Cell Receptor Sequencing Research Articles

IMMU-23. NEOADJUVANT ANTI-PD-1 AND ANTI-CTLA-4 COMBINATION THERAPY INDUCED INTRATUMORAL IMMUNOLOGIC ALTERATIONS IN RECURRENT GBM PATIENTS

Abstract Glioblastoma (GBM) is a highly aggressive brain tumor that responds poorly to checkpoint blockade inhibitors (ICIs), such as anti-PD-1. We previously reported that neoadjuvant anti-PD-1 therapy activated and recruited T cells into recurrent GBM (rGBM) tumors, but also increased immune checkpoint interactions between T cells and myeloid cells. To overcome this resistance mechanism, we are currently conducting a phase 1B clinical trial of neoadjuvant anti-PD-1 (Nivolumab, BMS) +/- anti-CTLA-4 (Ipilimumab, BMS) combination therapy for rGBM patients (NCT04606316). To evaluate the local tumor microenvironment effects of these checkpoint inhibitors, we used multiplex immunofluorescence (mIF), bulk RNA sequencing, and T cell receptor sequencing to analyze the intratumoral immune profile of tumors. Using mIF, we are able to determine the density of key immune cell phenotypes within the section of tumor sample as well as the proximity between phenotypes to better understand their interactions. Patients who received the combination therapy (n=15) trended to have a higher median density of cytotoxic T cells (CD45+CD8+; median density of 27.13 cells/mm2 for combination therapy and 13.78 cells/mm2 for anti-PD-1), helper T cells (CD45+CD4+; median density of 33.73 cells/mm2 for combination therapy and 9.82 cells/mm2 for anti-PD-1), monocytes (CD14+), and HLA-DR+ macrophages (CD45+CD14+HLA-DR+) while patients treated with anti-PD-1 alone (n=16) trended to have densities similar to placebo treated patients (n=6). Patients treated with combination therapy are also trending to have a higher density of macrophages in close proximity (≤20μm) to T cells, particularly HLA-DR+ macrophages, suggesting a higher frequency of interactions within their tumor microenvironments. This data set is currently incomplete, but the final data set will be completed by the meeting. Nonetheless, these preliminary results, as well as the bulk RNA sequencing and T cell receptor sequencing, suggest distinct immunologic effects of monotherapy vs. combination neoadjuvant anti-PD-1 and anti-CTLA-4 therapy in the tumor microenvironment of rGBM patients.

IMMU-71. CRANIOENCEPHALIC FUNCTIONAL LYMPHOID UNITS IN GLIOBLASTOMA

Abstract The general accessibility of the brain to immune cells has been a subject of intense debate and reinterpretation within the last decade. While glioblastoma’s brain tumor ecosystem is typically considered immunosuppressed, we report the presence of a tumor-reactive immune cell niche within the cranial bone marrow (CB) situated adjacent to treatment-naive tumors. At time of initial diagnosis, glioblastoma patients underwent clinical and anatomical imaging, which revealed an unexpected co-morbid association between the CB and the tumor. Following this observation, we conducted an extensive experimental study including multiplex immunofluorescence, immunoprofiling, functional assays, and single-cell analysis, which identified an active lymphoid niche within the patient’s CB. CD8+ T cell effector cells residing in this niche exhibited acute and durable antitumor responses, and, compared to cells from the distal bone marrow, showed increased expression of the lymphoid egress marker S1PR1. Single-cell T cell receptor sequencing revealed a rich reservoir of non-exhausted tumor-shared CD8+ T cell clonotypes within the CB lymphoid niche, suggesting re-circulation between the proximal bone and the tumor tissue. These clonotypes were characterized by a high predicted tumor-reactivity, significantly surpassing levels found in blood and distal bone marrow. We discovered the entire developmental spectrum of CD8+ T cells in the CB, indicating that anatomical proximity may play a critical role in the process of early anti-tumor response. Correspondingly, cranial enhancement of CXCR4 radiolabel may serve as a surrogate marker, indicating focal association with improved progression-free survival. Our findings support the preservation and further exploitation of these cranioencephalic units to improve clinical care in glioblastoma patients.

