T-cell Receptor Repertoire Analysis Research Articles

Abstract A075: Circulating T-cell receptor repertoire analysis improves cancer early detection

Abstract Introduction: Cancer screening by liquid biopsy promises to detect cancer early when it can be cured. However, current ctDNA-based approaches detect only a minority of early-stage cancers. For liquid biopsy to realize its full potential for cancer early detection, sensitivity for earliest stage disease must be improved. Methods: We set out to improve sensitivity of liquid biopsy by exploiting tumor recognition by T cells through sequencing of the circulating T-cell receptor repertoire. Using gDNA extracted from blood buffy coats, we studied a cohort of 471 lung cancer patients (85% stage I) and 600 subjects without cancer enriched for older individuals with a history of smoking. We performed TCR beta chain sequencing to yield a median of 101,899 TCR clonotypes per sample (median 81,602 cancer / 112,966 controls) and built a TCR sequence similarity graph to cluster clonotypes into TCR repertoire functional units (RFUs). The TCR frequencies of RFUs were tested for association with cancer status and significantly associated RFUs were combined using an SVM model into a cancer score. Cancer RFU levels were also correlated to imputed subject HLA types for 32 HLA Class I and 38 Class II alleles. The model was evaluated by 10-fold cross-validation and compared to a ctDNA panel of 237 mutation hotspots in 154 lung cancer driver genes and to 17 lung cancer related protein biomarkers in 86 subjects. Results: We identified 372 cancer-associated TCR RFUs at FDR≤0.1, including 198 enriched in cancer samples (fold-change range 1.03 to 3.13) and 174 enriched in controls (fold- change 0.96 to 0.30). Levels of 265/372 (71%) RFUs were correlated to presence of an HLA allele at FDR ≤ 0.1. Of the total 2,770 significant HLA-RFU associations, 479 (17%) involved an HLA Class I allele, while 2,291 (83%) involved an HLA Class II allele, highlighting a potential role for CD4 helper or regulatory T cell antigen recognition in the cancer RFUs found. The RFU cancer score achieved a cross-validated test ROC AUC of 0.71 for cancer status prediction (0.71 Stage I / 0.70 Stage II-IV) with 49% of Stage I cancers detected at a specificity of 80%. Importantly, the cancer score was not dependent on sample source site, technical or biological variation in TCR repertoire depth, or lung cancer histology subtype. The TCR RFU score also distinguished lung cancer patients from control subjects with other conditions such as benign lung nodules and COPD. We observed an up to ∼20%-point gain in sensitivity for stage I cancer when TCR RFUs were added to ctDNA and proteins in a potential multi-analyte cancer screening test. By contrast, TCR analysis did not provide additional sensitivity for stage II-IV disease. Conclusion: We demonstrate detection of a significant fraction of early lung cancer cases from blood by analyzing the circulating TCR repertoire and show that this signal is complementary to established analytes such as proteins and ctDNA. A similar study for the detection of early colorectal cancer and advanced adenomas is under way. Citation Format: Yilong Li, Michelle Nahas, Dennis Stephens, Kate Froburg, Emma Hintz, Devin Champagne, Amaneet Lochab, Markus Brown, Jasper Braun, María Antonia Fortuño, María-del-Mar Ocón, Andrea Pasquier, Inés María Luque, Christopher W. Seder, Jeffrey A. Borgia, Luis Miguel Seijo, Luis M Montuenga, Roman Yelensky. Circulating T-cell receptor repertoire analysis improves cancer early detection [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A075.

T-cell Receptor Repertoire Analysis in the Context of Transarterial Chemoembolization Synergy with Systemic Therapy for Hepatocellular Carcinoma

T-cell Receptor Repertoire Analysis in the Context of Transarterial Chemoembolization Synergy with Systemic Therapy for Hepatocellular Carcinoma

Characterization of the T-cell receptor repertoire associated with lymph node metastasis in colorectal cancer

