Low T-cell Receptor Research Articles

Genetic Alterations in Chromatin Regulatory Genes in Upper Tract Urothelial Carcinoma and Urothelial Bladder Cancer.

Upper tract urothelial carcinoma (UTUC) and urothelial carcinoma of the bladder (UCB) share histomorphological and therapeutic features but distinct epidemiologic and clinicopathologic characteristics. We examined alterations of chromatin regulatory genes in molecular subtypes, clonal relatedness, and T-cell receptor (TCR) diversity in UTUC and UCB. Targeted next-generation sequencing or whole-exome DNA sequencing and TCR sequencing were conducted with 34 UTUC and 49 UCB specimens from 63 patients. Tumors were subtyped based on the expression of CK5 and GATA3. Results of tissue microarray of 78 muscle-invasive bladder cancer (MIBC) samples were used as prognostic factors of different subtypes of MIBC. Chromatin regulatory genes were frequently mutated in both UTUC and UCB. Rapid relapse and progression of non-MIBC are correlated with alterations of KMT2C and EP300. Frequency of alterations in chromatin regulatory genes is higher in UTUC patients with SBS22 and SBS2 signatures and lower in UCB patients with SBS2 and SBS6 signatures. GATA3 and CK5 double-positive patients with higher frequencies of SMARCA4, ARID1A, and EP300 mutations have better prognoses than patients with basal subtypes. Although UTUC and UCB in the same patient can be either clonally related or developed independently, mutated genes in chromatin pathway were enriched in the related clones. Compared to UTUC, UCB had more deleterious mutations in DNA damage repair (DDR) genes, higher levels of tumor mutation burden (TMB) and copy number variations (CNVs), as well as higher TCR clonality and lower TCR diversity. Since genetic alterations of the chromatin pathway genes are important in both UTUC and UCB, they could serve as potential biomarkers for predicting disease progression and therapeutic targets. Differences in mutation frequencies of DDR pathway, TMB, CNV, and TCR might be the contributing factors for the distinct responses to immune checkpoint inhibitor (ICI) between UTUC and UCB.

IL-7-dependent and -independent lineages of IL-7R-dependent human T cells.

Infants with biallelic IL7R loss-of-function variants have severe combined immune deficiency (SCID) characterized by the absence of autologous T lymphocytes, but normal counts of circulating B and NK cells (T-B+NK+ SCID). We report 6 adults (aged 22 to 59 years) from 4 kindreds and 3 ancestries (Colombian, Israeli Arab, Japanese) carrying homozygous IL7 loss-of-function variants resulting in combined immunodeficiency (CID). Deep immunophenotyping revealed relatively normal counts and/or proportions of myeloid, B, NK, and innate lymphoid cells. By contrast, the patients had profound T cell lymphopenia, with low proportions of innate-like adaptive mucosal-associated invariant T and invariant NK T cells. They also had low blood counts of T cell receptor (TCR) excision circles, recent thymic emigrant T cells and naive CD4+ T cells, and low overall TCR repertoire diversity, collectively indicating impaired thymic output. The proportions of effector memory CD4+ and CD8+ T cells were high, indicating IL-7-independent homeostatic T cell proliferation in the periphery. Intriguingly, the proportions of other T cell subsets, including TCRγδ+ T cells and some TCRαβ+ T cell subsets (including Th1, Tfh, and Treg) were little affected. Peripheral CD4+ T cells displayed poor proliferation, but normal cytokine production upon stimulation with mitogens in vitro. Thus, inherited IL-7 deficiency impairs T cell development less severely and in a more subset-specific manner than IL-7R deficiency. These findings suggest that another IL-7R-binding cytokine, possibly thymic stromal lymphopoietin, governs an IL-7-independent pathway of human T cell development.

Early FDG PET imaging and circulating T-cell repertoire biomarkers of response to chemotherapy and PD-1 checkpoint inhibitors in patients with stage IV NSCLC.