Inhibition of Bruton's tyrosine kinase with PD-1 blockade modulates T cell activation in solid tumors.

BACKGROUNDInhibition of Bruton's tyrosine kinase with ibrutinib blocks the function of myeloid-derived suppressor cells (MDSC). The combination of ibrutinib and nivolumab was tested in patients with metastatic solid tumors.METHODSSixteen patients received ibrutinib 420 mg p.o. daily with nivolumab 240 mg i.v. on days 1 and 15 of a 28-day cycle. The effect of ibrutinib and nivolumab on MDSC, the immune profile, and cytokine levels were measured. Single-cell RNA-Seq and T cell receptor sequencing of immune cells was performed.RESULTSCommon adverse events were fatigue and anorexia. Four patients had partial responses and 4 had stable disease at 3 months (average 6.5 months, range 3.5-14.6). Median overall survival (OS) was 10.8 months. Seven days of Bruton's tyrosine kinase (BTK) inhibition significantly increased the proportion of monocytic-MDSC (M-MDSC) and significantly decreased chemokines associated with MDSC recruitment and accumulation (CCL2, CCL3, CCL4, CCL13). Single-cell RNA-Seq revealed ibrutinib-induced downregulation of genes associated with MDSC-suppressive function (TIMP1, CXCL8, VEGFA, HIF1A), reduced MDSC interactions with exhausted CD8+ T cells, and decreased TCR repertoire diversity. The addition of nivolumab significantly increased circulating NK and CD8+ T cells and increased CD8+ T cell proliferation. Exploratory analyses suggest that MDSC and T cell gene expression and TCR repertoire diversity were differentially affected by BTK inhibition according to patient response.CONCLUSIONIbrutinib and nivolumab were well tolerated and affected MDSC and T cell function in patients with solid metastatic tumors.TRIAL REGISTRATIONClinicalTrials.gov NCT03525925.FUNDINGNIH; National Cancer Institute Cancer; National Center for Advancing Translational Sciences; Pelotonia.

Immune responses in checkpoint myocarditis across heart, blood and tumour.

Immune checkpoint inhibitors are widely used anticancer therapies1 that can cause morbid and potentially fatal immune-related adverse events such as immune-related myocarditis (irMyocarditis)2-5. The pathogenesis of irMyocarditis and its relationship to antitumour immunity remain poorly understood. Here we sought to define immune responses in heart, tumour and blood in patients with irMyocarditis by leveraging single-cell RNA sequencing coupled with T cell receptor (TCR) sequencing, microscopy and proteomics analyses of samples from 28 patients with irMyocarditis and 41 unaffected individuals. Analyses of 84,576 cardiac cells by single-cell RNA sequencing combined with multiplexed microscopy demonstrated increased frequencies and co-localization of cytotoxic T cells, conventional dendritic cells and inflammatory fibroblasts in irMyocarditis heart tissue. Analyses of 366,066 blood cells revealed decreased frequencies of plasmacytoid dendritic cells, conventional dendritic cells and B lineage cells but an increased frequency of other mononuclear phagocytes in irMyocarditis. Fifty-two heart-expanded TCR clones from eight patients did not recognize the putative cardiac autoantigens α-myosin, troponin I or troponin T. Additionally, TCRs enriched in heart tissue were largely nonoverlapping with those enriched in paired tumour tissue. The presence of heart-expanded TCRs in a cycling blood CD8 T cell population was associated with fatal irMyocarditis case status. Collectively, these findings highlight crucial biology driving irMyocarditis and identify putative biomarkers.

Landscape of the Peripheral Immune Response Induced by Intraoperative Radiotherapy Combined with Surgery in Early Breast Cancer Patients.

