TCR Diversity Research Articles

CTIM-18. HIGH LEVEL OF MUTATIONAL LOAD AND DIVERSITY OF TUMOR INFILTRATING T LYMPHOCYTES ARE FAVORABLE PROGNOSTIC MARKERS FOR CANCER SURVIVAL IN CHILDREN WITH RECURRENT MEDULLOBLASTOMA DURING ADOPTIVE CELLULAR THERAPY

Abstract BACKGROUND Adoptive cellular therapy (ACT) has shown remarkable efficacy in some patients with solid tumors, resulting in durable complete responses. A key mechanism behind this success is the targeting of neoantigens by T cells. There is culminating evidence that tumor-infiltrating lymphocytes (TILs) have a major effect on the clinical attributes of human cancer and can influence the tumor response to various therapy regimens. We have been sequencing RNA isolated from patient’s tumors to evaluate their mutational burden and identify tumor-specific neoantigens. Additionally, we have identified the entire repertoire of tumor-infiltrating lymphocytes in patients with recurrent medulloblastoma. METHODS Total tumor RNA was extracted from resected brain tumor specimens and after we applied in-house bulk RNA-Seq and TCR-Seq techniques using rapid amplification of cDNA ends (RACE) technology with the addition of a common adapter at the 5’ end. RESULTS & CONCLUSIONS Analysis of the 22 tumor tissues revealed that the frequency of non-synonymous mutations varied dramatically more than 10-fold among patients with recurrent medulloblastoma undergoing adoptive cellular therapy (Re-MATCH protocol, FDA IND BB-14058). Patients with shorter overall survival exhibited non-synonymous mutation frequencies between 0.113 and 0.335 mutations per megabase (Mb). Children with prolonged overall survival had higher mutation frequencies, ranging from 0.6 to 1.1 mutations per Mb. These findings highlight the potential of using mutational burden as a biomarker for patient selection and treatment customization, ultimately aiming to improve outcomes in ACT for recurrent medulloblastoma. Up to 2000 unique TCR clones were found among TIL populations in patients who responded well to ACT. Patients with poor outcomes had significantly lower TCR diversity, with only 10-40 unique TCR clones among TILs. The correlation between TCR diversity among TILs and clinical outcomes in pediatric brain tumors underscores the importance of a diverse T cell repertoire for the success of ACT.

Genomic and immune heterogeneity of multiple synchronous lung adenocarcinoma at different developmental stages

Multiple synchronous lung cancers (MSLCs) constitute a unique subtype of lung cancer. To explore the genomic and immune heterogeneity across different pathological stages of MSLCs, we analyse 16 MSLCs from 8 patients using single-cell RNA-seq, single-cell TCR sequencing, and bulk whole-exome sequencing. Our investigation indicates clonally independent tumours with convergent evolution driven by shared driver mutations. However, tumours from the same individual exhibit few shared mutations, indicating independent origins. During the transition from pre-invasive to invasive adenocarcinoma, we observe a shift in T cell phenotypes characterized by increased Treg cells and exhausted CD8+ T cells, accompanied by diminished cytotoxicity. Additionally, invasive adenocarcinomas exhibit greater neoantigen abundance and a more diverse TCR repertoire, indicating heightened heterogeneity. In summary, despite having a common genetic background and environmental exposure, our study emphasizes the individuality of MSLCs at different stages, highlighting their unique genomic and immune characteristics.

Nucleos(T)ide Analogue Treatment Has a More Pronounced Impact on Immune Repertoires of CHB Patients Compared to HCC Patients.

Nucleos(t)ide analogues (NAs) as the first-line treatment for chronic hepatitis B (CHB) have been shown to partially restore the antiviral immunity of the patients. However, hepatitis B virus (HBV) related hepatocellular carcinoma (HCC) patients have a relatively longer duration of HBV infection and lower level of HBV DNA. Whether NAs treatments have a different effect on their immune repertoires between CHB and HCC patients remains to be determined. In this study, 126 CHB patients and 85 HBV-related HCC patients who received or did not receive NAs treatment, as well as 361 healthy individuals were enrolled to analyze the effect of NAs treatment on T cell receptor β chain (TCRβ) and B cell receptor heavy chain (BCRh) repertoires in peripheral blood of the patients. We found that after NAs therapy, the richness and evenness of TCRβ and BCRh repertoires in CHB patients were significantly lower than those in untreated patients and healthy controls, while the diversity of TCRβ and BCRh repertoires was stable in HCC patients. The alanine aminotransferase and HBV DNA levels were not correlated with the TCR or BCR diversity in CHB and HCC patients. The results suggest that NAs therapy could influence the overall T cell and B cell repertoires diversity in CHB patients but has minimal impact on HCC patients, indicating a significant difference in the potential to restore antiviral immunity between CHB and HCC patients by NAs treatment.

Aging trajectories of memory CD8 + T cells differ by their antigen specificity.

Memory T cells are a highly dynamic and heterogeneous population that is maintained by cytokine-driven homeostatic proliferation interspersed with episodes of antigen-mediated expansion and contraction which affect their functional state and their durability. This heterogeneity complicates studies on the impact of aging on global human memory cells, specifically, it is unclear how aging drives memory T cell dysfunction. Here, we used chronic infection with Epstein-Barr virus (EBV) to assess the influence of age on memory states at the level of antigen-specific CD8 + T cells. We find that in young adults (<40 years), EBV-specific CD8 + T cells assume preferred differentiation states depending on their peptide specificity. By age >65-years, different T cell specificities had undergone largely distinct aging trajectories, which had in common a loss in adaptive and a gain in innate immunity signatures. No evidence was seen for cellular senescence or exhaustion. While naïve/stem-like EBV-specific T cells disappeared with age, T cell diversity of EBV-specific memory cells did not change or even increased. In summary, by controlling for antigen specificity we uncover age-associated shifts in gene expression and TCR diversity that have implications for optimizing vaccination strategies and adoptive T cell therapy.

