotypes Research Articles

TIL the end: Tracking T cell clonotype dynamics during adoptive cell therapy

Understanding the factors that lead to the therapeutic success of adoptive cell therapies using tumor-infiltrating lymphocytes (TIL-ACT) will improve current treatment protocols. In this issue of Immunity, Chiffelle etal. comprehensively compare the dynamics of CD8+ Tcell clonotypes during the course of ACT between responding and non-responding patients.

Single cell and TCR analysis of immune cells from AAV gene therapy-dosed Duchenne muscular dystrophy patients

Clinical trials for Duchenne muscular dystrophy (DMD) are assessing the therapeutic efficacy of systemically delivered adeno-associated virus (AAV) carrying a modified DMD transgene. High vector doses (>1E14 vg/kg) are needed to globally transduce skeletal muscles; however, such doses trigger immune-related adverse events. Mitigating these immune responses is crucial for widespread application of AAV-based therapies. We used single-cell RNA-sequencing and T-cell receptor (TCR)-sequencing on peripheral blood mononuclear cells from five participants prior to, and after, dosing. One subject in the high-dose cohort experienced thrombotic microangiopathy (TMA). Few changes in cell frequencies occurred after treatment; however, differential gene expression demonstrated induction of interferon response genes in most T-cell types. T-cell clonotype and clumping analysis showed the expansion or appearance of groups of related TCR sequences in the post-treatment samples. Three of these expanded clumps could be assigned to prior Human Herpesvirus infections, two of which were present in the participant that exhibited TMA. These data provide insight on the mechanistic basis of human immune-AAV interactions and lay a foundation for improved understanding of why TMA arises in some patients and not others.

Personalized neoantigen vaccines as early intervention in untreated patients with lymphoplasmacytic lymphoma: a non-randomized phase 1 trial

Lymphoplasmacytic lymphoma (LPL) is an incurable low-grade lymphoma with no standard therapy. Nine asymptomatic patients treated with a first-in-human, neoantigen DNA vaccine experienced no dose limiting toxicities (primary endpoint, NCT01209871). All patients achieve stable disease or better, with one minor response, and median time to progression of 72+ months. Post-vaccine single-cell transcriptomics reveal dichotomous antitumor responses, with reduced tumor B-cells (tracked by unique B cell receptor) and their survival pathways, but no change in clonal plasma cells. Downregulation of human leukocyte antigen (HLA) class II molecules and paradoxical upregulation of insulin-like growth factor (IGF) by the latter suggest resistance mechanisms. Vaccine therapy activates and expands bone marrow T-cell clonotypes, and functional neoantigen-specific responses (secondary endpoint), but not co-inhibitory pathways or Treg, and reduces protumoral signaling by myeloid cells, suggesting favorable perturbation of the tumor immune microenvironment. Future strategies may require combinations of vaccines with agents targeting plasma cell subpopulations, or blockade of IGF-1 signaling or myeloid cell checkpoints.

Microbiota dictate T cell clonal selection to augment graft-versus-host disease after stem cell transplantation

Allogeneic Tcell expansion is the primary determinant of graft-versus-host disease (GVHD), and current dogma dictates that this is driven by histocompatibility antigen disparities between donor and recipient. This paradigm represents a closed genetic system within which donor Tcells interact with peptide-major histocompatibility complexes (MHCs), though clonal interrogation remains challenging due to the sparseness of the Tcell repertoire. We developed a Bayesian model using donor and recipient Tcell receptor (TCR) frequencies in murine stem cell transplant systems to define limited common expansion of Tcell clones across genetically identical donor-recipient pairs. A subset of donor CD4+ Tcell clonotypes differentially expanded in identical recipients and were microbiota dependent. Microbiota-specific Tcells augmented GVHD lethality and could target microbial antigens presented by gastrointestinal epithelium during an alloreactive response. The microbiota serves as a source of cognate antigens that contribute to clonotypic Tcell expansion and the induction of GVHD independent of donor-recipient genetics.