A comprehensive analysis of the immune response triggered by intraoperative radiation therapy (IORT) remains incomplete. In this study, single-cell RNA sequencing and single-cell T cell receptor sequencing are conducted on peripheral blood mononuclear cells (PBMCs) from patient with early-stage breast cancer before and after IORT. Following IORT combined with surgery (defined as IORT+Surgery), PBMC counts remained stable, with increased proportions of T cells, mononuclear phagocytes, and plasma cells, and a reduction in neutrophil proportions. The cytotoxic score of CD8Teff_GZMK cells increased significantly post-IORT. Communication between CD8Teff_GZMK cells and other immune cells via MIF_CD74 and MIF_TNFRSF14 is decreased after IORT. cDCs showed an upregulation of the MCH II signaling pathway, while memory B cells exhibited enhanced activation of the B cell pathway. T cell clones expanded significantly after treatment. IORT+Surgery demonstrated the ability to partially suppress the anti-tumor effects of neutrophils. Flow cytometry analysis and co-culture experiments are utilized to delve deeper into the functional alterations in T cells. IORT+Surgery significantly enhanced T cell cytotoxic activity. Blockade of PD-1 of post-IORT PBMCs shows higher T-cell activity than that of pre-IORT PBMCs. This research highlights IORT's impact on immune cells, offering insights for targeting immune responses in breast cancer.

Scifer: An R/Bioconductor package for large-scale integration of Sanger sequencing and flow cytometry data of index-sorted single cells

Sanger sequencing remains widely used in various experimental contexts, often in combination with flow cytometry for indexing specific cell populations. However, existing software lacks the capability to automate quality control (QC) of raw Sanger sequencing data and integrate it with flow cytometry information on a large scale. Here, we introduce scifer, an R package now available in the latest release of Bioconductor (3.20) showcasing its effectiveness in seamlessly integrating these types of data as demonstrated by analyses of B cell and T cell receptor sequences. Scifer preprocesses raw data from index sorts and immune receptor Sanger sequencing. It identifies high-quality sequences based on selected parameters, such as length, Phred scores, and heavy-chain complementarity-determining region 3 (HCDR3) quality. As a result, the quality of germline assignments is significantly increased and spurious variable gene mutations are reduced. Scifer is automated and can process thousands of sequences in less than an hour. Its output provides quality control reports, FASTA files, summarized tables, and electropherograms for manual inspection. In summary, scifer is a user-friendly software that speeds up the analysis of immune receptor repertoire sequences, offering wide applicability.

Physiological and pathogenic T cell autoreactivity converge in type 1 diabetes

Autoimmune diseases result from autoantigen-mediated activation of adaptive immunity; intriguingly, autoantigen-specific T cells are also present in healthy donors. An assessment of dynamic changes of this autoreactive repertoire in both health and disease is thus warranted. Here we investigate the physiological versus pathogenic autoreactive processes in the context of Type 1 diabetes (T1D) and one of its landmark autoantigens, glutamic acid decarboxylase 65 (GAD65). Using single cell gene expression profiling and tandem T cell receptor (TCR) sequencing, we find that GAD65-specific true naïve cells are present in both health and disease, with GAD65-specific effector and memory responses showing similar ratios in healthy donors and patients. Deeper assessment of phenotype and TCR repertoire uncover differential features in GAD65-specific TCRs, including lower clonal sizes of healthy donor-derived clonotypes in patients. We thus propose a model whereby physiological autoimmunity against GAD65 is needed during early life, and that alterations of these physiological autoimmune processes in predisposed individuals trigger overt Type 1 diabetes.

Prediction of cardiovascular outcomes by subtle changes in the peripheral immune cell landscape - insights from the LURIC single cell RNA-sequencing study (LuRNA)