PurposeColorectal cancer (CRC) is a leading cause of cancer-related mortality worldwide, with lymph node (LN) metastasis playing a pivotal role in disease progression. This study aimed to explore the T-cell receptor (TCR) repertoire among CRC patients, distinguishing those with LN metastasis from those without, in order to uncover potential biomarkers for predicting metastasis.MethodsWe analyzed the TCR repertoire in CRC patients with and without LN metastasis. A classification model utilizing random forest analysis was developed to assess the predictive potential of the TCR repertoire.ResultsThe findings demonstrated a significant increase in the number of V-J combinations and immune CDR3 sequences in patients with LN metastasis compared to the control group. The classification model achieved high accuracy in differentiating patients with LN metastasis, with AUC values ranging from 0.514 to 0.794. Specific V-J combinations and CDR3 sequences were identified as significant predictors of the model’s predictive accuracy.ConclusionThese results suggest that the TCR repertoire is altered in CRC patients exhibiting LN metastasis, potentially influencing disease progression. This study highlights the importance of TCR repertoire analysis as a non-invasive biomarker for predicting LN metastasis in CRC patients.

Unravelling T-cell dynamics and immune responses in initial and recurrent uveitis.

This study aimed to identify novel serological targets and investigate immune responses in patients with non-infectious uveitis, focusing on differences between initial onset and recurrent episodes. Differential gene expression analysis, immunocyte typing and T-cell receptor (TCR) gene analysis were conducted on RNA-sequenced peripheral blood samples from healthy individuals (n = 6) and non-infectious uveitis patients (n = 12), divided into 6 patients each at initial onset and recurrent stages. Peripheral blood T-cell types were analysed using flow cytometry. Bioinformatics methods included tools for RNA sequencing data processing, CIBERSORT for immune cell type prediction and specialized software for TCR repertoire analysis. Findings indicated that individuals with recurrent uveitis demonstrated a stronger adaptive immune response and a more pronounced immune imbalance compared to those with initial onset. Memory T cells were predominant in recurrent episodes, suggesting their potential role as biomarkers for disease progression. Significant differences in TCR diversity and V(D)J gene usage were observed between the various uveitis groups and healthy controls. Importantly, 38 uveitis-specific TCR sequences showed substantial expansion in the uveitis patients compared to controls. An elevated expansion of these specific TCR sequences was associated with an increased risk of uveitis development. The study highlights the critical role of adaptive immune responses and specific immune cell types in the pathogenesis of recurrent uveitis. Identification of the uveitis-specific TCR repertoire set could provide deeper insights into the disease and facilitate the development of targeted therapies for uveitis patients.

Predicting Antigen-Specificities of Orphan T Cell Receptors from Cancer Patients with TCRpcDist.

Approaches to analyze and cluster T-cell receptor (TCR) repertoires to reflect antigen specificity are critical for the diagnosis and prognosis of immune-related diseases and the development of personalized therapies. Sequence-based approaches showed success but remain restrictive, especially when the amount of experimental data used for the training is scarce. Structure-based approaches which represent powerful alternatives, notably to optimize TCRs affinity toward specific epitopes, show limitations for large-scale predictions. To handle these challenges, TCRpcDist is presented, a 3D-based approach that calculates similarities between TCRs using a metric related to the physico-chemical properties of the loop residues predicted to interact with the epitope. By exploiting private and public datasets and comparing TCRpcDist with competing approaches, it is demonstrated that TCRpcDist can accurately identify groups of TCRs that are likely to bind the same epitopes. Importantly, the ability of TCRpcDist is experimentally validated to determine antigen specificities (neoantigens and tumor-associated antigens) of orphan tumor-infiltrating lymphocytes (TILs) in cancer patients. TCRpcDist is thus a promising approach to support TCR repertoire analysis and TCR deorphanization for individualized treatments including cancer immunotherapies.

Abstract PO1-25-06: Integrated analysis of single-cell RNA and VDJ-sequencing data to identify T cell marker expressed on antigen-specific T cells