8622 Background: Chemotherapy and immune checkpoint inhibitors (chemoICI) for stage IV NSCLC without driver mutations produce variable response patterns and outcomes. Early assessment could establish selective benefit from treatment (de)escalation, consolidation, or adaptation. We evaluated FDG PET imaging and circulating T cell repertoire biomarkers of chemoICI response for clinical decision support. Methods: 35 patients with stage IV NSCLC prospectively enrolled on PET BRIGHT (NCT04151940) from 2019-2023 and received first-line carboplatin-pemetrexed-pembrolizumab. Synchronous FDG PET/CT and peripheral blood draws were performed prior to and after first dose of chemoICI at week 3. Imaging biomarkers included standardized uptake value (SUV) and total lesion glycolysis (TLG) metrics within the primary metabolic tumor volume and across lung lesions. Immunophenotyping included T cell receptor (TCR) sequencing from which TCR diversity metrics were extracted. Spearman correlation of FDG PET and TCR biomarkers with bi-dimensional size reduction after initial chemoICI was estimated. ROC analysis of PET and TCR biomarkers to discriminate chemoICI response status was benchmarked against PD-L1 testing, along with Mann-Whitney testing of groupwise differences. Results: The majority tested negative for PD-L1 (58% TPS<1%). Most patients (63%) had partial response (PR) to initial chemoICI versus 33% with stable disease (SD) and 4% with progressive disease (PD). FDG PET primary tumor peak SUV decreased by a median of 15% [-82%, 63%] at week 3, while TLG decreased by 36% [-55%, 83%]. Higher baseline peak SUV and TLG, along with greater decline after first dose, were correlated with post-chemoICI size reduction (R 0.43-0.64, p=0.001-0.036). PR patients had higher baseline peak SUV (12 vs 6 g/mL, p=0.014) and greater week 3 decline in TLG (39% vs 7%, p=0.032) relative to SD/PD patients. Across lung lesions, PR-patient maximum SUV decreased while SD/PD-patient maximum SUV increased at week 3 (20% vs -19%, p=0.010). FDG PET biomarkers were stronger discriminators of chemoICI response status (AUC 0.75-0.82, p=0.009-0.046) than PD-L1 (AUC 0.60, p=0.42). Higher baseline blood TCR Simpson clonality was positively correlated with size reduction (R 0.70, p=0.004), whereas TCR Pielou evenness was negatively correlated (R -0.68, p=0.006). Higher baseline TCR clonality / lower TCR evenness discriminated chemoICI response status (AUC 0.80-0.84, p=0.028-0.049). TCR diversity changes were uncorrelated with response. Conclusions: FDG PET imaging prior to and after first dose chemoICI, along with baseline circulating T cell repertoire biomarkers, predict future response. Further investigation of these patient- and lesion-level biomarkers to guide personalized (de)escalation, consolidation, or adaptation of regimens for stage IV NSCLC is warranted. Clinical trial information: NCT04151940 .

Single-cell full-length TCR/BCR profiling assay with high sensitivity and CITE-seq compatibility enables deeper immune cell characterization

Abstract Single-cell full-length TCR/BCR assays provide the ability to profile the highly diverse T cell receptor (TCR) and B cell receptor (BCR) repertoires of the adaptive immune response. The identification of T cell clonotype can be challenging due to insufficient sensitivity to detect low abundance TCR transcripts. Furthermore, cell type identification can be limited using only transcriptomic profiles. Here, we present data with high resolution of TCR clonotype using a new highly sensitive TCR/BCR assay combined with CITE-seq using the BD™ AbSeq Assay. We profiled human and mouse T cells alongside whole transcriptome analyses with and without CITE-seq. The enhanced assay showed high clonotype detection with transcriptome-only profiling and with CITE-seq. Results also showed high gene expression correlation (R2>0.97), consistent library quality and mapping, and better resolution of cell subtype clustering with CITE-seq. Compared to transcriptome-only profiling, the coupling of surface protein expression with transcriptomic and clonal information of each cell provides deeper insights of the immune repertoire and enhances detection of cell subsets within a complex population. The results demonstrate the utility of our improved immune profiling assay with highly sensitive TCR/BCR detection for in-depth multiomics analyses of single cells. For Research Use Only. Not for use in diagnostic or therapeutic procedures. © 2023 BD. All rights reserved.

Clonal expansion of shared T cell receptors reveals the existence of immune commonality among different lesions of synchronous multiple primary lung cancer

The increase in the detection rate of synchronous multiple primary lung cancer (MPLC) has posed remarkable clinical challenges due to the limited understanding of its pathogenesis and molecular features. Here, comprehensive comparisons of genomic and immunologic features between MPLC and solitary lung cancer nodule (SN), as well as different lesions of the same patient, were performed. Compared with SN, MPLC displayed a lower rate of EGFR mutation but higher rates of BRAF, MAP2K1, and MTOR mutation, which function exactly in the upstream and downstream of the same signaling pathway. Considerable heterogeneity in T cell receptor (TCR) repertoire exists among not only different patients but also among different lesions of the same patient. Invasive lesions of MPLC exhibited significantly higher TCR diversity and lower TCR expansion than those of SN. Intriguingly, different lesions of the same patient always shared a certain proportion of TCR clonotypes. Significant clonal expansion could be observed in shared TCR clonotypes, particularly in those existing in all lesions of the same patient. In conclusion, this study provided evidences of the distinctive mutational landscape, activation of oncogenic signaling pathways, and TCR repertoire in MPLC as compared with SN. The significant clonal expansion of shared TCR clonotypes demonstrated the existence of immune commonality among different lesions of the same patient and shed new light on the individually tailored precision therapy for MPLC.

Abstract 16: CD4+ and CD8+ TCRαβ-deficient bioluminescent reporter T cells for screening and characterization of neoantigen-specific TCRs