A pan-cancer gamma delta T cell repertoire.

This report presents the largest collection of gamma-delta T cell receptor (γδ TCR) reads in human cancer to date, analyzing about 11,000 patient tumor samples across 33 cancer types using the TRUST4 algorithm. Despite γδ T cells being a small fraction of the T cell population, they play a key role in both innate and adaptive immunity. Our comprehensive analysis reveals their significant presence across all cancer types, specifically highlighting the diverse spectrum and clonality patterns of their γδ receptors. This research highlights the complex roles of γδ T cells in tumor tissues and their potential as prognostic biomarkers. We also demonstrate the utility of T cell receptor gamma (TRG) and delta (TRD) gene expression values from standard RNA-seq data. Ultimately, our work establishes a fundamental resource for future tumor-infiltrating γδ T cell research and may facilitate the development of novel γδ-T-cell-based therapeutic strategies. Together, we demonstrate the strong diversity and prognostic potential of γδ T cells in multiple cancer types.

Peripheral blood immune parameters, response, and adverse events after neoadjuvant chemotherapy plus durvalumab in early-stage triple-negative breast cancer.

We evaluated T- and B-cell receptor (TCR and BCR) repertoire diversity and 38 serum cytokines in pre- and post-treatment peripheral blood of 66 patients with triple-negative breast cancer (TNBC) who received neoadjuvant chemotherapy plus durvalumab and assessed associations with pathologic response and immune-related adverse events (irAEs) during treatment. Genomic DNA was isolated from buffy coat for TCR and BCR clonotype profiling using the Immunoseq platform and diversity was quantified with Pielou's evenness index. MILLIPLEX MAP Human Cytokine/Chemokine Magnetic Bead Panel was used to measure serum cytokine levels, which were compared between groups using moderated t-statistic with Benjamini-Hochberg correction for multiple testing. TCR and BCR diversity was high (Pielou's index > 0.75) in all samples. Baseline receptor diversities and change in diversity pre- and post-treatment were not associated with pathologic response or irAE status, except for BCR diversity that was significantly lower post-treatment in patients who developed irAE (unadjusted p = 0.0321). Five cytokines increased after treatment in patients with pathologic complete response (pCR) but decreased in patients with RD, most prominently IL-8. IFNγ, IL-7, and GM-CSF levels were higher in pre-treatment than in post-treatment samples of patients who developed irAEs but were lower in those without irAEs. Baseline peripheral blood cytokine levels may predict irAEs in patients treated with immune checkpoint inhibitors and chemotherapy, and increased post-treatment B-cell clonal expansion might mediate irAEs.

Impact of concurrent antibiotics on survival with immunotherapy in metastatic colorectal and pancreatic cancer.

2609 Background: The interplay between antibiotics and the microbiome has been hypothesized to modify the immune response to cancer and may negatively affect immune checkpoint inhibitor (ICI) efficacy. Methods: We retrospectively reviewed concurrent antibiotics received in the randomized phase II Canadian Cancer Trials Group (CCTG) CO.26 and PA.7 clinical trials. CO.26 evaluated dual programmed death-ligand 1 (PD-L1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) inhibition (with durvalumab and tremelimumab) versus best supportive care only (BSC) in metastatic refractory colorectal cancer (CRC). PA.7 evaluated gemcitabine and nab-paclitaxel +/- durvalumab and tremelimumab in metastatic pancreatic ductal adenocarcinoma. In CO.26, T cell receptor sequencing (TCR-seq) was available at baseline and week 8. Results: In CO.26 (n=180), median (range) age was 65 (36-87) years, 33% (n=59) were female, and 29% (n=50) were ECOG 0. Concurrent exposure to antibiotics was associated with improved overall survival (OS) (7.4 vs 6.2 months, adjusted hazard ratio (aHR) 0.66 (95% confidence interval (CI) 0.46-0.93), p=0.018) in patients with metastatic CRC who received ICIs, but not in patients who received BSC (3.5 vs 4.5 months, HR 1.49 (95% CI 0.81-2.73), p= 0.20); (p-interaction=0.094). In antibiotic class analysis, concurrent exposure of ICIs to fluoroquinolones (9.4 vs 6.2 months, aHR 0.50 (95% CI 0.30-0.82), p=0.0067), but not penicillins or cephalosporins, was associated with improved OS. There was no difference in RAS/RAF status or tumor mutation burden in patients who received antibiotics/fluoroquinolones or not. The results were consistent when also controlling for TMB (antibiotics aHR 0.63 (0.44, 0.91), p=0.013; fluoroquinolones aHR 0.51 (0.31, 0.84), p=0.009). Patients treated with antibiotics did not have a different TCR diversity or clonality between baseline and 8 weeks (p=0.37 and p=0.47) compared to those who did not receive antibiotics (p=0.26 and p=0.60). In PA.7 (n=180), median (range) age was 65 (29-84) years, 48% (n=87) were female, and 24% (n=44) were ECOG 0. Antibiotics were not associated with OS in patients who did/did not receive ICIs (9.7 vs 10.8 months, HR 0.97 (95% CI 0.64, 1.46), p=0.87; 7.4 vs 10.3 months HR 1.22 (95% CI 0.72, 2.09), p=0.46, respectively). Fluoroquinolones were also not associated with OS regardless of whether or not the patient received ICI therapy (10.9 vs 9.8 months, HR 0.74 (95% CI 0.47, 1.15), p=0.18; 7.2 vs 10.2 months, HR 1.18 (95% CI 0.62, 2.27), p=0.62). Conclusions: Concurrent exposure to antibiotics, and specifically fluoroquinolones during ICI therapy was associated with a statistically significant improvement in OS in patients with metastatic CRC, but not metastatic pancreatic cancer. This is discrepant from prior reports in other tumor types and suggests that the gut microbiome may impact ICI efficacy uniquely in patient with CRC. Clinical trial information: NCT02870920 , NCT02879318 .