T-cell immunity induced and reshaped by an anti-HPV immuno-oncotherapeutic lentiviral vector

We recently developed an immuno-oncotherapy against human papillomavirus (HPV)-induced tumors based on a lentiviral vector encoding the Early E6 and E7 oncoproteins of HPV16 and HPV18 genotypes, namely “Lenti-HPV-07”. The robust and long-lasting anti-tumor efficacy of Lenti-HPV-07 is dependent on CD8+ T-cell induction and remodeling of the tumor microenvironment. Here, we first established that anti-vector immunity induced by Lenti-HPV-07 prime has no impact on the efficacy of a homologous boost to amplify anti-HPV T-cell immunity. To longitudinally monitor the evolution of the T-cell repertoire generated after the prime, homologous or heterologous boost with Lenti-HPV-07, we tracked T-cell clonotypes by deep sequencing of T-Cell Receptor (TCR) variable β and α chain mRNA, applied to whole peripheral blood cells (PBL) and a T cell population specific of an immunodominant E7HPV16 epitope. We observed a hyper-expansion of clonotypes post prime, accompanied by increased frequencies of HPV-07-specific T cells. Additionally, there was a notable diversification of clonotypes post boost in whole PBL, but not in the E7HPV16-specific T cells. We then demonstrated that the effector functions of such Lenti-HPV-07-induced T cells synergize with anti-checkpoint inhibitory treatments by systemic administration of anti-TIM3 or anti-NKG2A monoclonal antibodies. While Lenti-HPV-07 is about to enter a Phase I/IIa clinical trial, these results will help better elucidate its mode of action in immunotherapy against established HPV-mediated malignancies.

Characterization of T-cell receptor repertoire and correlation with tumor mutational landscape in lung cancer.

2577 Background: T-cell receptor (TCR) repertoire represents an overall immune condition which is closely related to pathogens- or tumor-associated T cell responses. Increasing evidence suggests that TCR repertoire is expected to undergo cancer-specific changes during tumorigenesis, supporting that TCR characteristics can serve as novel markers to indicate early cancer progression. Here, we performed a systematic T cell repertoire analysis in lung cancer. We identified cancer-enriched TCR signatures and constructed a comprehensive lung cancer database integrating TCR diversity, tumor mutational profiles, immune markers expression and HLA genotypes. Methods: A total of 988 tissue samples and 3,360 blood samples were obtained from patients with lung cancer, in addition to 2,699 blood samples from healthy individuals. Multiplex-PCR-based sequencing of the CDR3 regions of TCR-β chains was applied to the tissue and blood samples. TCR repertoires were analyzed using MiXCR and VDJtools. Whole exome sequencing was performed on partial tumor tissues to profile the mutational landscape and HLA genotypes. An in-house pipeline, considering the frequency and distribution of CDR3, was utilized to identify cancer-enriched CDR3 sequences. The PD-L1 expression was evaluated and quantified using CPS. Furthermore, an enrich score was developed to measure the content of cancer-related TCRs by aligning against the built cancer-enriched CDR3 dataset. Spearman's rank correlation was used to assess relationships between variables. Results: Lung cancer exhibited significantly lower Shannon index, Simpson index and evenness index in TCR repertoire (p<0.001), both in tissue and blood samples, when compared with healthy blood samples. Approximate 3% T-cell clonotypes within lung tissues were detected in blood TCR repertoires. A correlation was observed between the decreased TCR diversity and a higher tumor mutational burden (TMB)(r = -0.15, p < 0.01), as well as a higher variant allele frequency (VAF) (r = -0.19, p < 0.01). In this study, 3,652 and 3,840 CDR3 sequences were identified to be enriched in lung cancer tissue and blood respectively. A significant difference in cancer-enriched score was observed between cancer and healthy TCR repertoires (p < 0.001). The cancer-enriched score showed a significant positive correlation with TMB, particularly in TP53- mutant tumors (r = 0.27, p < 0.001). A higher CPS (PD-L1) was associated with a higher cancer-enriched score (r = 0.18 ,p < 0.01). Conclusions: Cancer-related T-cell clonotypes in the tumor microenvironment and peripheral blood informs the changes in anti-tumor responses, supporting the potential application of TCR signatures in early cancer detection. This study identified the lung cancer-related TCR signatures and provided a comprehensive lung cancer TCR database, revealing the relationship between TCR characteristics and mutational profiles. Clinical trial information: ChiCTR2200055761.