Abstract Background and Aims Atherosclerosis and its clinical sequelae, myocardial infarction and stroke, represent the main causes of death worldwide. Although preclinical evidence has suggested the existence of an inflammatory response driving disease, the extend of cellular alterations in human atherosclerosis remains enigmatic. Here, we employ single cell RNA-sequencing (scRNA-seq) on peripheral blood mononucleated cells (PBMCs) in a well-defined cardiovascular risk cohort from the "Ludwigshafen Risk and Cardiovascular Health (LURIC) trial" to define changes in the immune cell landscape in atherosclerotic patients. Methods and Results Of 3317 patients enrolled in the LURIC trial between 1997 and 2000, we selected individuals with stable coronary-artery disease (CAD) (n=31) and healthy patients (no CAD) (n=16) by propensity score matching, adjusted for CRP, NTproBNP, Troponin T, LDL-C, and Cystatin-C, in a case-control design. scRNA-seq was performed by droplet sequencing on PBMCs stored in liquid nitrogen. Individual data sets were integrated and changes between healthy individuals and patients with CAD were evaluated on numeric and transcriptional level. Single cell transcriptomes were annotated using an established CITE-seq reference atlas. While bulk RNA-sequencing of PBMC preparations revealed only minor changes between both conditions, we identified several leukocyte populations on a single cell level with specific transcriptomes that were differentially regulated between both conditions. Interestingly, we found that several subsets of Natural Killer (NK) cells, T cells with an enrichment of proliferation-associated pathways, and a population resembling hematopoietic stem cells were more frequent in patients with CAD, while naïve phenotypes of B and T cells were overrepresented in the absence of CAD. Consistently, most T and B cell populations in patients with CAD were enriched in pathways associated with cell activation, immune receptor signalling, and differentiation. In addition, we detected restricted repertoires of T cell receptor sequences in several adaptive immune cell subtypes from patients with CAD, suggestive of increased cellular clonality and antigen-specificity. Finally, in a comparison of cellular frequencies with cardiovascular outcomes over more than 15 years, several immune cell subsets and cell-type specific transcriptional programs predicted cardiovascular death in this case-control scenario in multi-variate adjusted regression analysis. Notably, addition of scRNA-seq derived parameters was superior to traditional risk prediction with clinical characteristics and soluble biomarkers alone. Conclusion Employing scRNAseq, we demonstrate that atherosclerosis is associated with a profound change in the circulating immune cell landscape even in the absence of measurable differences in inflammatory biomarkers. These findings propose the usage of scRNAseq for the discovery of outcome-relevant cellular biomarkers in the future.

Paired single-cell RNA sequencing and T cell receptor sequencing reveals both pro- and anti-inflammatory roles of T cells in patients with calcific aortic valve disease

Abstract Background Calcified aortic valve disease (CAVD) is a chronic unresolvable inflammatory disorder that affects 25% of adults over 65 years old, leading to aortic stenosis, heart failure, and death [1]. Since the molecular mechanisms of its pathogenesis are not well understood, therapies to treat root causes of the disease remain to be developed, with valve replacement as the major intervention strategy currently. Increasing evidence indicates that substantial T cells infiltrate in the aortic valve during calcification, and clonal expanded CD8+ T cells have been identified in the calcificed aortic valve by bulk T cell receptor (TCR) repertoire sequencing [2]. Purpose To decipher T cell heterogenity and understand the precise mechanisms of T cell subsets involving CAVD progression as a basis for novel therapeutic targets. Methods In Cohort 1, we collected aortic valves from CAVD (n=3) and healthy (n=2) patients for single-cell RNA sequencing (scRNA-seq). In Cohort 2, we performed paired scRNA-seq and TCR sequencing (scTCR-seq) on aortic valves obtained from CAVD (n=3) and healthy (n=3) patients to evaluate both gene expression and clonality. Pathway enrichment analysis and cell-cell interaction analysis elucidated the function of T cells in the calcificied valves. Immunohistochemistry and fluorescence-activated cell sorting were recruited to verify the key findings. Results scRNA-seq demonstrated a conversion from anti-inflammatory regulatory T (Treg) cells towards pro-inflammatory T helper 17 (Th17) cells in the calcificied aortic valve. Paired scRNA-seq and scTCR-seq revealed CAVD-related signaling pathways (osteoclast differentiation, NOD-like receptor signaling pathways, IL-17 signaling pathways) significantly upregulated in highly expanded CD8+ T cells in the calcified valve, with upregulation of pro-inflammatory mediators such as IFNG, CCL4 and CCL5, suggesting pro-inflammation functions in clonally expanded T cells. Moreover, we characterized tissue-resident memory T cells, which resembled similar transcriptome and TCR clonality as exhausted T cells in calcified valves. These cells up-regulated anti-inflammation-related transcriptomes but their cellular fraction decreased in calcified valves, implying anti-inflammatory functions of these tissue-resident memory and exhausted T cells. Conclusion This study elucidates transcriptomic and immune profiling of T cells in the calcified aortic valve, therefore shedding light on the pathophysiological mechanisms underlying aortic valve calcification from the novel perspective of tissue-specific T lymphocytes, providing promising targets for immune checkpoint-based therapeutic interventions.