Abstract Background In recent years, the advancement of adoptive cell transfer-based immunotherapies has significantly contributed to the evolution of cancer treatment. The crucial step in achieving successful treatment lies in the identification of T cells that exhibit reactivity towards tumor-specific antigens. However, the heterogeneous composition of tumor antigen-specific T cells poses a challenge as there are no standardized biomarkers available for their identification. To overcome this limitation, we investigated alternative approaches, employing single cell RNA and VDJ sequencing to identify antigen-specific T cells. Methods First, peripheral blood mononuclear cells (PBMCs) obtained from healthy human donors were stained with carboxyfluorescein succinimidyl ester (CFSE) to facilitate the identification of proliferating antigen-specific T cells. Then the PBMCs were exposed to 1 ug/ml of CMV pp65 peptide. After a 2-day incubation period with the CMV peptide, interleukin-2 (IL-2) was administered to stimulate T cell expansion. At various time points (0, 1, 2, 3, 5, and 7 days) following the CMV peptide treatment, CD8+ T cells were isolated through fluorescence-activated cell sorting (FACS) analysis. On day 7, the CD8+ T cells were further segregated into CFSE- and CFSE+ cell populations. The single-cell transcriptomes and T cell receptor (TCR) repertoire of the CD8+ T cells were subsequently examined using the Chromium Single Cell Reagent Kit. Results The TCR repertoire analysis of CD8+ CFSE- T cells revealed the dominance of 10 major clones, accounting for 93.9% of the total cells. These clones were confirmed to be CMV-specific through various in vitro assays, including NFAT-luciferase, IFN-γ ELISA, and cytotoxicity assay. In contrast, CD8+ CFSE+ T cells did not exhibit significant overlap with the major TCR clones found in CFSE- CD8+ T cells, comprising only 0.02% of the total cells. Gene set enrichment analysis demonstrated that the upregulated genes in CFSE- CD8+ T cells, when compared to CFSE+ T cells, were related to cell cycle proliferation and T cell cytotoxicity. Furthermore, these genes exhibited a matching upregulation pattern with CFSE- T cells from another batch of the same donor, as well as from two other healthy donors. In addition, there was a moderate increase in the expression of the upregulated genes in CFSE- CD8+ T cells at day 3 compared to day 0-2. In this end, we selected a set of nine genes that were consistently upregulated in CFSE- CD8+ T to serve as markers for antigen-specific T cells, employing a logistic regression model. This gene set showed high sensitivity (97.7%) and specificity (95.6%) in identifying T cell clones that respond to CMV antigen at day 3. Conclusions We have discovered potential markers for antigen-specific CD8+ T cells. These results can offer the promising opportunity for earlier isolation of T cells that exhibit reactivity towards tumor-specific antigens. This breakthrough has significant implications, as it can potentially contribute to cost savings in adoptive cell transfer-based immunotherapies. Citation Format: Byung-Kwan Jeong, Young-Ae Kim, Jungwook Park, Baknoon Ham, Jiheyong Kim, Chae Lyul Lim, Hee Jin Lee. Integrated analysis of single-cell RNA and VDJ-sequencing data to identify T cell marker expressed on antigen-specific T cells [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-25-06.

Single-Cell RNA Sequencing Reveals an Immune Landscape of CD4+ T Cells in Coronary Culprit Plaques With Acute Coronary Syndrome in Humans-Brief Report.

Acute coronary syndrome (ACS) involves plaque-related thrombosis, causing primary ischemic cardiomyopathy or lethal arrhythmia. We previously demonstrated a unique immune landscape of myeloid cells in the culprit plaques causing ACS by using single-cell RNA sequencing. Here, we aimed to characterize T cells in a single-cell level, assess clonal expansion of T cells, and find a therapeutic target to prevent ACS. We obtained the culprit lesion plaques from 4 patients with chronic coronary syndrome (chronic coronary syndrome plaques) and the culprit lesion plaques from 3 patients with ACS (ACS plaques) who were candidates for percutaneous coronary intervention with directional coronary atherectomy. Live CD45+ immune cells were sorted from each pooled plaque samples and applied to the 10× platform for single-cell RNA sequencing analysis. We also extracted RNA from other 3 ACS plaque samples and conducted unbiased TCR (T-cell receptor) repertoire analysis. CD4+ T cells were divided into 5 distinct clusters: effector, naive, cytotoxic, CCR7+ (C-C chemokine receptor type 7) central memory, and FOXP3 (forkhead box P3)+ regulatory CD4+ T cells. The proportion of central memory CD4+ T cells was higher in the ACS plaques. Correspondingly, dendritic cells also tended to express more HLAs (human leukocyte antigens) and costimulatory molecules in the ACS plaques. The velocity analysis suggested the differentiation flow from central memory CD4+ T cells into effector CD4+ T cells and that from naive CD4+ T cells into central memory CD4+ T cells in the ACS plaques, which were not observed in the chronic coronary syndrome plaques. The bulk repertoire analysis revealed clonal expansion of TCRs in each patient with ACS and suggested that several peptides in the ACS plaques work as antigens and induced clonal expansion of CD4+ T cells. For the first time, we revealed single cell-level characteristics of CD4+ T cells in patients with ACS. CD4+ T cells could be therapeutic targets of ACS. URL: https://upload.umin.ac.jp/cgi-open-bin/icdr_e/ctr_view.cgi?recptno=R000046521; Unique identifier: UMIN000040747.