The effect of distance reiki on patient reported quality of life, correlated with changes in immune repertoires among patients with multiple myeloma: A pilot study.

e23199 Background: Psychological distress and diminished health related quality of life (HRQOL) is associated with multiple myeloma (MM). Complementary and alternative medicine (CAM) is an adjunct to traditional medicine. T-cells are essential to the adaptive immune system. The complementarity determining region (CDR3) of the T cell receptor formed by combination of the V,D,J regions is highly variable and critical for antigen specificity. PCR is used to analyze the TCR-CDR3 to monitor T-cell clonotypes as a measure of antigen specific T cell responses and thereby cancer immunity. Reiki is a Japanese energy healing technique involving noninvasive light touch to modulate energy fields. The primary aim was feasibility and acceptability of Reiki within a structured medical paradigm. Secondary aims were changes in HRQOL and T-cell receptor repertoires with Reiki. Methods: Adult MM patients (pts) on a stable regimen were eligible. Pts randomized in a 1:1:1 to weekly True Reiki (Treiki), Simulated Reiki (Sreiki) or no intervention (NI). The PROMIS-29® assessed HRQOL. A “Was it Worth it” (WIWI) questionnaire was administered post intervention to assess the experience. TCR-CDR3 sequencing was performed on PBMCs at enrollment and end of therapy to determine immune repertoire changes. Samples underwent library preparation and next generation sequencing for TCR repertoire analysis by iRepertoire® Results: Thirty pts were randomized into 3 arms between 12/2020 to 4/2022. Median age 62.5 (range 27-76), 18/30 (60%) female, median time to diagnosis was 48 mo (Range 7-221), 50% had high risk FISH,(90%) had prior ASCT and received median 2 (range 1-13) lines of therapy prior to enrollment. No pts relapsed or progressed during study period. Using WIWI 100% of pts in Treiki and 75% in Sreiki arms found the therapy useful and would do it again; 100% Treiki pts and 87.5% in the Sreiki arm would recommend the study to others. No statistical difference between groups in any HRQOL domains. Among TCR repertoires, no statistically significant difference was observed between groups with respect to Richness, Shannon entropy, Clonality, Diversity 50 (D50) and Inverse Simpson indexes. Each pt showed substantial variability in certain TCR-CDR3 clones and clonal diversity. Conclusions: We successfully demonstrated acceptability and feasibility of Reiki among MM pts, although no impact on HRQOL was shown. Reiki intervention is an easy non-invasive additional option for pts’ to their conventional therapies. The TCR-CDR3 repertoires showed no difference with the reiki intervention but had significant variability. Further research comparing TCR repertoires between MM and age/sex matched healthy controls is ongoing. T-cell directed therapies are evolving in MM, we plan to analyze if changing diversity of TCR-CDR3 repertoires could define how best to sequence these treatments. Clinical trial information: NCT04783038 .

Abstract 5006: Discovery of immunogenic neo-peptides from actionable RNA fusions for developing cancer vaccines

Abstract The global surge in cancer cases, with a staggering 20 million new instances reported in 2023 alone, underscores an urgent demand for advanced and reliable cancer therapeutics. Immunotherapies centered around neoantigens have emerged as a promising avenue for enhancing treatment efficacy and have become a focal point in cancer research. We hypothesize that the tumor cells present a rich neoantigenic repertoire and the immunogenic neoantigens arising from the junctions of proteins translated from chimeric RNAs present viable targets for peptide and mRNA vaccines, offering a promising approach to impede tumor progression. To validate our hypothesis, we acquired a dataset of RNA fusions detected through RNAseq of tissue extracted from patient-derived xenograft (PDX) models established by XenoSTART, a global non clinical oncology contract research organization. This comprehensive dataset encompassed 985 PDX models across 27 cancer types, revealing three fusions associated with the neuregulin-1 (NRG1) gene. NRG1 gene fusions have been identified as a clinically actionable target for multiple solid tumors as they result in ErbB-mediated pathway activation. CD74-NRG1 has emerged as the most frequently reported NRG1 fusion across many cancers such as pancreatic ductal adenocarcinoma, triple negative breast cancer and ovarian cancer. The CD74-NRG1 fusion was identified in two PDX models harboring lung cancer (ST2891 and ST3204) and in an ovarian cancer model (ST088). The lung cancer PDXs displayed a notable abundance of fusion junction-crossing reads. We generated the CD74 (Exon 1-6) -NRG1 (Exon 6-12) fusion transcript model, where the first 6 exons of CD74 fused to exon 6 of NRG1 leading to an in-frame fusion. Validation of this fusion in PDX tissue was accomplished through PCR and Sanger sequencing of the PCR product. We assessed the affinity of the 9-mer neo peptide sequences formed by the translated junction of CD74-NRG1 to Major Histocompatibility Complex (MHC) Class I molecules using the in-silico prediction pipelines MHCNuggets and MixMHCPred-2 and found HLA-A*68:23 to be the MHC allele with highest binding affinity. HLA-Arena was used to investigate the binding affinity between the peptides and the MHC molecules via molecular docking, which revealed 4 peptides to be strong binders to HLA-A*68:23.The immunogenicity of these neoantigenic peptides to HLA-A*68:23 matched Peripheral Blood Mononuclear Cells (PBMCs) will be assessed by the IFN-γ Enzyme Linked Immunosorbent Spot (ELISpot) assay. To elucidate the specific T-cell clonotypes responding to the neo peptides, the 10X Genomics single-cell 5’ Gene Expression Assay, coupled with T Cell Receptor mapping, will be employed. The objective of the study is to establish a robust combinatorial therapeutic strategy leveraging peptide and mRNA vaccine technology to support an effective framework for personalized cancer treatment. Citation Format: Sakuni Rankothgedera, Micah Castillo, Shiyanth Thevasagayampillai, Cole Woody, Aaranyah Kandasamy, Armando Diaz, Michael Wick, Preethi Gunaratne. Discovery of immunogenic neo-peptides from actionable RNA fusions for developing cancer vaccines [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 5006.