Abstract Adoptive antigen-specific T cell therapy is currently comprised of chimeric antigen receptor (CAR) and T cell receptor (TCR) engineered T cells. Clinical results from CAR-T cells have demonstrated promising results in treating leukemias, while TCR-engineered T cells, which have the advantage of recognizing intracellular tumor antigens, is still in early development. Here, we report the development of CD4+ or CD8+ TCRαβ-KO reporter T cell lines for the screening and characterization of transgenic TCRs. A TCRαβ-KO reporter T cell line was first developed by knocking out the endogenous TCR α and β chains in the reporter T cell line using CRISPR/Cas9. Successful knockout was confirmed by phenotypic assays and TCR v chain locus sequencing. We demonstrated that re-introduction of influenza hemagglutinin (HA) peptide-specific TCR (HA1.7) α and β chains into TCRαβ-KO reporter T cell lines results in HA peptide-dependent TCR activation and luciferase reporter expression when HA peptide is presented by a MHCII+ cell line. Similarly, re-introduction of MART-1-specific TCR (DMF5) resulted in MART-1-dependent activation in the presence of MHCI+ cells. The select expression of CD4- or CD8-expressing variants of the TCRαβ-KO reporter T cell line enables the development of transgenic TCRs for both MHCI- and MHCII-restricted tumor antigen targets. Further development of CD4/CD8 double-positive and double-negative cell lines enables screening of low and high affinity TCRs without MHC bias. Together, this TCRαβ-KO platform represents a powerful tool for the screening and characterization of neoantigen-specific TCRs. Citation Format: Mei Cong, Jamison Grailer, Michael Slater, Pete Stecha, Jim Hartnett. CD4+ and CD8+ TCRαβ-deficient bioluminescent reporter T cells for screening and characterization of neoantigen-specific TCRs [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 16.

Abstract 5216: Peripheral T cell receptor repertoire diversity is associated with outcome in bladder cancer

Abstract Bladder cancer is the sixth most common cancer overall, and treatment often involves removal of the bladder, which comes with a significant reduction in quality of life. Within this cancer type, an easily applicable prognostic biomarker may have clinical utility, potentially stratifying patients into bladder-sparing treatments. Expansion of T cell populations targeting cancer-specific neoantigens is an early response to malignancy. To investigate if the T cell receptor (TCR) repertoire could serve as a biomarker for cancer progression, we analyzed the TCR landscape in a cohort of 119 patients with muscle-invasive bladder cancer, treated with neoadjuvant chemotherapy before undergoing radical cystectomy. Peripheral and tumor TCR repertoires were produced using amplicon-based sequencing of the TCR beta chain. Additionally, T cell fraction was determined from whole exome sequencing data using TcellExTRECT, and latent viral DNA was investigated in plasma whole genome sequencing data using Kraken2. Antigen targets were estimated using GLIPH2. To explore the T cell repertoire subtypes, single-cell sequencing was performed on four fresh blood samples. In pre-treatment peripheral blood, low-diversity TCR repertoires were associated with worse survival (HR=2.3, p=0.024). Particularly poor outcome was observed in a subset of patients with both low TCR diversity and low fraction of circulating T cells (HR=4.7, p=5e-4). Low-diversity repertoires were dominated by expanded T cell clones. TCR target annotation revealed that expanded T cell clones disproportionately targeted latent viral infections (p=2e-13), and cytomegalovirus DNA detection was strongly associated with decreased TCR diversity (p=5e-5). Additionally, tumor TCR sequencing revealed that expanded clones were rarely found in the tumor. Single-cell sequencing of peripheral blood found that most expanded clones consisted of cytotoxic T cells, while non-expanded clones mostly consisted of naive T cells. This indicates that low TCR diversity may represent a contraction of the naive T cell compartment, resulting in reduced immune competence. Finally, analysis of patient-matched longitudinal samples revealed that chemotherapy and cystectomy reduced TCR diversity in patients with high pre-treatment diversity (p=0.001), indicating that treatment negatively affected the immune system in the most immune-competent patients. Our results suggest that both low TCR diversity and low T cell fraction represent decreased immune competency. This could be associated with an elevated risk of progression due to a reduced ability to control cancer growth and limit systemic cancer cell dissemination. These findings underline that immune health is an important factor during malignant disease, even when patients are not treated with immunotherapies. Therefore, improving immune health could be a potential future goal for cancer treatment and prevention. Citation Format: Asbjørn Kjær, Nanna Kristjánsdóttir, Iver Nordentoft, Randi Istrup Juul, Karin Birkenkamp-Demtröder, Johanne Ahrenfeldt, Darren Hodgson, Christopher Abbosh, Hugo JWL Aerts, Mads Agerbæk, Jørgen Bjerggaard Jensen, Nicolai J Birkbak, Lars Dyrskjøt. Peripheral T cell receptor repertoire diversity is associated with outcome in bladder 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 5216.

Predictors for low T-cell receptor excision circles (TREC) in infants without severe combined immunodeficiency (SCID) or thymic defects

Predictors for low T-cell receptor excision circles (TREC) in infants without severe combined immunodeficiency (SCID) or thymic defects

LFA-1 nanoclusters integrate TCR stimulation strength to tune T-cell cytotoxic activity

T-cell cytotoxic function relies on the cooperation between the highly specific but poorly adhesive T-cell receptor (TCR) and the integrin LFA-1. How LFA-1-mediated adhesion may scale with TCR stimulation strength is ill-defined. Here, we show that LFA-1 conformation activation scales with TCR stimulation to calibrate human T-cell cytotoxicity. Super-resolution microscopy analysis reveals that >1000 LFA-1 nanoclusters provide a discretized platform at the immunological synapse to translate TCR engagement and density of the LFA-1 ligand ICAM-1 into graded adhesion. Indeed, the number of high-affinity conformation LFA-1 nanoclusters increases as a function of TCR triggering strength. Blockade of LFA-1 conformational activation impairs adhesion to target cells and killing. However, it occurs at a lower TCR stimulation threshold than lytic granule exocytosis implying that it licenses, rather than directly controls, the killing decision. We conclude that the organization of LFA-1 into nanoclusters provides a calibrated system to adjust T-cell killing to the antigen stimulation strength.