Decision model for durable clinical benefit from front- or late-line immunotherapy alone or with chemotherapy in large population of non-small cell lung cancer.

e20533 Background: Predictive biomarkers and models of immune checkpoint inhibitors (ICI) have been extensively studied in NSCLC. However, evidence for many biomarkers remains inconclusive, and the opaqueness of machine learning models hinders their practicality. We aimed to provide compelling evidence for biomarkers and develop a transparent decision-tree model. Methods: We consolidated data from ICI-treated patients with NSCLC (n = 3,288) across real-world multicentre studies, public cohorts, and the Choice-01 trial (NCT03607539). Over 50 features were gathered to examine the predictive capacity of durable clinical benefits (DCB) from mono-ICIs and ICIs plus chemotherapy. Noteworthy biomarkers were identified to establish a decision tree model. We also explored the tumour microenvironment and peripheral PD-1+ CD8+ T-cell receptor profiles. Results: Multivariate logistic regression analysis identified tumour histology, PD-L1 expression, tumour mutational burden, line, and regimen of ICI treatment as significant factors. Mutation subtypes of EGFR, KRAS, KEAP1, STK11, and disruptive TP53 mutations demonstrated significant association with DCB. The decision-tree (DT10) model, using the aforementioned five clinicopathological markers and five genomic mutations, predicted DCB with superior performance in the training set (N = 286, AUC = 0.82), and consistently outperformed the logistic regression, random forest, and support vector machine models, as well as FDA-approved biomarkers of PD-L1 and TMB in the internal and three external test sets (Table). We visualized the decision routes for DCB from ICI-based treatment based on the DT10 model, which was interpretable for clinical use. Furthermore, DT10 predicted-DCB patients manifested significantly longer progression-free survival and overall survival than -NDB patients across the train and test tests (HR &lt; 1, p &lt; 0.05). Additionally, the DT10 predicted-DCB group presented an enriched inflamed tumour immune phenotype (67%) and higher peripheral TCR diversity, whereas the -NDB group showed an enriched desert immune phenotype (86%) and higher peripheral TCR clonality. Conclusions: Based on the large population, we provided persuasive evidence for the predictive reliability of biomarkers in NSCLC. The development of the DT10 model introducing an easily interpretable, visually engaging, and clinically relevant model, which assisted the decision making in varied clinical scenarios. [Table: see text]

Little ones can do big things: Small molecule inhibitors target PTPN2/PTPN1 for tumor immunotherapy.

AC484 was developed by designing compounds based on the PTPN2 protein structure. AC484 enhances antitumor immunity through multiple mechanisms: increasing tumor sensitivity to IFN-γ, improving T-cell functions, stimulating tumor microenvironment inflammation, expanding TCR diversity, and preventing T-cell exhaustion. Interestingly, the efficacy of AC484 was also mediated by CD8+ and NK cells.

Single-cell molecular signature of pathogenic T helper subsets in type 2-associated disorders in humans.

To unravel the heterogeneity and molecular signature of effector memory Th2 cells (Tem2), we analyzed 23 individuals' PBMCs of filaria-infected (Filaria+) and 24 healthy volunteers (Filaria-), with or without coincident house dust mite (HDM) allergic sensitization. Flow cytometry revealed 3 CD4+ Tem subsets - CCR4+CCR6+CRTH2- Tem17, CCR4+CCR6-CRTH2+ Tem2, and CCR6+CCR4+CRTH2+ Tem17.2 - markedly enriched in Filaria+ individuals. These subsets were sorted and analyzed by multiomic single-cell RNA immunoprofiling. SingleR-annotated Th2 cells from Tem2 and Tem17.2 cell subsets had features of pathogenic Th2 effector cells based on their transcriptional signatures, with downregulated CD27 and elevated expression levels of ITGA4, IL17RB, HPGDS, KLRB1, PTGDR2, IL9R, IL4, IL5, and IL13 genes. When the Filaria+ individuals were subdivided based on their allergic status, Tem2 cells in HDM+Filaria+ individuals showed an overall reduction in TCR diversity, suggesting the occurrence of antigen-driven clonal expansion. Moreover, HDM+Filaria+ individuals showed not only an expansion in the frequency of both Tem2 and Tem17.2 cell subsets, but also a change in their molecular program by overexpressing GATA3, IL17RB, CLRF2, and KLRB1, as well as increased antigen-induced IL-4, IL-5, and IL-13 production, suggesting that aeroallergens reshape the transcriptional and functional programming of Th2 cell subsets in human filarial infection toward a pathogenic immunophenotype.