Bivalent mRNA COVID vaccines elicit predominantly cross-reactive CD4+ T cell clonotypes

Bivalent COVID vaccines containing mRNA for ancestral and Omicron BA.5 spike proteins do not induce stronger Tcell responses to Omicron BA.5 spike proteins than monovalent vaccines that contain only ancestral spike mRNA. The reasons for this finding have not been elucidated. Here, we show that healthy donors (HDs) and people living with HIV (PLWH) on antiretroviral therapy mostly target Tcell epitopes that are not affected by BA.5 mutations. We use the functional expansion of specific Tcells (FEST) assay to determine the percentage of CD4+ Tcells that cross-recognize both spike proteins and those that are monoreactive for each protein. We show a predominance of cross-reactive CD4+ Tcells; less than 10% percent of spike-specific CD4+ Tcell receptors were BA.5 monoreactive in most HDs and PLWH. Our data suggest that the current bivalent vaccines do not induce robust BA.5-monoreactive Tcell responses.

Perturbations of the T-cell receptor repertoire in response to SARS-CoV-2 in immunocompetent and immunocompromised individuals

Functional T-cell responses are essential for virus clearance and long-term protection after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, whereas certain clinical factors, such as older age and immunocompromise, are associated with worse outcome. We sought to study the breadth and magnitude of T-cell responses in patients with coronavirus disease 2019 (COVID-19) and in individuals with inborn errors of immunity (IEIs) who had received COVID-19 mRNA vaccine. Using high-throughput sequencing and bioinformatics tools to characterize the T-cell receptor β repertoire signatures in 540 individuals after SARS-CoV-2 infection, 31 IEI recipients of COVID-19 mRNA vaccine, and healthy controls, we quantified HLA class I- and class II-restricted SARS-CoV-2-specific responses and also identified several HLA allele-clonotype motif associations in patients with COVID-19, including a subcohort of anti-type 1 interferon (IFN-1)-positive patients. Our analysis revealed that elderly patients with COVID-19 with critical disease manifested lower SARS-CoV-2 T-cell clonotype diversity as well as T-cell responses with reduced magnitude, whereas the SARS-CoV-2-specific clonotypes targeted a broad range of HLA class I- and class II-restricted epitopes across the viral proteome. The presence of anti-IFN-I antibodies was associated with certain HLA alleles. Finally, COVID-19 mRNA immunization induced an increase in the breadth of SARS-CoV-2-specific clonotypes in patients with IEIs, including those who had failed to seroconvert. Elderly individuals have impaired capacity to develop broad and sustained T-cell responses after SARS-CoV-2 infection. Genetic factors may play a role in the production of anti-IFN-1 antibodies. COVID-19 mRNA vaccines are effective in inducing T-cell responses in patients with IEIs.

Abstract A001: Reshaping the immune microenvironment after temozolomide priming in metastatic colorectal cancer patients in ARETHUSA clinical trial