Post-Transplant T Cell Clonotype Diversity Is Associated with Survival in Patients with TP53-Mutated Acute Myeloid Leukemia

Background TP53 mutations are associated with unfavorable outcomes in various cancer types and present an obstacle to achieving sustained remission in acute myeloid leukemia (AML). Even with allogeneic hematopoietic stem cell transplantation (HSCT), the risk of relapse approximates 80%, with dismal long-term survival. This could imply methods of immune escape in TP53 mutated leukemias that allow them to evade the graft-versus-leukemia (GVL) effect, which is a critical mechanism of disease control with HSCT. This study investigates the dynamics of immune reconstitution at single-cell resolution in TP53-mutated AML at pre- and multiple post-HSCT timepoints in patients who either remain in long-term remission or relapse following HSCT. Methods We collected 26 longitudinal bone marrow aspirates and 1 peripheral blood sample from 12 patients with TP53-mutated AML who underwent HSCT at the Dana-Farber Cancer Institute between 2015-21. All patients received HLA-matched T-cell replete HSCT in morphologic remission, with standard tacrolimus/methotrexate-based prophylaxis and peripheral blood stem cells. We categorized the patients into two cohorts based on their treatment response: long-term remission (>3.5 years, cohort 1, n=4) or relapse (cohort 2, n=8). We performed paired 5' single-cell RNA and T cell receptor (TCR) sequencing on sorted viable mononuclear from each sample and CD3+ T cells from selected samples using 10x Genomics procedures. In total, we analyzed 44 samples (27 mononuclear, 17 CD3+), resulting in 250,640 high-quality single-cell transcriptomes, including 137,649 T cells and NK cells. Data analysis was performed using Cell Ranger Multi (10x Genomics) and R 4.3 with the tidyverse 2.0, Seurat 5.1, and scRepertoire 1.1 packages. Results We focused our initial analysis on the 2-6-month post-HSCT period that is critical for immune reconstitution. This included eight samples from seven patients in the two cohorts (1: remission, 2: relapse, pink box in Figure 1). Of these 7 patients, 4 were in the relapse cohort, with these relapses occurring >4 months post-HSCT. No significant difference in T-cell chimerism was seen between these 7 patients in this 2-6 month period. Based on canonical gene expression signatures, we annotated cell types and selected T cells for further evaluation (Figure 2). First, we found that the CD4/CD8 ratio differed between the cohorts, indicating a relative CD8+ T cell expansion in the relapse cohort (cohort 1: 1.69, cohort 2: 0.55, P<0.0001). Second, we found lower TCR diversity in the relapse cohort (inverse Simpson index, cohort 1: 234.6, cohort 2: 83.0, P<0.0001). This difference remained significant when subsampling the same number of T cells per patient and when restricting all samples to the 3 to 4-month timepoint. Considering the disparity in CD8 T cell numbers between the cohorts, we also analyzed repertoire diversity, specifically in CD8+ T cells, and found that the observed significance persisted. To further investigate TCR diversity, we overlaid T cell clonotypes (cells that share the same TCR sequence) in a UMAP visualization, which highlighted increased hyperexpanded CD8+ T cells in the relapse cohort (Figure 2). Finally, we found that greater clonotype expansion was correlated with the expression of key T cell genes, including CD8A, CD8B, PRF1, NKG7, and GZMA. These results suggest that cytotoxic CD8+ T cells link lower TCR diversity to the likelihood of disease relapse. Conclusions By performing deep analysis of single-cell gene expression linked to TCR clonotypes, we discover new insights into the biology of hematopoietic reconstitution after HSCT of patients with TP53-mutated AML. Despite having analyzed a limited number of samples, we find that patients who remain in long-term remission have significantly higher CD8+ TCR diversity between 2-6 months post-HSCT, while those who relapsed between 5-18 months post-HSCT showed lower TCR diversity and hyperexpanded CD8+ clones at the same timepoints. This work suggests that early post-HSCT TCR diversity could be a biomarker associated with GVL and warrants further study. In ongoing and future analyses, we will mine the current dataset for further insights into the factors influencing survival in TP53-mutated AML patients. We also plan to extend the measurement of TCR diversity as a predictor of survival for validation in a larger cohort.

Heterozygote advantage at HLA class I and II loci and reduced risk of colorectal cancer.