Abstract 1192: Diversity of TCR repertoire on peripheral CD8+PD-1+ T-cell predicting the PFS of chemoradiotherapy followed by durvalumab maintenance in unresectable locally advanced NSCLC patients

Abstract Background: Chemoradiotherapy (CRT) followed by Durvalumab consolidation has been the standard treatment for unresectable locally advanced non-small cell lung cancer (LA-NSCLC) patients. This study aimed to evaluate how CRT changes TCR repertoire on peripheral CD8+PD-1+ T-cells, and the change affects the clinical outcomes of CRT followed by durvalumab treatment. Methods: This was a prospective cohort study conducted from November 2019 to May 2021 in three institutes (National Cancer Center Hospital, Kansai Medical University Hospital, and Aichi Cancer Center Hospital) in Japan. We revealed the diversity of TCR repertoire (diversity evenness 50 [DE50]) on PD-1+CD8+ T cells, and CD8+ T cell phenotype in PBMCs before and after CRT treatment. Results: A total of 40 patients treated with CRT were included. Of these patients, 32 patients received durvalumab consolidation treatment after CRT (Durva cohort). The diversity and usage of T cell Receptor Beta Variable (TRBV) and T-cell receptor beta joining gene (TRBJ) usage were significantly positively correlated before and after CRT (TRBV usage: r=0.62, p&amp;lt;0.001, TRBJ usage: r=0.55, p&amp;lt;0.001). Regarding the association of TCR repertoire diversity with CD8+ T cell phenotype, DE50 and frequencies of CD8 naive were significantly positively correlated before CRT (p=0.049), while there was no correlation between PD-L1 expression and TCR repertoire before CRT (r=0.03, p=0.89) In Durva cohort, DE50 in patients with non-recurrence was higher than those with recurrence both before and after CRT (Before CRT: 0.0032 vs. 0.0019, p=0.13, After CRT: 0.0026 vs. 0.0014, p=0.06). The progression-free survival (PFS) of patients with DE50High before CRT was significantly greater than those with DE50Low (NR vs. 14.3 months; p=0.01). Moreover, when the patients were exploratory classified based on the PD-L1 expression status and the diversity of TCR repertoire, the PFS of the CRT followed by durvalumab apparently differed (DE50High, PD-L1 TPS≥1%, NR, and DE50Low, PD-L1 TPS&amp;lt;1%, 5.9 (4.6-7.3) months, p=0.001). Conclusions: TCR diversity of peripheral CD8+PD-1+ T-cell predicted the recurrence in unresectable locally advanced NSCLC patients treated with chemoradiotherapy followed by durvalumab maintenance. Additionally, the TCR diversity combined with PD-L1 expression status could more accurately predict the efficacy of the CRT followed by durvalumab. Citation Format: Masayuki Shirasawa, Tatsuya Yoshida, Yuki Takeyasu, Takaji Matsutani, Shigehiro Yagishita, Shigehisa Kitano, Hiroaki Kuroda, Toyoaki Hida, Takayasu Kurata, Yuichiro Ohe. Diversity of TCR repertoire on peripheral CD8+PD-1+ T-cell predicting the PFS of chemoradiotherapy followed by durvalumab maintenance in unresectable locally advanced NSCLC patients [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 1192.

Abstract 6593: Rejuvenation of tumor-infiltrating lymphocytes (TIL): A novel strategy to revitalize TIL antitumor function for cell therapy

Abstract TIL therapy is a promising approach for the treatment of metastatic solid tumors, but its efficacy is limited by T-cell exhaustion and terminal differentiation. In addition, recent studies have underscored the adverse effects of aging on T cells. Reprogramming T cells into induced pluripotent stem cells (iPSC) and differentiating them back to the T-cell lineage has proven to be complex and time-consuming. In particular for TIL applications, de novo T cell production via iPSCs requires each TCR in the final product to be derived from an independently established iPSC clone. To overcome these barriers, we developed a novel technology called Rejuvenation, which bypasses the need for reprogramming of T cells into iPSCs. Rejuvenated TIL products retain their TCR diversity while offering several significant advantages, including reduced T-cell epigenetic age, improved expansion capacity, stem cell-like phenotype, and enhanced secretion of cytokines upon activation by target antigens. In this study, we used TIL derived from patients with melanoma and NSCLC. Rejuvenated TIL were generated by the transient expression of transcription factors in a process called ‘Partial Reprogramming’ and returned back to a T-cell lineage phenotype by a process called ‘Redirection’. Partially reprogrammed TIL showed an altered morphology resembling adherent stromal cells and down-regulated conventional T-cell markers including CD3, CD4, and CD8. Redirected T cells reacquired a conventional CD4/CD8 phenotype including morphology, surface markers and function, and demonstrated decreased epigenetic age (&amp;gt;7 years younger, N=3). We also observed a higher proliferative capacity, improved metabolic state, and increased expression of biomarkers associated with T-cell stemness, including CCR7 and CD62L, when compared with untreated TIL. Rejuvenated TIL demonstrated improved cytokine secretion, including IFNγ, TNFα and IL-2 in functional assays. Immune repertoire analysis revealed a significant polyclonal population of putative tumor-reactive TCRs. In vivo functionality was also evaluated using a xenograft model of engineered A375 melanoma cell line that agonizes T-cells in hIL2-NOG mice. Adoptive transfer of rejuvenated TIL resulted in significantly improved suppression of tumor growth and prolonged survival compared with TIL expanded by conventional methods. These results were also confirmed in other surrogate models, including rejuvenated PBMC, TCR and CAR T cells, demonstrating the broad applicability of Rejuvenation technology. Our results indicate TIL Rejuvenation enhances the functionality and antitumor characteristics of T cells, while preserving a broad TCR repertoire. This technology holds substantial promise and may pave the way for the development of the first rejuvenated autologous polyclonal TIL therapy for treatment of advanced solid tumors. Citation Format: Raul Vizcardo, Yin Huang, Jessica Fioravanti, Takuya Maeda, Naritaka Tamaoki, Yasuhiro Yamazaki, Burak Kutlu, Kriti Bahl, Biao Wang, Zheng Zhong, Shobha Potluri, Nicholas P. Restifo, Gary Lee. Rejuvenation of tumor-infiltrating lymphocytes (TIL): A novel strategy to revitalize TIL antitumor function for cell therapy [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 6593.