Abstract Mismatch repair (MMR) proficient (MMRp) tumors, known for their weak immune response, often exhibit limited effectiveness in immunotherapy. In contrast, responsive mismatch repair deficient (MMRd) tumors, characterized by heightened immune activity, show a stronger response to immune checkpoint blockade (ICB). In the ARETHUSA clinical trial (NCT03519412), we revealed that treatment with temozolomide (TMZ) can pharmacologically deactivate the MMR machinery, as indicated by mutational signature analysis and the emergence of MMR gene mutations. We focused our investigation on a subset of initially MMRp CRC patients who experienced sustained disease stabilization with ICB after receiving TMZ priming treatment. This treatment resulted in the emergence of an inactivating MSH6 mutation and the TMZ mutational signature. Additionally, TMZ treatment induced diverse genomic changes, leading to the identification of three distinct subtypes through analysis of blood and tissue samples. Notably, we observed a dose-dependent accumulation of mutations with a specific molecular signature visible at the clonal level (subtype B2) in 10% of patients, at the subclonal level (subtype B1) in 71% of patients, and the absence of these mutations (subtype A) in 19% of patients within our cohort of 21 patients. In conjunction with previous genetic findings, in this new study we performed an analysis of T-cell receptors (TCRs) in the tumor microenvironment following TMZ treatment. Our results revealed expanded clonotypes in tumors that exhibited a clonal increase in mutations following priming treatment. Furthermore, the diversity of TCRs within the immune infiltrate confirmed the categorization of tumors into three distinct classes based on Tumor Mutational Burden (TMB) and mutational signatures. We also established a significant linear correlation (p-value 0.0048) between the diversity of T-cells in the microenvironment and the number of mutations induced by TMZ. Based on these clinically-oriented preliminary findings, we propose that increasing the number of TMZ-induced mutations could enhance the likelihood of TMZ-driven neoantigens triggering clonal expansion of specific T-cell clonotypes within the tumor. Interestingly, the subset of TMZ-treated tumors displaying an acquired MSH6 mutation, TMZ mutational signature, and increased TMB not only achieved temporary disease stabilization with ICB but also exhibited clonal expansion of T-cells in the tumor microenvironment. These clinically relevant findings suggest that the inactivation of MMR achieved through TMZ priming has the potential to reshape the immune microenvironment modulating the immune response in metastatic colorectal cancer. Citation Format: Giovanni Crisafulli, Andrea Sartore-Bianchi, Luca Lazzari, Filippo Pietrantonio, Paolo Battuello, Alice Bartolini, Federica Di Nicolantonio, Silvia Marsoni, Salvatore Siena, Alberto Bardelli. Reshaping the immune microenvironment after temozolomide priming in metastatic colorectal cancer patients in ARETHUSA clinical trial [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr A001.

Early Intervention and Favorable Biologic Effects of Personalized Neoantigen Vaccines on the Tumor Immune Microenvironment in Smoldering Waldenstrom Macroglobulinemia

Lymphoplasmacytic lymphoma (LPL) is an incurable low-grade B-cell lymphoma, characterized by the presence of clonal tumor cells infiltrating the bone marrow. Despite a cumulative risk of progression, there is no approved therapy for patients in the asymptomatic phase. We conducted a first-in-human clinical trial of a novel individualized therapeutic DNA idiotype neoantigen vaccine as an early intervention in nine patients with untreated asymptomatic LPL. The vaccine platform encoded the autologous LPL-derived Ig single chain variable fragment fused to human chemokine CCL20 (macrophage inflammatory protein-3, MIP-3a), which was designed to trigger T-cell immunity by targeting delivery of the expressed fusion protein to antigen presenting cells. The vaccine was well tolerated with no dose limiting toxicities. One patient achieved a minor response, and all remaining patients experienced stable disease, with median time to disease progression of 61+ months, which compares favorably to median time to progression of 3.9 years in published series. Direct interrogation of the tumor microenvironment by single-cell transcriptome analysis revealed explanations for lack of more robust objective clinical responses. Comparing paired pre- and post- vaccine bone marrow samples, we observed a striking dichotomous pattern of significantly reduced numbers of clonal tumor cells, identified by their unique BCR sequences, and downregulation of genes involved in signaling pathways critical for B-cell survival among the mature B cells subpopulation post-vaccine in the majority of patients, but no change in clonally related plasma cell-like clusters of any patient (Figure 1). Downregulation of HLA molecule expression suggested intrinsic resistance by tumor plasma cell subpopulations and cell-cell interaction analyses predicted paradoxical upregulation of IGF signaling post vaccine by plasma cell, but not mature B-cell subpopulations, suggesting a potential mechanism of acquired resistance. Vaccine therapy induced dynamic changes in bone marrow T-cells, including upregulation of signaling pathways involved in T-cell activation, expansion of T-cell clonotypes (Figure 2), increased T-cell clonal diversity, and functional tumor idiotype-specific cytokine production, with little change in co-inhibitory pathways or Treg. Vaccine therapy also globally altered cell-cell communication networks across various bone marrow cell types. CellChat analysis inferred significant downregulation of signaling pathways post-vaccine that likely directly promote growth of LPL cells, such as APRIL and IL-6 which are known to promote B- or plasma cell survival. Other pathways were reduced post-vaccine, such as RESISTIN, which has a known role in supporting proliferation of solid cancers. A role for RESISTIN in supporting LPL has not been previously inferred, but it has been reported to induce multidrug resistance in multiple myeloma. Bioinformatic analysis also identified a predominant role for myeloid cells in the tumor microenvironment as a source of vaccine-induced, downregulated pro-tumoral signaling to LPL cells. The pathways affected were primarily associated with monocytic, rather than granulocytic or dendritic cell subpopulations, particularly non-classical CD14 -CD16 + monocytes, and to a lesser extent classical CD14 +CD16 - monocytes . Recent reports indicate that an increased pro-inflammatory myeloid signature is an early step in the development of LPL and in monoclonal gammopathy of undetermined significance. Taken together, these results suggest that this prototype neoantigen vaccine favorably perturbed the tumor immune microenvironment, resulting in reduction of clonal tumor mature B-cell, but not plasma cell subpopulations. Future functional studies of the pathways affected are needed to confirm mechanisms of resistance elucidated and to design combination strategies to circumvent them. Such strategies could include adding IFN gamma or epigenetic drugs, designed to increase HLA molecule expression on plasma cell-like LPL subpopulations and combining neoantigen vaccines with agents that specifically target plasma cells or pathways known to promote their growth, such as IGF-1 receptor inhibitors. Finally, our data suggest that combinations of these vaccines with myeloid cell checkpoint blockade may be worthwhile.