Reduced diversity at Human Leukocyte Antigen (HLA) loci may adversely affect the host's ability to recognize tumor neoantigens and subsequently increase disease burden. We hypothesized that increased heterozygosity at HLA loci is associated with a reduced risk of developing colorectal cancer (CRC). We imputed HLA class I and II four-digit alleles using genotype data from a population-based study of 5,406 cases and 4,635 controls from the Molecular Epidemiology of Colorectal Cancer Study (MECC). Heterozygosity at each HLA locus and the number of heterozygous genotypes at HLA class -I (A, B, and C) and HLA class -II loci (DQB1, DRB1, and DPB1) were quantified. Logistic regression analysis was used to estimate the risk of CRC associated with HLA heterozygosity. Individuals with homozygous genotypes for all loci served as the reference category, and the analyses were adjusted for sex, age, genotyping platform, and ancestry. Further, we investigated associations between HLA diversity and tumor-associated T cell repertoire features, as measured by tumor infiltrating lymphocytes (TILs; N=2,839) and immunosequencing (N=2,357). Individuals with all heterozygous genotypes at all three class I genes had a reduced odds of CRC (OR: 0.74; 95% CI: 0.56-0.97, p= 0.031). A similar association was observed for class II loci, with an OR of 0.75 (95% CI: 0.60-0.95, p= 0.016). For class-I and class-II combined, individuals with all heterozygous genotypes had significantly lower odds of developing CRC (OR: 0.66, 95% CI: 0.49-0.87, p= 0.004) than those with 0 or one heterozygous genotype. HLA class I and/or II diversity was associated with higher T cell receptor (TCR) abundance and lower TCR clonality, but results were not statistically significant. Our findings support a heterozygote advantage for the HLA class-I and -II loci, indicating an important role for HLA genetic variability in the etiology of CRC.

Colorectal cancer–associated T cell receptor repertoire abnormalities are linked to gut microbiome shifts and somatic cell mutations

ABSTRACT As with many diseases, tumor formation in colorectal cancer (CRC) is multifactorial and involves immune, environmental factors and various genetics that contribute to disease development. Accumulating evidence suggests that the gut microbiome is linked to the occurrence and development of CRC, and these microorganisms are important for immune maturation. However, a systematic perspective integrating microbial profiling, T cell receptor (TCR) and somatic mutations in humans with CRC is lacking. Here, we report distinct features of the expressed TCRβ repertoires in the peripheral blood of and CRC patients (n = 107) and healthy donors (n = 30). CRC patients have elevated numbers of large TCRβ clones and they have very low TCR diversity. The metagenomic sequencing data showed that the relative abundance of Fusobacterium nucleatum (F. nucleatum), Escherichia coli and Dasheen mosaic virus were elevated consistently in CRC patients (n = 97) compared to HC individuals (n = 30). The abundance of Faecalibacterium prausnitzii and Roseburia intestinalis was reduced in CRC (n = 97) compared to HC (n = 30). The correlation between somatic mutations of target genes (16 genes, n = 79) and TCR clonality and microbial biomarkers in CRC had been investigated. Importantly, we constructed a random forest classifier (contains 15 features) based on microbiome and TCR repertoires, which can be used as a clinical detection method to screen patients for CRC. We also analysis of F. nucleatum-specific TCR repertoire characteristics. Collectively, our large-cohort multi-omics data aimed to identify novel biomarkers to inform clinical decision-making in the detection and diagnosis of CRC, which is of possible etiological and diagnostic significance.

T cell receptor and B cell receptor exhibit unique signatures in tumor and adjacent non-tumor tissues of hepatocellular carcinoma.

The tumor microenvironment in hepatocellular carcinoma (HCC) is complicated. Tumor-infiltrating T and B cells play a pivotal role in anti-tumor immunity. T cell receptor (TCR) and B cell receptor (BCR) features may reflect the disease-associated antigen response. By combining bulk TCR/BCR-sequencing, RNA-sequencing, whole exome-sequencing, and human leukocyte antigen-sequencing, we examined the immune repertoire (IR) features of tumor and adjacent non-tumor tissues obtained from 64 HCC patients. High IR heterogeneity with weak similarity was discovered between tumor and non-tumor tissues. Higher BCR diversity, richness, and somatic hypermutation (SHM) were found in non-tumor tissues, while TCRα and TCRβ diversity and richness were comparable or higher in tumor. Additionally, lower immune infiltration was found in tumor than non-tumor tissues; the microenvironment in tumor appeared to keep stably inhibited and changed slightly with tumor progression. Moreover, BCR SHM was stronger, whereas TCR/BCR diversity declined with HCC progression. Importantly, we found that higher IR evenness in tumor and lower TCR richness in non-tumor tissues indicated better survival in HCC patients. Collectively, the results revealed that TCR and BCR exhibited distinct features in tumor and non-tumor tissues. We demonstrated that IR features vary between different tissues of HCC. IR features may represent a biomarker for the diagnosis and treatment of HCC patients, providing references for subsequent immunotherapy research and strategy selection.