Abstract 4959: Immunopipe: A comprehensive and flexible scRNA-seq and scTCR-seq data analysis pipeline

Abstract Introduction: Single-cell RNA-sequencing (scRNA-seq) pairing with single-cell T-Cell-Receptor-sequencing (scTCR-seq) enables profiling of gene expression and TCR repertoires in individual T cells, elevating our understanding of T-cell-mediated immunity. Here, we present immunopipe, a comprehensive and flexible pipeline (https://github.com/pwwang/immunopipe) for analyzing the paired data. Besides the command-line tool, it offers a user-friendly web interface that allows users with varying programming expertise to configure, initiate, and monitor the pipeline. By combining extensive functionality with ease of use, immunopipe empowers users to unlock valuable insights from the data, which notably advances our understanding of immune response and fosters the development of innovative immunotherapies. Results: Immunopipe is a comprehensive analytical pipeline for scRNA-seq and scTCR-seq data, offering various modules leveraging existing tools and novel methods to ensure data quality and enable downstream analytics. It performs quality control for scRNA-seq data, clusters cells based on gene expression to facilitate the identification of cell populations and provides insights into T-cell heterogeneity and functional diversity through T-cell selection and clustering. Enrichment analysis identifies markers and pathways for T-cell characterization. Our automated T-cell annotation tool annotates cell types accurately and hierarchically. For scTCR-seq data, immunopipe analyzes TCR clones, including clonality, diversity, gene usage, and repertoire overlap. Clone residency analysis examines the presence of clones across samples. Integrative analyses combine scRNA-seq and scTCR-seq data, providing a comprehensive understanding of the cellular landscape. Immunopipe incorporates diverse cell group information to analyze T-cell populations, which enables granular perspectives of the data for comprehensive insights. Immunopipe offers a flexible platform, allowing users to customize the analyses as needed. It supports both core and optional analyses through configuration, where users have control over the adjustable parameters. The pipeline relies on cell grouping information that can be referenced from metadata or modified to generate new variables for analysis and data filtering. Immunopipe can be executed locally or on scheduler platforms, and a docker image is provided for compatibility and simplified installation. It also offers a web interface that facilitates configuration, execution monitoring, and access to results. Conclusion: Immunopipe is a flexible and user-friendly pipeline for analyzing scRNA-seq and scTCR-seq data. It empowers researchers to gain valuable insights that could enhance the understanding of immune responses and facilitate the development of innovative immunotherapies. Citation Format: Panwen Wang, Haidong Dong, Yue Yu, Shuwen Zhang, Zhifu Sun, Jean-Pierre A. Kocher, Junwen Wang, Lin Yi, Ying Li. Immunopipe: A comprehensive and flexible scRNA-seq and scTCR-seq data analysis pipeline [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 4959.

Abstract 1516: Large scale, transcriptome-based analysis of TCR clonality reveals functional immunity in non-small cell lung cancer

Abstract Tumor-related T-cell activation and subsequent clonal expansion are essential for an effective anti-cancer immune response. Thus, quantifying the clonal distribution of T-cells in cancer tissue from patients might provide prognostic and predictive information, particularly in high immunogenic tumors like non-small cell lung cancer (NSCLC). We evaluated TCR clonality in the tumor microenvironment of NSCLC, its relation to the in situ immune phenotype, and its clinical impact in a cohort of 182 resected NSCLC patients. The α and β TCR clones were determined based on RNA sequencing data, and the Gini index was used to weight individual clonal distribution. We used matched FFPE cancer tissue for multiplex immunofluorescence cell analysis and in-situ sequencing of T-cell clones. The analysis revealed a broad spectrum of TCR clonality patterns, including tissue with high TCR diversity and high evenness (low Gini coefficient) and tissue with clonal dominance with low evenness (high Gini coefficient). Highly expended clones were detected in 15/182 patients, whereas 43/182 revealed no clonal enrichment. A low TCR clone evenness (high Gini coefficient) was significantly more frequent in normal lung areas than in the corresponding tumor areas (n=20, p=0.02). In the cancer tissue, a high Gini index further correlated with high tumor mutational burden, indicating neoantigen-induced T cell clone expansions. Correspondingly, high TCR clonality was associated with an inflamed tumor phenotype associated with PD-1+ immune cells, CD3+, CD8A+, CD163+, and CD138+ cells, as well as higher expression of genes connected to immune activation (PRF1, GZMB, GZMH) and T cell exhaustion (PD-L1, LAG3, TIGIT). Cancer tissue with dominant T-cell clones also showed higher numbers of mature tertiary lymphoid structures with germinal centers. In situ sequencing suggested a close spatial relation between specific T cell clones and tumor cells. Finally, NSCLC patients with dominant T-cell clones showed strong responses and longer survival when treated with immune checkpoint inhibitors. This suggests that TCR clonality analysis is a strong candidate for precision diagnostics. Plausibly, the dominant clones found in NSCLC are responsible for a specific but malfunctioning anti-tumor response that can be unleashed by immune checkpoint inhibition. Citation Format: Hui Yu, Anastasia Magoulopoulou, Masafumi Hori, Amanda Lindberg, Max Backman, Hans Brunström, Millaray Marincevic Zuniga, Johanna Mattsson, Akira Saito, Karin Leandersson, Mats Nilsson, Rose-Marie Amini, Patrick Micke, Carina Strell. Large scale, transcriptome-based analysis of TCR clonality reveals functional immunity in non-small cell lung cancer [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 1516.