Innate-like T cell subset commitment in the murine thymus is independent of TCR characteristics and occurs during proliferation

How T-cell receptor (TCR) characteristics determine subset commitment during T-cell development is still unclear. Here, we addressed this question for innate-like T cells, mucosal-associated invariant T (MAIT) cells, and invariant natural killer T (iNKT) cells. MAIT and iNKT cells have similar developmental paths, leading in mice to two effector subsets, cytotoxic (MAIT1/iNKT1) and IL17-secreting (MAIT17/iNKT17). For iNKT1 vs iNKT17 fate choice, an instructive role for TCR affinity was proposed but recent data argue against this model. Herein, we examined TCR role in MAIT and iNKT subset commitment through scRNAseq and TCR repertoire analysis. In our dataset of thymic MAIT cells, we found pairs of T-cell clones with identical amino acid TCR sequences originating from distinct precursors, one of which committed to MAIT1 and the other to MAIT17 fates. Quantitative in silico simulations indicated that the number of such cases is best explained by lineage choice being independent of TCR characteristics. Comparison of TCR features of MAIT1 and MAIT17 clonotypes demonstrated that the subsets cannot be distinguished based on TCR sequence. To pinpoint the developmental stage associated with MAIT sublineage choice, we demonstrated that proliferation takes place both before and after MAIT fate commitment. Altogether, we propose a model of MAIT cell development in which noncommitted, intermediate-stage MAIT cells undergo a first round of proliferation, followed by TCR characteristics-independent commitment to MAIT1 or MAIT17 lineage, followed by an additional round of proliferation. Reanalyzing a published iNKT TCR dataset, we showed that this model is also relevant for iNKT cell development.

Unraveling the chicken T cell repertoire with enhanced genome annotation.

T cell receptor (TCR) repertoire sequencing has emerged as a powerful tool for understanding the diversity and functionality of T cells within the host immune system. Yet, the chicken TCR repertoire remains poorly understood due to incomplete genome annotation of the TCR loci, despite the importance of chickens in agriculture and as an immunological model. Here, we addressed this critical issue by employing 5' rapid amplification of complementary DNA ends (5'RACE) TCR repertoire sequencing with molecular barcoding of complementary DNA (cDNA) molecules. Simultaneously, we enhanced the genome annotation of TCR Variable (V), Diversity (D, only present in β and δ loci) and Joining (J) genes in the chicken genome. To enhance the efficiency of TCR annotations, we developed VJ-gene-finder, an algorithm designed to extract VJ gene candidates from deoxyribonucleic acid (DNA) sequences. Using this tool, we achieved a comprehensive annotation of all known chicken TCR loci, including the α/δ locus on chromosome 27. Evolutionary analysis revealed that each locus evolved separately by duplication of long homology units. To define the baseline TCR diversity in healthy chickens and to demonstrate the feasibility of the approach, we characterized the splenic α/β/γ/δ TCR repertoire. Analysis of the repertoires revealed preferential usage of specific V and J combinations in all chains, while the overall features were characteristic of unbiased repertoires. We observed moderate levels of shared complementarity-determining region 3 (CDR3) clonotypes among individual birds within the α and γ chain repertoires, including the most frequently occurring clonotypes. However, the β and δ repertoires were predominantly unique to each bird. Taken together, our TCR repertoire analysis allowed us to decipher the composition, diversity, and functionality of T cells in chickens. This work not only represents a significant step towards understanding avian T cell biology, but will also shed light on host-pathogen interactions, vaccine development, and the evolutionary history of avian immunology.

Decade-long WT1-specific CTLs induced by WT1 peptide vaccination.