Immune Infiltration in Tumor and Adjacent Non-Neoplastic Regions Codetermines Patient Clinical Outcomes in Early-Stage Lung Cancer

IntroductionIn recent years, the proportion of patients with NSCLC diagnosed at an early stage has increased continuously. MethodsIn this study, we analyzed samples and data collected from 119 samples from 67 early stage patients with NSCLC, including 52 pairs of tumor and adjacent non-neoplastic samples, and performed RNA-sequencing analysis with high sequencing depth. ResultsWe found that immune-related genes were highly enriched among the differentially expressed genes and observed significantly higher inferred immune infiltration levels in adjacent non-neoplastic samples than in tumor samples. In survival analysis, the infiltration of certain immune cell types in tumor, but not adjacent non-neoplastic, samples were associated with overall patient survival, and excitingly, the differential infiltration between paired samples (tumor minus non-neoplastic) was more prognostic than expression in either non-neoplastic or tumor tissues. We also performed B cell receptor (BCR) and T cell receptor (TCR) repertoire analysis and observed more BCR/TCR clonotypes and increased BCR clonality in tumor than in non-neoplastic samples. Finally, we carefully quantified the fraction of the five histologic subtypes in our adenocarcinoma samples and found that higher histologic pattern complexity was associated with higher immune infiltration and low TCR clonality in the tumor-proximal regions. ConclusionsOur results indicated significantly differential immune characteristics between tumor and adjacent non-neoplastic samples and suggested that the two regions provided complementary prognostic values in early-stage NSCLCs.

T cell receptor signaling mediates enhanced IFNγ production by γδ intraepithelial lymphocytes in response to type I interferon

Abstract Intraepithelial lymphocytes (IEL) expressing the γδ T cell receptor (TCR) provide a rapid response to limit enteric pathogen invasion. Despite constant γδTCR triggering in vivo, these IELs remain immunologically quiescent until their activation threshold is surpassed. Activated γδ IELs limit viral replication by producing type I interferons (IFN), such as IFNα, yet the extent to which IFNα activates γδ IELs remains unknown. To this end, murine small intestinal γδ IELs were stimulated ex vivowith 1 ug/mL αCD3, 10 ng/mL IFNα or both for 5 h. We observed a 26%±10 increase in IFNγ +γδ IELs treated with αCD3 and IFNα compared to αCD3 treatment alone (p<0.0001) and a 2-fold increase in IFNγ MFI (p<0.0001). Using Nur77-GFP mice, we found that only Nur77 +γδ IELs produced IFNγ (p<0.0001), indicating that TCR signaling is necessary for IFNα-induced effector function. Suboptimal αCD3 stimulation (0.1 ug/mL) was sufficient to increase the frequency of IFNγ +γδ IELs following IFNα exposure relative to αCD3 alone (p<0.001). To interrogate the molecular mechanism involved in IFNα co-stimulation, γδ IELs were treated with PMA and/or ionomycin in the presence or absence of IFNα. Ionomycin and IFNα increased the frequency of IFNγ +γδ IELs by 16%±7 compared to ionomycin alone (p<0.0001), whereas PMA had no effect. Pharmacological inhibition of NFAT signaling (10 uM INCA6) abrogated both TCR- or IFNα-induced γδ IEL IFNγ production (p<0.01 or p<0.0001, respectively). Lastly, we observed that the co-stimulatory effect of IFNα was lost in STAT4-deficient γδ IELs compared to WT (p<0.0001). Together, these data indicate that low level TCR signaling through NFAT is required for IFNα to rapidly enhance γδ IEL IFNγ production in a STAT4-dependent manner. This work was supported by the National Institutes of Health R01 DK119349, the New Jersey Commission on Cancer Research, Busch Biomedical Research grant and the RBHS Chancellor Scholar Award (KLE).

Abstract 2271: Characteristics of peripheral blood T cell receptor repertoire and correlation with response to chemotherapy in patients with breast cancer

Abstract Purpose: The success of immune checkpoint inhibitors therapy in triple negative breast cancer highlights the potential immunogenic characteristics of breast cancer. Increasing evidence have suggested that conventional chemotherapy can exert anti-cancer activity through various immune-based mechanisms. T cell receptor (TCR) diversity is crucial for immune responses and TCR repertoire has been reported acting as predictive and prognostic markers for cancer outcome. Here, we reported the TCR repertoire profiling in breast cancer and its association with chemotherapy and treatment response in breast cancer patients. Experimental design: This study is part of VGH-TAYLOR study, a comprehensive precision medicine study protocol on the heterogeneity of Taiwanese breast cancer patients (NCT04626440). A total of 856 female patients with luminal A, luminal B1, luminal B2, HER2-enriched, or triple-negative breast cancers were recruited. The enrolled subjects contains patients who receive surgery as the first-line treatment followed by adjuvant therapy and who receive neoadjuvant therapy as the first-line treatment followed by surgery. The blood samples from patients during chemotherapy were collected for TCR sequencing. The Oncomine TCR Beta-LR Assay was applied to determine the dynamic change in TCR repertoire. We examined TCR repertoire in terms of clonality, TCR richness/evenness and convergence. Results: Higher TCR clonality in breast cancer patients was associated with lower clonal richness. TCR clonality was positively correlated with age and stage but not tumor size. Patients with recurrence had higher TCR clonality and lower Shannon diversity. Patients with luminal B2 breast cancer showed lower TCR diversity compared to luminal A and triple-negative breast cancer. However, there were no significant differences in CDR3 length and the usage of variable and joining genes among breast cancer subtypes. Notably, both of adjuvant and neoadjuvant chemotherapies increased convergence and clonality while decreased Shannon diversity of TCR. In neoadjuvant settings, lower post-treatment blood TCR richness was associated with complete pathologic response. Conclusion: These data suggested that TCR repertoire is associated with clinicopathological characteristics including stage and recurrence. TCR clonal richness might provide prognostic value for patients receiving neoadjuvant chemotherapy. Citation Format: Chun-Yu Liu, Chi-Cheng Huang, Ji-Lin Chen, Yi-Fang Tsai, Ta-Chung Chao, Pei-Ju Lien, Yen-Shu Lin, Chin-Jung Feng, Yen-Jen Chen, Jiun-I Lai, Jen-Hwey Chiu, Chih-Yi Hsu, Ling-Ming Tseng. Characteristics of peripheral blood T cell receptor repertoire and correlation with response to chemotherapy in patients with breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2271.