Abstract 1176: Identification of circulating and spatial dynamics of immune cells response to to immune checkpoint inhibitors in triple-negative breast cancer at single-cell resolution

Abstract Background: Triple-negative breast cancer (TNBC) have had significantly worse survival outcome compared to other BC subtypes even though treatment strategies of TNBC have been advanced. The advent of immune checkpoint inhibitors (ICIs), especially pembrolizumab, a PD-1 inhibitor, has started to revolutionize TNBC treatment, showing encouraging effects in improving survival outcomes regardless of treatment settings. In neoadjuvant setting, pembrolizumab with cytotoxic chemotherapy achieved 13.6 % of pathologic complete response rate improvement and 36% of three-year event reduction. Therefore, pembrolizumab has highlighted new dimensions of TNBC's immunogenic characteristics. However, no immunogenic characteristics to predict the response of pembrolizumab have not been revealed. Methods: This study was designed as prospective, translational research for stage II or III TNBC patients scheduled for neoadjuvant chemotherapy with or without pembrolizumab followed by curative surgery. Tissue and blood samples were collected at baseline, 1 or 3 weeks after the first treatment initiation and curative surgery. We employed various single-cell and bulk RNA techniques for our multiomic analysis, including peripheral blood mononuclear cell (PBMC) single-cell RNA sequencing, T cell receptor sequencing (scTCR), high-resolution single-cell spatial transcriptomics (Xenium) targeting 380 genes, and bulk PBMC and tissue TCR analysis Results: From March 2023 to October 2023, 37 patients were enrolled in this study. Of 37 patients, we collected 36 blood and BC tissue samples at baseline, as well as 35 blood and 14 BC tissues at follow-up. These samples were analyzed and also compared with sex and age-matched healthy controls (n = 65). Of 37 patients, two patients were treated with only cytotoxic chemotherapy without pembrolizumab. We observed substantial increases in both systemic and localized immune responses, primarily marked by an upsurge in activated T cells and a decrease in regulatory T cells. A notable rise in TCR diversity post one week of treatment was followed by clonal expansion of activated T cells after three weeks in both blood and tissue. In addition, we observed a significant enrichment of pathways related to inflammatory response in classical monocytes following treatment. Conclusion: The findings from this study significantly enhance our understanding of the dynamics of circulating and local immune cells in TNBC, analyzed at a single-cell level with spatial information. These insights reveal how pembrolizumab alters the immune landscape and correlates these changes with treatment responses. Importantly, the data provide valuable indicators for predicting which TNBC patients are more likely to respond to neoadjuvant pembrolizumab therapy. Citation Format: Eun Seop Seo, Hae Hyun Jung, Kibyung Kim, Kyunghee Park, Eun Young Ko, Byung Joo Chae, Jeong Eon Lee, Eun Yoon Cho, Ji-Yeon Kim, Woong-Yang Park. Identification of circulating and spatial dynamics of immune cells response to to immune checkpoint inhibitors in triple-negative breast cancer at single-cell resolution [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 1176.

Abstract 5486: Spatial profiling reveals unique immune-transcriptomic features of tertiary lymphoid structures in melanoma