The peptide-based cancer vaccine targeting Wilms' tumor 1 (WT1) is a promising immunotherapeutic strategy for hematological malignancies. It remains unclear how long and to what extent the WT1-specific CD8 + cytotoxic T cell (CTL) persist after WT1 peptide vaccination. The WT1 peptide vaccine was administered with written consent to a patient with CML in the chronic phase who did not respond well to imatinib, and the patient was followed for 12 years after vaccination. Immune monitoring was performed by specific amplification of WT1-specific CTLs using a mixed lymphocyte peptide culture. T-cell receptors (TCRs) of amplified WT1-specific CTLs were analyzed using next-generation sequencing. This study was approved by the Institutional Review Board of our institution. WT1-specific CTLs, which were initially detected during WT1 peptide vaccination, persisted at a frequency of less than 5 cells per 1,000,000 CD8 + T cells for more than 10 years. TCR repertoire analysis confirmed the diversity of WT1-specific CTLs 11 years after vaccination. CTLs exhibited WT1 peptide-specific cytotoxicity in vitro. The WT1 peptide vaccine induced an immune response that persists for more than 10 years, even after cessation of vaccination in the CML patient.

TCRosetta: An Integrated Analysis and Annotation Platform for T-cell Receptor Sequences.

T cells and T-cell receptors (TCRs) are essential components of the adaptive immune system. Characterization of the TCR repertoire offers a promising and highly informative source for understanding the functions of T cells in the immune response and immunotherapy. Although TCR repertoire studies have attracted much attention, there are few online servers available for TCR repertoire analysis, especially for TCR sequence annotation or advanced analyses. Therefore, we developed TCRosetta, a comprehensive online server that integrates analytical methods for TCR repertoire analysis and visualization. TCRosetta combines general feature analysis, large-scale sequence clustering, network construction, peptide-TCR binding prediction, generation probability calculation, and k-mer motif analysis for TCR sequences, making TCR data analysis as simple as possible. The TCRosetta server accepts multiple input data formats and can analyze ∼ 20,000 TCR sequences in less than 3 min. TCRosetta is the most comprehensive web server available for TCR repertoire analysis and is freely available at https://guolab.wchscu.cn/TCRosetta/.

The spike-specific TCRβ repertoire shows distinct features in unvaccinated or vaccinated patients with SARS-CoV-2 infection

BackgroundThe evolving variants of SARS-CoV-2 may escape immunity from prior infections or vaccinations. It’s vital to understand how immunity adapts to these changes. Both infection and mRNA vaccination induce T cells that target the Spike protein. These T cells can recognize multiple variants, such as Delta and Omicron, even if neutralizing antibodies are weakened. However, the degree of recognition can vary among people, affecting vaccine efficacy. Previous studies demonstrated the capability of T-cell receptor (TCR) repertoire analysis to identify conserved and immunodominant peptides with cross-reactive potential among variant of concerns. However, there is a need to extend the analysis of the TCR repertoire to different clinical scenarios. The aim of this study was to examine the Spike-specific TCR repertoire profiles in natural infections and those with combined natural and vaccine immunity.MethodsA T-cell enrichment approach and bioinformatic tools were used to investigate the Spike-specific TCRβ repertoire in peripheral blood mononuclear cells of previously vaccinated (n = 8) or unvaccinated (n = 6) COVID-19 patients.ResultsDiversity and clonality of the TCRβ repertoire showed no significant differences between vaccinated and unvaccinated groups. When comparing the TCRβ data to public databases, 692 unique TCRβ sequences linked to S epitopes were found in the vaccinated group and 670 in the unvaccinated group. TCRβ clonotypes related to spike regions S135-177, S264-276, S319-350, and S448-472 appear notably more prevalent in the vaccinated group. In contrast, the S673-699 epitope, believed to have super antigenic properties, is observed more frequently in the unvaccinated group. In-silico analyses suggest that mutations in epitopes, relative to the main SARS-CoV-2 variants of concern, don’t hinder their cross-reactive recognition by associated TCRβ clonotypes.ConclusionsOur findings reveal distinct TCRβ signatures in vaccinated and unvaccinated individuals with COVID-19. These differences might be associated with disease severity and could influence clinical outcomes.Trial registration: FESR/FSE 2014–2020 DDRC n. 585, Action 10.5.12, noCOVID19@UMG.

Alteration of γδ T cell subsets in non-human primates transplanted with GGTA1 gene-deficient porcine blood vessels.