Abstract 6547: Features of the TCR repertoire associate with patient’s clinical and molecular characteristics in acute myeloid leukemia

Abstract Allogeneic hematopoietic stem cell transplant (allo-HSCT) remains the most effective strategy for high risk patients with acute myeloid leukemia (AML). Leukemia specific neoantigens when presented by the major histocompatibility complexes (MHCs) are recognized by the T cell receptors (TCR) triggering the graft versus leukemia effect. Unique TCR signature is generated by a complex rearrangement process of an array of variable (V), diversity (D), and joining (J) exons to form a functional receptor with complementary determining regions 3 responsible for the interaction between the TCR and the peptide-MHC. The generated TCR repertoire undergoes dynamic changes with the onset and progression of leukemia and with the course of treatment. To better understand how the TCR repertoire impacts disease progression and patient’s clinical outcome, we leveraged RNA-seq data from The Cancer Genome Atlas (TCGA) to extract the TCR sequences using TRUST4 and MIXCR and examined the association between features of the TCR repertoire in patients with AML and their clinical and molecular characteristics. Data were analyzed by normalizing the number of unique clones to the number of total RNA-seq reads specific for each sample and one minus peripheral blood blast percentage. Both TRUST4 and MIXCR data showed a strong correlation between the number of unique clones for TCRA and TCRB per patient (r=0.944, p <0.001 for TRUST4, r=0.941, p <0.001 for MIXCR). Only 1389 TCRA clones (28.9%; TRUST4: 3450, MIXCR: 2747) and 1327 TCRB (31.3%; TRUST4: 3986, MIXCR: 1584) unique clonotypes were shared by the two computational tools. Nevertheless, we were able to identify the majority of the known TRAV and TRBV segments. Our analysis shows that the normalized number of unique TCRA and TCRB clones were lower in patients carrying FLT3 mutations (TRUST4: TCRA: p=0.013; TCRB: p=0.058; MIXCR: TCRA: p=0.043; TCRB: p=0.030), and higher in patients carrying IDH1 mutations (TRUST4: TCRA: p=0.037; TCRB: p=0.038; MIXCR: TCRA: p=0.037; TCRB: p=0.002) compared with patients carrying the wild type gene. No significant difference was observed for DNMT3A, NPM1, TET2, RUNX1, CEBPA IDH2, NRAS, WT1 and TP53 compared with wild type. Although the normalized number of unique TCR clones was not significantly associated with patient’s overall survival, we found that patients that shared more than five TCRB clones with other patients had an improved overall survival compared with patients that shared less than 5 clones (median OS: 55.4 vs 17 months, p=0.058). Despite the limitations of low TCR gene coverage associated with RNAseq data and the small sample size, we were able to find associations between TCR repertoire and clinical and molecular features in patients with AML using two different computational tools. This study further supports the need for TCR-Seq analyses to provide the depth needed to characterize the TCR repertoire in patients with AML. Citation Format: Mateusz Pospiech, Mukund Tamizharasan, Yu-Chun Wei, Advaith M. Kumar, Mimi Lou, Joshua Milstein, Houda Alachkar. Features of the TCR repertoire associate with patient’s clinical and molecular characteristics in acute myeloid leukemia. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6547.

Characterizing the immune phenotype of FGFR3 mutated upper tract urothelial carcinoma (UTUC) using single-cell (sc)RNA-sequencing (seq).