T cell receptor sequences are the dominant factor contributing to the phenotype of CD8+ T cells with specificities against immunogenic viral antigens

Antigen-specific CD8+ Tcells mediate pathogen clearance. Tcell phenotype is influenced by Tcell receptor (TCR) sequences and environmental signals. Quantitative comparisons of these factors in human disease, while challenging to obtain, can provide foundational insights into basic Tcell biology. Here, we investigate the phenotype kinetics of 679 CD8+ Tcell clonotypes, each with specificity against one of three immunogenic viral antigens. Data were collected from a longitudinal study of 68 COVID-19 patients with antigens from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), cytomegalovirus (CMV), and influenza. Each antigen is associated with a different type of immune activation during COVID-19. We find TCR sequence to be by far the most important factor in shaping Tcell phenotype and persistence for populations specific to any of these antigens. Our work demonstrates the important relationship between TCR sequence and Tcell phenotype and persistence and helps explain why Tcell phenotype often appears to be determined early in an infection.

Peripheral T-Cell Priming and Micrometastatic Disease Control with Metastasis-Directed Therapy: Multidimensional Immunogenomic Profiling of Oligometastatic Prostate Cancer in the EXTEND Trial

Comprehensive metastasis-directed therapy (MDT) for oligometastatic prostate cancer extended progression-free survival (PFS) and time to new lesion formation in the intermittent hormone therapy (HT) basket of EXTEND. To better understand the mechanism of MDT benefit, we pooled the intermittent and continuous HT baskets of EXTEND and tested the hypothesis that adding MDT to HT would program systemic T-cells to control micrometastatic disease. A total of 174 men were randomized to HT with or without MDT to up to 5 sites of metastases. HT was given for 6 months (intermittent basket, n = 87) or indefinitely (continuous basket, n = 87). Peripheral blood samples were drawn at enrollment, at the end of MDT, at 3 months follow-up (3 mo F/U), and at progression and then analyzed by flow cytometry, T-cell receptor (TCR)-β CDR3 variable region sequencing, multiplex cytokine profiling, and next-generation circulating tumor DNA (ctDNA) sequencing. TCR clonal expansion was determined using a published betabinomial model. Repertoire changes were assessed by Morisita's index, and dominant TCR repertoire motifs were characterized with ImmunoMap. Associations between blood markers and PFS were evaluated with Cox regression adjusted hazard ratios (aHR) accounting for randomization arm and stratifying for intermittent vs continuous HT. Randomization to MDT+HT was associated with T-cell activation, proliferation, and clonal expansion. This response was first observed at end-MDT as upregulated expression of T-cell activation and inhibition markers (i.e., ICOS, Tim-3, and LAG-3) and increases in highly proliferative CD4+ and CD8+ Ki67hi T-cells (all P<0.05). TCR sequencing of 7,678,911 T-cells revealed that MDT+HT was associated with TCR clonal expansion, remodeling of the TCR repertoire, and changes in dominant TCR motifs at end-MDT and 3 mo F/U (all P<0.05). Observed T-cell priming could be driven by signaling networks of canonical T-cell stimulatory cytokines (IL-2, IL-12, and IL-15), which were upregulated at end-MDT and persisted at 3 mo F/U (all P<0.05). This modulation of T-cell phenotype, clonotype, and cytokine concentrations was not observed in the HT-monotherapy arm. At end-MDT, systemic T-cell responses were associated with improved PFS, most notably CD8+ T-cell expression of LAG-3 (aHR 0.22, 95% CI 0.03-0.91) and high TCR clonal expansion (aHR 0.13, 95% CI 0.02-0.52). High ctDNA burden at end-MDT correlated with worse PFS (aHR 1.41, 95% CI 1.04-2.54), as did CD8+ T-cell expression of inhibitory receptor TIGIT at 3 mo F/U (aHR 1.03, 95% CI 1.01-1.06). The addition of MDT to HT induced systemic T-cell activation and expansion, which was not observed in the HT-only arm. This systemic immune response was independently associated with improved PFS. In addition to cytoreduction of macroscopic disease, MDT-induced immune education may be an important complementary mechanism of micrometastatic control in oligometastatic prostate cancer.