αGal-deficient xenografts are protected from hyperacute rejection during xenotransplantation but are still rejected more rapidly than allografts. Despite studies showing the roles of non-Gal antibodies and αβ T cells in xenograft rejection, the involvement of γδ T cells in xenograft rejection has been limitedly investigated. Six male cynomolgus monkeys were transplanted with porcine vessel xenografts from wild-type (n=3) or GGTA1 knockout (n=3) pigs. We measured the proportions and T cell receptor (TCR) repertoires of blood γδ T cells before and after xenotransplant. Grafted porcine vessel-infiltrating immune cells were visualized at the end of experiments. Blood γδ T cells expanded and infiltrated into the graft vessel adventitia following xenotransplantation of α-Gal-deficient pig blood vessels. Pre- and post-transplant analysis of γδ TCR repertoire revealed a transition in δ chain usage post-transplantation, with the expansion of several clonotypes of δ1, δ3, or δ7 chains. Furthermore, the distinctions between pre- and post-transplant δ chain usages were more prominent than those observed for γ chain usages. γδ TCR repertoire was significantly altered by xenotransplantation, suggesting the role of γδ T cells in sustained xenoreactive immune responses.

Genetically corrected RAG2-SCID human hematopoietic stem cells restore V(D)J-recombinase and rescue lymphoid deficiency

AbstractRecombination-activating genes (RAG1 and RAG2) are critical for lymphoid cell development and function by initiating the variable (V), diversity (D), and joining (J) (V(D)J)-recombination process to generate polyclonal lymphocytes with broad antigen specificity. The clinical manifestations of defective RAG1/2 genes range from immune dysregulation to severe combined immunodeficiencies (SCIDs), causing life-threatening infections and death early in life without hematopoietic cell transplantation (HCT). Despite improvements, haploidentical HCT without myeloablative conditioning carries a high risk of graft failure and incomplete immune reconstitution. The RAG complex is only expressed during the G0-G1 phase of the cell cycle in the early stages of T- and B-cell development, underscoring that a direct gene correction might capture the precise temporal expression of the endogenous gene. Here, we report a feasibility study using the CRISPR/Cas9-based “universal gene-correction” approach for the RAG2 locus in human hematopoietic stem/progenitor cells (HSPCs) from healthy donors and RAG2-SCID patient. V(D)J-recombinase activity was restored after gene correction of RAG2-SCID-derived HSPCs, resulting in the development of T-cell receptor (TCR) αβ and γδ CD3+ cells and single-positive CD4+ and CD8+ lymphocytes. TCR repertoire analysis indicated a normal distribution of CDR3 length and preserved usage of the distal TRAV genes. We confirmed the in vivo rescue of B-cell development with normal immunoglobulin M surface expression and a significant decrease in CD56bright natural killer cells. Together, we provide specificity, toxicity, and efficacy data supporting the development of a gene-correction therapy to benefit RAG2-deficient patients.

Alterations in Intratumoral Immune Response before and during Early-On Nivolumab Treatment for Unresectable Advanced or Recurrent Gastric Cancer

We investigated the tumor immune response in gastric cancer patients receiving third-line nivolumab monotherapy to identify immune-related biomarkers for better patient selection. Nineteen patients (10 males, median age 67 years) who received nivolumab as a third- or later-line therapy were enrolled. We analyzed the tumor immune response in durable clinical benefit (DCB) and non-DCB patients. Pre-treatment and early-on-treatment tumor transcriptomes were examined, and gene expression profiles, immunograms, and T cell receptor (TCR) repertoire were analyzed. DCB was observed in 15.8% of patients, with comparable secondary endpoints (ORR; objective response rate, OS; overall survival, PFS; progression-free survival) to previous trials. The immunograms of individual subjects displayed no significant changes before or early in the treatment, except for the regulatory T cell (Treg) score. Moreover, there were no consistent alterations observed among cases experiencing DCB. The intratumoral immune response was suppressed by previous treatments in most third- or later-line nivolumab recipients. TCR repertoire analysis revealed newly emerged clonotypes in early-on-treatment tumors, but clonal replacement did not impact efficacy. High T cell/Treg ratios and a low UV-radiation-response gene signature were linked to DCB and treatment response. This study emphasizes the tumor immune response’s importance in nivolumab efficacy for gastric cancer. High T cell/Treg ratios and specific gene expression signatures show promise as potential biomarkers for treatment response. The tumor-infiltrating immune response was compromised by prior treatments in third-line therapy, implying that, to enhance immunotherapeutic outcomes, commencing treatment at an earlier stage might be preferable. Larger cohort validation is crucial to optimize immune-checkpoint inhibitors in gastric cancer treatment.