Single-cell sequencing reveals immune features of treatment response to neoadjuvant immunochemotherapy in esophageal squamous cell carcinoma

Neoadjuvant immunochemotherapy (nICT) has dramatically changed the treatment landscape of operable esophageal squamous cell carcinoma (ESCC), but factors influencing tumor response to nICT are not well understood. Here, using single-cell RNA sequencing paired with T cell receptor sequencing, we profile tissues from ESCC patients accepting nICT treatment and characterize the tumor microenvironment context. CXCL13+CD8+ Tex cells, a subset of exhausted CD8+ T cells, are revealed to highly infiltrate in pre-treatment tumors and show prominent progenitor exhaustion phenotype in post-treatment samples from responders. We validate CXCL13+CD8+ Tex cells as a predictor of improved response to nICT and reveal CXCL13 to potentiate anti-PD-1 efficacy in vivo. Post-treatment tumors from non-responders are enriched for CXCL13+CD8+ Tex cells with notably remarkable exhaustion phenotype and TNFRSF4+CD4+ Tregs with activated immunosuppressive function and a significant clone expansion. Several critical markers for therapeutic resistance are also identified, including LRRC15+ fibroblasts and SPP1+ macrophages, which may recruit Tregs to form an immunosuppressive landscape. Overall, our findings unravel immune features of distinct therapeutic response to nICT treatment, providing a rationale for optimizing individualized neoadjuvant strategy in ESCC.

Single-Cell Transcriptomic Analysis of Normal and Malignant B Cells.

In the past decade, single-cell (sc) transcriptomics has overcome the limitations of bulk analysis by measuring gene expression in individual cells, not just a population average. This can identify diverse cell types and states within a sample with high resolution, even without prior purification. Various technologies exist, each with its own capture, barcoding, and library preparation methods. This chapter focuses on the analysis of normal and malignant mature B cells using the 10× Genomics 5' sc-gene expression in parallel with B cell immune repertoire profiling. By integrating the gene expression data from similar cells, the complete transcriptome for each population can be reconstructed, while the identification of the expressed immunoglobulin genes allows investigating clonotype evolution and the detection of tumor clones that share the same clonally rearranged B cell receptor sequence. Researchers are guided through both the experimental protocols and data analysis with a comprehensive, step-by-step walkthrough of how to use some of the more popular single-cell software tools.

Single-cell transcriptomics reveal potent extrafollicular B cell response linked with granzyme K+ CD8 T cell activation in lupus kidney

ObjectivesB and T cells constitute the majority of infiltrating lymphocytes in the kidney and represent the local perpetrators in lupus nephritis (LN), but the underlying pathogenic mechanisms are not well...

B cell immune repertoire sequencing in tobacco cigarette smoking, vaping, and chronic obstructive pulmonary disease in the COPDGene cohort.

Cigarette smoking (CS) impairs B cell function and antibody production, increasing infection risk. The impact of e-cigarette use ('vaping') and combined CS and vaping ('dual-use') on B cell activity is unclear. To examine B cell receptor sequencing (BCR-seq) profiles associated with CS, vaping, dual-use, COPD-related outcomes, and demographic factors. BCR-seq was performed on blood RNA samples from 234 participants in the COPDGene study. We assessed multivariable associations of B cell function measures (immunoglobulin heavy chain (IGH) subclass expression and usage, class-switching, V-segment usage, and clonal expansion) with CS, vaping, dual-use, COPD severity, age, sex, and race. We adjusted for multiple comparisons using the Benjamini-Hochberg method, identifying significant associations at 5% FDR and suggestive associations at 10% FDR. Among 234 non-Hispanic white (NHW) and African American (AA) participants, CS and dual-use were significantly positively associated with increased secretory IgA production, with dual-use showing the strongest associations. Dual-use was positively associated with class switching and B cell clonal expansion, indicating increased B cell activation, with similar trends in those only smoking or only vaping. We observed significant associations between race and IgG antibody usage. AA participants had higher IgG subclass proportions and lower IgM usage compared to NHW participants. CS and vaping additively enhance B cell activation, most notably in dual-users. Self-reported race was strongly associated with IgG isotype usage. These findings highlight associations between B cell activation and antibody transcription, suggesting potential differences in immune and vaccine responses linked to CS, vaping, and race.