Single-cell sequencing on CD8+ TILs revealed the nature of exhausted T cells recognizing neoantigen and cancer/testis antigen in non-small cell lung cancer

BackgroundCD8+tumor infiltrating lymphocytes (TILs) are often observed in non-small cell lung cancers (NSCLC). However, the characteristics of CD8+ TILs, especially T-cell populations specific for tumor antigens, remain poorly understood.MethodsHigh throughput...

Virus-Specific T Cells From Cryopreserved Blood During an Emergent Virus Outbreak for a Potential Off-the-Shelf Therapy

During the first outbreak of an emergent virus, methods need to be developed to rapidly establish suitable therapies for patients with high risk of severe disease caused by the pathogen. Considering the importance of the T-cell response in controlling viral infections, adoptive cell therapy with virus-specific T cells has been used as a safe and effective antiviral prophylaxis and treatment for immunocompromised patients. The main objective of this study was to establish an effective and safe method to cryostore whole blood as starting material and to adapt a T-cell activation and expansion protocol to generate an off-the-shelf antiviral therapeutic option. Additionally, we studied how memory T-cell phenotype, clonality based on T-cell receptor, and antigen specificity could condition characteristics of the final expanded T-cell product. Twenty-nine healthy blood donors were selected from a database of convalescent plasma donors with a confirmed history of SARS-CoV-2 infection. Blood was processed using a fully automated, clinical-grade, and 2-step closed system. Eight cryopreserved bags were advanced to the second phase of the protocol to obtain purified mononucleated cells. We adapted the T-cell activation and expansion protocol, without specialized antigen-presenting cells or presenting molecular structures, in a G-Rex culture system with IL-2, IL-7, and IL-15 cytokine stimulation. The adapted protocol successfully activated and expanded virus-specific T cells to generate a T-cell therapeutic product. We observed no major impact of post-symptom onset time of donation on the initial memory T-cell phenotype or clonotypes resulting in minor differences in the final expanded T-cell product. We showed that antigen competition in the expansion of T-cell clones affected the T-cell clonality based on the T-cell receptor β repertoire. We demonstrated that good manufacturing practice of blood preprocessing and cryopreserving is a successful procedure to obtain an initial cell source able to activate and expand without a specialized antigen-presenting agent. Our 2-step blood processing allowed recruitment of the cell donors independently of the expansion cell protocol timing, facilitating donor, staff, and facility needs. Moreover, the resulting virus-specific T cells could be also banked for further use, notably maintaining viability and antigen specificity after cryopreservation.