Abstract Background:Previous studies have shown that intratumoral immune aggregates called tertiary lymphoid structures (TLS) are associated with exceptional response to immune checkpoint blockade (ICB) in several cancer types. However, the specific mechanisms through which TLS contribute to ICB-response remain poorly characterized in part due to technical limitations with isolating TLS. Here, we use the NanoString GeoMx digital spatial profiler to interrogate TLS with deep spatial and transcriptional resolution and identify cellular and molecular features of response-associated TLS in melanoma. Methods:Twelve neoadjuvant ICB-treated high-risk resectable melanoma patients [NCT02519322] with formalin-fixed paraffin embedded (FFPE) surgical resections were included in this study. Major pathologic response (MPR) defined as &amp;lt;10% viable tumor at time of surgery was used to evaluate response (R – responder; NR – non-responder). TLS were sampled (via H&amp;E) using 1mm core punches and assembled into a tissue microarray (TMA). The TLS TMA was probed using the human whole transcriptome atlas (huWTA) and the human T-cell receptor (huTCR) atlas. Melanoma (S100B) and immune markers (CD45 and CD20) were used to guide region-of-interest (ROI) selection of classical TLS. Differential gene expression was calculated using a linear mixed model and corrected with Benjamini-Hochberg. Immune deconvolution was performed using CIBERSORTx with FDR cutoff of &amp;lt; 0.25. Results:Fifty percent (6/12) of patients included in this study demonstrated MPR following treatment with neoadjuvant ICB. Principal component analysis of huWTA probes revealed distinct transcriptional states of R TLS vs NR TLS, with PC1 strongly correlating with response status (r=0.88, p&amp;lt;0.0001). Differential immune analysis showed that R TLS harbored more CD8+ T-cells (p&amp;lt;0.001), NK cells (p=0.004), γδ T-cells (p=0.017) and memory B-cells (p=0.026) and preferentially upregulated inflammatory genes such as pattern recognition receptors (TLR8 and KLRC2) and immune-stimulatory markers (CD27 and CD103). Immune-repertoire analysis revealed a trend towards increased TCR diversity in R TLS (p = 0.3) and distinct TCR signatures in R TLS underscored by elevated expression of TRDJ4 (p&amp;lt;0.001), TRGJ1/2(p&amp;lt;0.001), TRGV4(p&amp;lt;0.001) and TRAV1-2(p=0.018) genes amongst others. Conclusions:These findings demonstrate that, although melanomas from both R and NR patients contain TLS, the immune-phenotype of R TLS is more cytotoxic and characterized by a more diverse TLS-resident T-cell population. These findings present new insights into the therapeutic underpinnings of TLS in cancer and will inform future investigations evaluating their formation/function in solid tumors. Citation Format: Manoj Chelvanambi, Brenda Melendez, Elise F. Nassif, Rossana N. Lazcano, Matthew J. Lastrapes, Bharat S. Bhushan, Sarah B. Johnson, Khalida Wani, Davis R. Ingram, Y David Seo, Beth A. Helmink, Michael G. White, Russell G. Witt, Laurence P. Diggs, Golnaz Morad, Monika Zelazowska, Somnath Paul, Florentia Dimitriou, Ashish V. Damania, Matthew C. Wong, Neeta Somaiah, Nadim J. Ajami, Emily Z. Keung, Wolf H. Fridman, James P. Allison, Padmanee Sharma, Kevin M. McBride, Tina Cascone, Christina L. Roland, Alexander J. Lazar, Jennifer A. Wargo. Spatial profiling reveals unique immune-transcriptomic features of tertiary lymphoid structures in melanoma [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 5486.

Abstract 3557: A pancancer gamma delta T cell repertoire atlas

Abstract While representing only a small portion of T cell population, Gamma delta (γδ) T cells have unique contributions to both innate and adaptive immunity. Compared to αβ T, γδ T cells have a more diverse TCR repertoire, allowing them to recognize a broader range of tumor antigens. Recent breakthrough discoveries indicate that the antitumor efficacy of γδ T cells can be potentiated or rescued through targeted therapeutic approaches. Despite their functional and translational significance, a comprehensive understanding of the γδ T cell repertoire within tumor tissues has yet to be fully achieved.In this work, we generated a pan-cancer γδ T cell repertoire Atlas through reanalyzing RNA-seq data from more than 11,000 tumors in TCGA utilizing TRUST4, an advanced computational method for reconstruction of TCR repertoires. This allowed us to offer the most accurate and comprehensive examination of the γδ TCR landscape spanning 33 cancer types, marking it the largest collection of γδ clones on human cancer to date. We processed 660 billion RNAseq reads, identified ~3 million CDR3 reads, applied a series of stringent filtering criteria to remove biologically implausible, ambiguous sequences, and out-of-frame TCRs, which resulted in 22,205 unique γδ TCR clones from 6,751 tumors. TCR gene enrichment scores were calculated to represent overall expression of γδ in each tumor. The γδ enrichment is highly variable between and within cancer types. As expected, tumors traditionally classified as immune-cold (UVM and GBM) show the lowest scores. Cancers intrinsically related to lymphatic system (LAML, THYM and DLBC), exhibit the highest scores. Beyond THYM, the top five solid-tumor cancers carrying the highest enrichment are PRAD, KIRC, TGCT, LUAD and KICH. We further profiled the clone counts, spectrum of clone sizes, CDR3 length, V-J pair usage, and clonality in 33 cancers. Overall, a dominant presence of large clone segment is not observed in most cancers. The most prevalent V-J pairs are TRGV10/9/2-TRGJ1 for γ, and TRDV1-TRDJ1 for δ chain. Cancers including LAML, KIRC, LUAD, SKCM, CESC, LUSC, STAD, display a relatively higher proportion of intermediate to larger clones. THYM exists the highest γδ diversity, with the lowest clonality and most diverse V-J pairs. In addition, we evaluated the prognostics value of γδ genes by multivariate Cox regression, adjusting for age, sex, stage, and tumor purity, and identified 38 γδ genes associated with patient survival with p&amp;lt;0.05 in 25 cancer types. Finally, within HNSC, we observed higher γδ gene enrichment (p=1.6e-9) and clone diversity (p=0.014) in HPV+ vs. HPV- tumors. The γδ gene enrichment is also found associated with patients’ survival (p=0.002) in HPV+, but not in HPV- HNSC. Similarly, COAD tumors with high Microsatellite Instability (MSI) shows higher γδ gene enrichment and clone diversity compared to MSI low COAD. Collectively, our findings serve as a foundational resource for γδ T cell research in oncology. . Citation Format: Xiaoqing Yu, Song Li, Ling Cen, Xuefeng Wang. A pancancer gamma delta T cell repertoire atlas [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 3557.