Multiomic single-cell sequencing defines tissue-specific responses in Stevens-Johnson syndrome and toxic epidermal necrolysis.

Stevens-Johnson syndrome and toxic epidermal necrolysis (SJS/TEN) is a rare but life-threatening cutaneous drug reaction mediated by human leukocyte antigen (HLA) class I-restricted CD8+ T cells. For unbiased assessment of cellular immunopathogenesis, here we perform single-cell(sc) transcriptome, surface proteome, and T cell receptor (TCR) sequencing on unaffected skin, affected skin, and blister fluid from 15 SJS/TEN patients. From 109,888 cells, we identify 15 scRNA-defined subsets. Keratinocytes express markers indicating HLA class I-restricted antigen presentation and appear to trigger the proliferation of and killing by cytotoxic CD8+ tissue-resident T cells that express granulysin, granzyme B, perforin, LAG3, CD27, and LINC01871, and signal through the PKM, MIF, TGFβ, and JAK-STAT pathways. In affected tissue, cytotoxic CD8+ T cells express private expanded and unexpanded TCRαβ that are absent or unexpanded in unaffected skin, and mixed populations of macrophages and fibroblasts express pro-inflammatory markers or those favoring repair. This data identifies putative cytotoxic TCRs and therapeutic targets.

Limits on inferring T cell specificity from partial information

A key challenge in molecular biology is to decipher the mapping of protein sequence to function. To perform this mapping requires the identification of sequence features most informative about function. Here, we quantify the amount of information (in bits) that T cell receptor (TCR) sequence features provide about antigen specificity. We identify informative features by their degree of conservation among antigen-specific receptors relative to null expectations. We find that TCR specificity synergistically depends on the hypervariable regions of both receptor chains, with a degree of synergy that strongly depends on the ligand. Using a coincidence-based approach to measuring information enables us to directly bound the accuracy with which TCR specificity can be predicted from partial matches to reference sequences. We anticipate that our statistical framework will be of use for developing machine learning models for TCR specificity prediction and for optimizing TCRs for cell therapies. The proposed coincidence-based information measures might find further applications in bounding the performance of pairwise classifiers in other fields.

High-dimensional profiling of immune responses to kidney transplant reveals heterogeneous T helper 1 and B cell effectors associated with rejection

Rejection is a primary cause of allograft dysfunction after kidney transplantation. The diversity of immune subpopulations involved in the different endotypes of rejection remains to be delineated at single-cell resolution. In a cohort of 76 kidney transplant recipients, we conducted high-dimensional immune phenotyping of blood CD4 T and B cells, single-cell RNA and T/B cell receptor sequencing, and plasma cytokine profiling. Phenotypic, transcriptional, and clonal states of CD4T and B cells could significantly distinguish stable allograft states from rejection. Patients undergoing T cell-mediated rejection displayed accumulation of clonally expanded cytotoxic T helper (Th)1 cells and Th17-like cells, associated with predominant naive B cell responses. In contrast, antibody-mediated rejection was characterized by clonal expansion of Th1-polarized T follicular helper cells and effector T-bet+ memory B cells, both of which strongly expressed interleukin 12 and tumor necrosis factor-signaling pathways. Plasma cytokine analysis confirmed mixed Th1/Th17 and Th1/T follicular helper cell-driven inflammatory profiles distinguishing T cell-mediated rejection and antibody-mediated rejection, respectively. CD4T and B cell subpopulations and signatures were validated using bulk RNA-seq analysis of matched kidney allografts and using an independent single-cell RNA-seq data set. These data improve mechanistic understanding of the immune pathogenesis of rejection and support the development of more specific immunosuppressive therapies to treat allograft rejection.

Intratumoral STING agonist reverses immune evasion in PD-(L)1-refractory Merkel cell carcinoma: mechanistic insights from detailed biomarker analyses

BackgroundAntibodies blocking programmed death (PD)-1 or its ligand (PD-L1) have revolutionized cancer care, but many patients do not experience durable benefits. Novel treatments to stimulate antitumor immunity are needed in...