CTIM-21. T-CELL RECEPTOR REPERTOIRE ANALYSIS IN PBMCS FROM PEDIATRIC PATIENTS WITH RECURRENT MEDULLOBLASTOMA DURING ADOPTIVE CELLULAR THERAPY

Abstract BACKGROUND Adoptive cellular therapy (ACT), which involves transferring tumor-specific lymphocytes, has shown promising clinical efficacy across various cancer types. However, accurately tracking these transferred cells has been challenging. To overcome this limitation, we utilized T-cell receptor (TCR) sequencing to investigate the TCR repertoire in peripheral blood mononuclear cells (PBMCs). Specifically, we examined PBMCs stimulated ex vivo using autologous dendritic cells (DCs) loaded with total tumor RNA, which we applied for infusion in pediatric patients. We also collected blood samples prior to and following immunotherapy to compare the TCR repertoire dynamics. METHODS We isolated total RNA from patient PBMCs obtained before the initiation of adoptive cellular therapy (ACT) and continued collection on a weekly basis for one month, followed by monthly collections after immunotherapy treatment. To generate cDNA, we used RACE technology with the addition of a common adapter at the 5' end. The raw sequencing data obtained was then preprocessed using MiXCR software. Subsequent TCR repertoire analysis was performed using the Immunarch R-package and VDJtools software. RESULTS& CONCLUSIONS The analysis of T-cell receptor (TCR) diversity across ex vivo extended T-cell samples showed a range of 593 to 34,000 unique CDR3 clones among 28 patients with recurrent medulloblastoma. The observed range suggests that different patients exhibit distinct levels of TCR diversity. Our results revealed significant instability in the TCR repertoire within the peripheral blood during the initial 28 days after ACT for all patients. Subsequently, the regeneration was mainly driven by de novo thymic production of naïve T-cells. TCR sequencing of PBMCs stimulated with autologous DCs loaded with total tumor RNA allowed us to gain a deeper understanding of the TCR repertoire in the context of immunotherapy. This approach enabled us to track and analyze the tumor-specific T-cells in the repertoire, providing valuable insights into their behavior and response to the treatment.

Lenvatinib activates anti-tumor immunity by suppressing immunoinhibitory infiltrates in the tumor microenvironment of advanced hepatocellular carcinoma

BackgroundLenvatinib, a multiple receptor tyrosine kinase inhibitor, might exert antitumor effects via tumor immune modulation. However, changes in the tumor immune microenvironment induced by lenvatinib are poorly understood. We investigated the effect of lenvatinib on immune features in clinical samples from patients with hepatocellular carcinoma.MethodsFifty-one patients with advanced hepatocellular carcinoma who received lenvatinib monotherapy as first-line treatment were enrolled. We collected blood sample (n = 51) and tumor tissue (n, baseline/four weeks after treatment initiation/post-progression = 50/8/12). DNA, RNA, and proteins extracted from the tissues were subjected to multi-omics analysis, and patients were classified into two groups according to baseline immune status. Each group was investigated in terms of the dynamics of tumor signaling. We also longitudinally analyzed circulating immune proteins and chemokines in peripheral blood.ResultsHere we show that lenvatinib has similar anti-tumor efficacy with objective response rate and progression-free survival in both Immune-Hot and Immune-Cold subtypes. Immune signatures associated with T-cell functions and interferon responses are enriched in the early phase of treatment, while signatures associated with immunoinhibitory cells are downregulated along with efficient vascular endothelial growth factor receptor and fibroblast growth factor receptor blockades. These findings are supported by imaging mass cytometry, T-cell receptor repertoire analysis and kinetics of circulating proteins. We also identify interleukin-8 and angiopoietin-2 as possible targets of intervention to overcome resistance to existing immunotherapies.ConclusionsOur findings show the ability of lenvatinib to modulate tumor immunity in clinical samples of hepatocellular carcinoma.