Genetic fusion of CCL11 to antigens enhances antigenicity in nucleic acid vaccines and eradicates tumor mass through optimizing T-cell response

Nucleic acid vaccines have shown promising potency and efficacy for cancer treatment with robust and specific T-cell responses. Improving the immunogenicity of delivered antigens helps to extend therapeutic efficacy and reduce dose-dependent toxicity. Here, we systematically evaluated chemokine-fused HPV16 E6/E7 antigen to improve the cellular and humoral immune responses induced by nucleotide vaccines in vivo. We found that fusion with different chemokines shifted the nature of the immune response against the antigens. Although a number of chemokines were able to amplify specific CD8 + T-cell or humoral response alone or simultaneously. CCL11 was identified as the most potent chemokine in improving immunogenicity, promoting specific CD8 + T-cell stemness and generating tumor rejection. Fusing CCL11 with E6/E7 antigen as a therapeutic DNA vaccine significantly improved treatment effectiveness and caused eradication of established large tumors in 92% tumor-bearing mice (n = 25). Fusion antigens with CCL11 expanded the TCR diversity of specific T cells and induced the infiltration of activated specific T cells, neutrophils, macrophages and dendritic cells (DCs) into the tumor, which created a comprehensive immune microenvironment lethal to tumor. Combination of the DNA vaccine with anti-CTLA4 treatment further enhanced the therapeutic effect. In addition, CCL11 could also be used for mRNA vaccine design. To summarize, CCL11 might be a potent T cell enhancer against cancer.

Mtb HLA-E-tetramer-sorted CD8+ T cells have a diverse TCR repertoire

HLA-E molecules can present self- and pathogen-derived peptides to both natural killer (NK) cells and Tcells. Tcells that recognize HLA-E peptides via their Tcell receptor (TCR) are termed donor-unrestricted Tcells due to restricted allelic variation of HLA-E. The composition and repertoire of HLA-E TCRs is not known so far. We performed TCR sequencing on CD8+ Tcells from 21 individuals recognizing HLA-E tetramers (TMs) folded with two Mtb-HLA-E-restricted peptides. We sorted HLA-E Mtb TM+ and TM- CD8+ Tcells directly exvivo and performed bulk RNA-sequencing and single-cell TCR sequencing. The identified TCR repertoire was diverse and showed no conservation between and within individuals. TCRs selected from our single-cell TCR sequencing data could be activated upon HLA-E/peptide stimulation, although not robust, reflecting potentially weak interactions between HLA-E peptide complexes and TCRs. Thus, HLA-E-Mtb-specific Tcells have a highly diverse TCR repertoire.

Immune Reconstitution after BTKi Treatment in Chronic Lymphocytic Leukemia

To analyze the immune reconstitution after BTKi treatment in patients with chronic lymphocytic leukemia (CLL). The clinical and laboratorial data of 59 CLL patients admitted from January 2017 to March 2022 in Fujian Medical University Union Hospital were collected and analyzed retrospectively. The median age of 59 CLL patients was 60.5(36-78). After one year of BTKi treatment, the CLL clones (CD5 +/CD19 +) of 51 cases (86.4%) were significantly reduced, in which the number of cloned-B cells decreased significantly from (46±6.1)×109/L to (2.3±0.4)×109/L (P =0.0013). But there was no significant change in the number of non-cloned B cells (CD19 + minus CD5 +/CD19 +). After BTKi treatment, IgA increased significantly from (0.75±0.09)g/L to (1.31±0.1)g/L (P <0.001), while IgG and IgM decreased from (8.1±0.2)g/L and (0.52±0.6)g/L to (7.1±0.1)g/L and (0.47±0.1)g/L, respectively (P <0.001, P =0.002). BTKi treatment resulted in a significant change in T cell subpopulation of CLL patients, which manifested as both a decrease in total number of T cells from (2.1±0.1)×109/L to (1.6±0.4)×109/L and NK/T cells from (0.11±0.1)×109/L to (0.07±0.01)×109/L (P =0.042, P =0.038), both an increase in number of CD4 + cells from (0.15±6.1)×109/L to (0.19±0.4)×109/L and CD8 + cells from (0.27±0.01)×109/L to (0.41±0.08)×109/L (both P <0.001). BTKi treatment also up-regulated the expression of interleukin (IL)-2 while down-regulated IL-4 and interferon (IFN)-γ. However, the expression of IL-6, IL-10, and tumor necrosis factor (TNF)-α did not change significantly. BTKi treatment could also restored the diversity of TCR and BCR in CLL patients, especially obviously in those patients with complete remission (CR) than those with partial remission (PR). Before and after BTKi treatment, Shannon index of TCR in patients with CR was 0.02±0.008 and 0.14±0.001 (P <0.001), while in patients with PR was 0.01±0.03 and 0.05±0.02 (P >0.05), respectively. Shannon index of BCR in patients with CR was 0.19±0.003 and 0.33±0.15 (P <0.001), while in patients with PR was 0.15±0.009 and 0.23±0.18 (P <0.05), respectively. BTKi treatment can shrink the clone size in CLL patients, promote the expression of IgA, increase the number of functional T cells, and regulate the secretion of cytokines such as IL-2, IL-4, and IFN-γ. BTKi also promote the recovery of diversity of TCR and BCR. BTKi treatment contributes to the reconstitution of immune function in CLL patients.