A prospective non-randomized phase I trial of dendritic cell–based cryoimmunotherapy combined with checkpoint inhibitors in mCRPC.

e14708 Background: Despite improvements, metastatic castration-resistant prostate cancer (mCRPC) still has dismal overall survival (OS). We treated 18 mCRPC patients with dendritic cell (DC)-based cryoimmunotherapy (CryoIT) as monotherapy or combined with checkpoint inhibitors to determine safety and immune response. Methods: mCRPC patients were biopsied before cryoablation, and subsequently injected intratumorally with autologous immature DCs. DC doses were escalated in a 3+3 design (n=9) followed by dose expansion combined with either ipilimumab (n=6) or pembrolizumab (n=3). The disease burden was assessed by PSA values, iRECISTv1.1 (PET/CT, MRI and bone scintigraphy at baseline, 14, 22 and 46 weeks) and circulating tumor cells (CTC; CellSearch). Immune response was monitored by ultradeep T-cell receptor sequencing. Results: The patients had aggressive cancer, with ISUP grade group 4 and 5 in 72% (13/18) at diagnosis and in 100% at inclusion. At baseline, higher ALP correlated with higher regulatory T cells (p=0.047), FoxP3+/CD3+ratios (p=0.014) and FoxP3+/CD8+ratios (p=0.004) in tissues. Median OS and progression-free survival (PFS) were 40.7 and 10.5 months, respectively. Of the 8 patients alive 43 to 85 months post-CryoIT, 7 had progressed and received other treatment. Early decreases in PSA (p=0.002) and LDH (p=0.01) levels 6 weeks after CryoIT indicated better outcomes at 22 weeks. Longer OS correlated with lower tissue ratios of CD4+/CD3+cells (p=0.002) and CD4+/CD8+cells (p=0.007). All patients with increased numbers of new T-cell clonotypes after CryoIT and CTC at the pretreatment level of 1-4 cells/ 7.5 mL blood changed to negative during follow-up up to 72 weeks. Conclusions: CryoIT demonstrates non-inferior OS and PFS compared to other mCRPC trials, and therapy response was suggested by decreased CTCs and induction of new T-cell clonotypes post-CryoIT. Lower CD4+cell fractions of lymphocytes in the TME indicate better OS after CryoIT. CryoIT appears not to decrease effects of post-treatment recurrence therapies. Clinical trial information: NCT02423928 . [Table: see text]

Cell surface marker-based capture of neoantigen-reactive CD8+ T-cell receptors from metastatic tumor digests

BackgroundCellular immunotherapies using autologous tumor-infiltrating lymphocytes (TIL) can induce durable regression of epithelial cancers in selected patients with treatment-refractory metastatic disease. As the genetic engineering of T cells with tumor-reactive...

Lineage tracing reveals clonal progenitors and long-term persistence of tumor-specific T cells during immune checkpoint blockade

Paired single-cell RNA and Tcell receptor sequencing (scRNA/TCR-seq) has allowed for enhanced resolution of clonal Tcell dynamics in cancer. Here, we report a scRNA/TCR-seq analysis of 187,650 Tcells from 31 tissue regions, including tumor, adjacent normal tissues, and lymph nodes (LN), fromthree patients with non-small cell lung cancer after immune checkpoint blockade (ICB). Regions with viable cancer cells are enriched for exhausted CD8+ Tcells, regulatory CD4+ Tcells (Treg), and follicular helper CD4+ Tcells (TFH). Tracking Tcell clonotypes across tissues, combined with neoantigen specificity assays, reveals that TFH and tumor-specific exhausted CD8+ Tcells are clonally linked to TCF7+SELL+ progenitors in tumor draining LNs, and progressive exhaustion trajectories of CD8+ T, Treg, and TFH cells with proximity to the tumor microenvironment. Finally, longitudinal tracking of tumor-specific CD8+ and CD4+ Tcell clones reveals persistence in the peripheral blood for years after ICB therapy.

Anti-tumor memory CD4 and CD8 T-cells quantified by bulk T-cell receptor (TCR) clonal analysis.

Simple, reliable methods to detect anti-tumor memory T-cells are necessary to develop a clinical tumor vaccination program. A mouse model of curative viral onco-immunotherapy found that peritoneal tumor challenge following cure identified an oligoclonal anti-tumor memory CD4 and CD8 T-cell response. Clonotypes differed among the challenged animals but were congruent in blood, spleen and peritoneal cells (PC) of the same animal. Adoptive transfer demonstrated that the high-frequency responding T-cells were tumor specific. Tetramer analysis confirmed that clonotype frequency determined by T-cell receptor (TCR)- chain (TRB) analysis closely approximated cell clone frequency. The mean frequency of resting anti-tumor memory CD4 T-cells in unchallenged spleen was 0.028% and of memory CD8 T-cells was 0.11% which was not high enough to distinguish them from background. Stimulation produced a mean ~10-fold increase in splenic and 100-fold increase in peritoneal anti-tumor memory T-cell clonotypes. This methodology can be developed to use blood and tissue sampling to rapidly quantify the effectiveness of a tumor vaccine or any vaccine generating therapeutic T-cells.