558 Background: Fibroblast growth factor 3 (FGFR3) is the most common mutation in UTUC and is altered in approximately 75% of tumors. Tumors harboring FGFR3 mutations (FGFR3-M) have a T-cell impaired tumor microenvironment (TME) which may explain the incomplete response to immune checkpoint blockade. We performed scRNA-seq on 8 untreated tumors to further characterize the T-cell immune phenotype of FGFR3-M tumors. Methods: scRNA-seq (10x Genomics platform) was performed on 8 UTUC tissue specimens from 8 different patients who had not received treatment (chemotherapy or immunotherapy) using an established institutional process. We also performed targeted gene sequencing (MSK-IMPACT) on all samples to identify mutational calls. We assessed the phenotype of defined cell clusters and the immune composition of each sample according to known marker gene expression as well as SingleR prediction. We then performed the gene set enrichment analysis over the differentially expressed genes with the Gene Ontology Biologic Process (GO:BP) to identify unique biologic processes and possible functional state of each immune cluster. Results: Among the 8 samples, 4 (50%) had altered FGFR3 (Table). We identified 19 immune cell clusters (8 T-cell clusters) with unique biologic function. Within the CD4 compartment, FGFR3-M was enriched with exhausted/active CD4 cells characterized with Th17 cell differentiation/immune regulatory function (cluster 4) and yet with lower frequency of naive-like CD4 cells possessing alpha-beta T cell activation functions and lower T-cell receptor (TCR) signaling (cluster 2). Regulatory T cells (cluster 5) were less frequently found in FGFR3-M tumors compared to their wild-type counterpart. In the CD8 compartment, FGFR3-M tumors had higher infiltration specifically in cluster 3 which corresponds to a naïve state with lower exhausted/active markers, lower cytotoxic activity, leukocyte apoptotic process, and alpha-beta T cell differentiation regulation. There was also a lower proportion among cluster 9, a mixture of NK and CD8 cytotoxic cells, which is characterized with response to interleukin (IL)-1, tumor necrosis factor (TNF), and NK cell chemotaxis. Additionally, this cluster had high cytotoxic activity and lower exhausted/active markers. Conclusions: FGFR3 mutated patients have a T-cell phenotype with more active/exhausted Th17-like CD4, lower Treg, and more CD8/cytotoxic cells in naïve state with lower response to IL-1 and TNF. scRNA-seq revealed enrichment of different functional states among T-cell compartments which may lead to improved therapeutic decision making in the future. [Table: see text]

Peripheral T-cell receptor repertoire dynamics in small cell lung cancer.

Identifying a circulating biomarker predictive of immune checkpoint inhibitor (ICI) benefit in patients with small cell lung cancer (SCLC) remains an unmet need. Characteristics of peripheral and intratumoral T-cell receptor (TCR) repertoires have been shown to predict clinical outcomes in non-small cell lung cancer (NSCLC). Recognizing a knowledge gap, we sought to characterize circulating TCR repertoires and their relationship with clinical outcomes in SCLC. SCLC patients with limited (n=4) and extensive (n=10) stage disease were prospectively enrolled for blood collection and chart review. Targeted next-generation sequencing of TCR beta and alpha chains of peripheral blood samples was performed. Unique TCR clonotypes were defined by identical CDR3, V gene, and J gene nucleotide sequences of the beta chain and subsequently used to calculate TCR diversity indices. Patients with stable versus progressive and limited versus extensive stage disease did not demonstrate significant differences in V gene usage. Kaplan-Meier curve and log-rank analysis did not identify a statistical difference in progression-free survival (PFS) (P=0.900) or overall survival (OS) (P=0.200) between high and low on-treatment TCR diversity groups, although the high diversity group exhibited a trend toward increased OS. We report the second study investigating peripheral TCR repertoire diversity in SCLC. With a limited sample size, no statistically significant associations between peripheral TCR diversity and clinical outcomes were observed, though further study is warranted.

Differences in tumor-associated T cell receptor repertoires between young-onset and average-onset colorectal cancer.

207 Background: The incidence of colorectal cancer (CRC) among individuals younger than age 50 (young-onset CRC; YOCRC) has increased, yet the etiology and molecular mechanisms underlying this alarming rise remain unclear. Some studies suggest that YOCRCs have more aggressive biology. Here, we compare tumor-associated T cell repertoires between patients with YOCRC and average-onset CRC (AOCRC). A robust, focused T cell response is a positive prognostic indicator; therefore, we hypothesized that YOCRCs demonstrate lower T cell abundance and greater T cell diversity than AOCRCs. Methods: The discovery cohort included 242 patients with histologically confirmed Stage I-IV CRC who underwent surgical resection at Cleveland Clinic from 2000 to 2020 and consented to a biorepository. YOCRC was defined as < 50 years at diagnosis (N = 126), and AOCRC as > 60 years (N = 116). DNA was extracted from frozen tumors, and T Cell Receptor (TCR) abundance and clonality were measured using immunoSEQ (Adaptive Biotechnologies). Following quality control, logistic regression models were used to evaluate the associations between TCR repertoire features and age of onset, adjusting for sex, race, tumor location, and stage. Findings were replicated in 152 YOCRC and 1,984 AOCRC cases from the Molecular Epidemiology of Colorectal Cancer Study with adjustment for sex, Jewish ethnicity, tumor location, and stage. Results: YOCRC patients were more likely to have left-sided tumors and be diagnosed at advanced stages in both cohorts. In the discovery cohort, YOCRC tumors had significantly lower TCR clonality (higher T cell diversity) compared to AOCRC tumors in a multivariable model adjusting for sex, race, tumor location and stage (OR: 0.38, 95% Confidence Interval (CI): 0.25-0.56, p < 0.0001). This association was also observed in the replication cohort (OR: 0.74, 95% CI: 0.62-0.89, p = 0.0013). In the replication cohort, further adjustment for microsatellite instability status did not substantially change the association (OR: 0.73, 95% CI: 0.61-0.88 p = 0.0012). When restricting to microsatellite stable tumors, clonality remained statistically significant in both the discovery and replication cohorts. No significant difference in TCR abundance was observed between YOCRC and AOCRC in either cohort. Conclusions: Higher T cell repertoire diversity, indicating a less focused intratumoral T cell response, is more frequently observed in YOCRC. Further studies are warranted to investigate the role of T cell diversity in the etiology and outcomes of YOCRC.