Activation of CD8+ T Cells in Chronic Obstructive Pulmonary Disease Lung.

Rationale: Despite the importance of inflammation in chronic obstructive pulmonary disease (COPD), the immune cell landscape in the lung tissue of patients with mild-moderate disease has not been well characterized at the single-cell and molecular level. Objectives: To define the immune cell landscape in lung tissue from patients with mild-moderate COPD at single-cell resolution. Methods: We performed single-cell transcriptomic, proteomic, and T-cell receptor repertoire analyses on lung tissue from patients with mild-moderate COPD (n = 5, Global Initiative for Chronic Obstructive Lung Disease I or II), emphysema without airflow obstruction (n = 5), end-stage COPD (n = 2), control (n = 6), or donors (n = 4). We validated in an independent patient cohort (N = 929) and integrated with the Hhip+/- murine model of COPD. Measurements and Main Results: Mild-moderate COPD lungs have increased abundance of two CD8+ T cell subpopulations: cytotoxic KLRG1+TIGIT+CX3CR1+ TEMRA (T effector memory CD45RA+) cells, and DNAM-1+CCR5+ T resident memory (TRM) cells. These CD8+ T cells interact with myeloid and alveolar type II cells via IFNG and have hyperexpanded T-cell receptor clonotypes. In an independent cohort, the CD8+KLRG1+ TEMRA cells are increased in mild-moderate COPD lung compared with control or end-stage COPD lung. Human CD8+KLRG1+ TEMRA cells are similar to CD8+ T cells driving inflammation in an aging-related murine model of COPD. Conclusions: CD8+ TEMRA cells are increased in mild-moderate COPD lung and may contribute to inflammation that precedes severe disease. Further study of these CD8+ T cells may have therapeutic implications for preventing severe COPD.

Homeostatic PD-1 signaling restrains EOMES-dependent oligoclonal expansion of liver-resident CD8 T cells

The co-inhibitory programmed death (PD)-1 signaling pathway plays a major role in the context of tumor-specific Tcell responses. Conversely, it also contributes to the maintenance of peripheral tolerance, as patients receiving anti-PD-1 treatment are prone to developing immune-related adverse events. Yet, the physiological role of the PD-1/PDL-1 axis in Tcell homeostasis is still poorly understood. Herein, we show that under steady-state conditions, the absence of PD-1 signaling led to a preferential expansion of CD8+ Tcells in the liver. These cells exhibit an oligoclonal Tcell receptor (TCR) repertoire and a terminally differentiated exhaustion profile. The transcription factor EOMES is required for the clonal expansion and acquisition of this differentiation program. Finally, single-cell transcriptomics coupled with TCR repertoire analysis support the notion that these cells arise locally from liver-resident memory CD8+ Tcells. Overall, we show a role for PD-1 signaling in liver memory Tcell homeostasis.

Spatiotemporal commonality of the TCR repertoire in a T-cell memory murine model and in metastatic human colorectal cancer.

Immune checkpoint inhibitors (ICIs) have shown superior clinical responses and significantly prolong overall survival (OS) for many types of cancer. However, some patients exhibit long-term OS, whereas others do not respond to ICI therapy at all. To develop more effective and long-lasting ICI therapy, understanding the host immune response to tumors and the development of biomarkers are imperative. In this study, we established an MC38 immunological memory mouse model by administering an anti-PD-L1 antibody and evaluating the detailed characteristics of the immune microenvironment including the T cell receptor (TCR) repertoire. In addition, we found that the memory mouse can be established by surgical resection of residual tumor following anti-PD-L1 antibody treatment with a success rate of > 40%. In this model, specific depletion of CD8 T cells revealed that they were responsible for the rejection of reinoculated MC38 cells. Analysis of the tumor microenvironment (TME) of memory mice using RNA-seq and flow cytometry revealed that memory mice had a quick and robust immune response to MC38 cells compared with naïve mice. A TCR repertoire analysis indicated that T cells with a specific TCR repertoire were expanded in the TME, systemically distributed, and preserved in the host for a long time period. We also identified shared TCR clonotypes between serially resected tumors in patients with colorectal cancer (CRC). Our results suggest that memory T cells are widely preserved in patients with CRC, and the MC38 memory model is potentially useful for the analysis of systemic memory T-cell behavior.