Abstract C156: Examining the role of T cell responses in colorectal cancer sex disparities

Abstract Purpose: Persistent sex disparities in colorectal cancer (CRC) incidence and mortality exist and vary by race and ethnicity. The overarching goal of this proposal is to quantify differences in the CRC tumor immune microenvironment by sex in the Latino Colorectal Cancer Consortium (LC3). Methods: The LC3 is a consortium of pooled individual-level data from Latino participants diagnosed with CRC from California, Florida, and Puerto Rico. Biospecimens, epidemiologic data, and clinical outcomes were collected from each contributing study. DNA was extracted from tumor tissue and paired germline or normal tissue samples. Whole exome sequencing was performed using established protocols. Global genetic ancestry was estimated from whole exome sequence data. High-throughput T cell receptor (TCR) sequencing was used to estimate features of the T cell receptor (TCR) repertoire (e.g. abundance, clonality), and tumor-infiltrating lymphocyte (TIL) counts were obtained through pathology review. We evaluated differences in TCR abundance and TILs by sex using linear models for continuous measurements and generalized linear models for binary or count measurements. Primary analyses were unadjusted, and secondary analyses adjusted for age at diagnosis, stage at diagnosis, tumor location (right colon vs. left colorectum), and microsatellite instability (MSI) status. To examine how genetic ancestry modified the relationship between sex and T cell responses, we fit separate models with an interaction term between each genetic ancestry proportion variable (i.e. African, European, Native American) and sex, and assessed effect modification using a likelihood ratio test. Results: To date, 240 participants had TCR repertoire profiling and whole exome sequencing data generated. 52% were male, with a mean age of 58. 17% of participants had rectal tumors. With respect to Hispanic ethnic heritage or country of origin, 49% of participants were of Mexican descent, followed by 13% of Central or South American descent, and 10% were of Puerto Rican descent. Mean T cell abundance was 0.18 (standard deviation (SD): 0.16) and mean clonality was 0.05 (SD: 0.05). Further analysis is underway. Conclusions: This project leverages within-Latino genetic diversity to disentangle the contributions of sex and genetic ancestry to variability in immune function and to understand how this may further sex disparities in CRC. We anticipate that results from this analysis will provide insight on the relationship between the tumor immune microenvironment and sex disparities in CRC. Citation Format: Nicole C. Loroña, Ya-Yu Tsai, Daniel Sobieski, Eric Cockman, Stephanie L. Schmit, Jane C. Figueiredo. Examining the role of T cell responses in colorectal cancer sex disparities [abstract]. In: Proceedings of the 17th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2024 Sep 21-24; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2024;33(9 Suppl):Abstract nr C156.

Abstract C020: Identification of KRAS-specific TCRs from human peripheral blood

Abstract Mutant KRAS is the predominant driver oncogene in pancreatic cancer accounting for approximately 90% of KRAS mutations. However, these mutant KRAS peptides exhibit weak binding affinity to most human leukocyte antigens (HLAs), impeding their recognition by the immune system and the subsequent generation of a corresponding T cell response. Identification of KRAS-mutant specific T cells would be useful to track tumor-specific T cell responses in patients and could also be used for adoptive transfer of TCR-transduced cells. Current efforts to identify KRAS-specific T cells have been limited by their restriction to specific HLA haplotypes or the provision of only a few T cell receptor (TCR) sequences. In this study, we developed a facile means of detecting KRAS-specific T cells from peripheral blood at frequencies as low as 1 in 1,000,000. We employ an α41BB agonist-selective lentivirus system combined with a KRAS peptide pool to facilitate the high-throughput discovery of KRAS-specific TCRs in peripheral blood mononuclear cells (PBMCs) derived from both patients with pancreatic cancer and healthy donors. Through in vitro stimulation and single-cell TCR analysis, we identified a series of T cell expansions sharing the same clonotype, indicating a response to KRAS-mutant peptide stimulation. We have quantified CD4 and CD8 KRAS-specific TCRs from patients with a diverse range of HLA haplotypes. Citation Format: Jiao Shen, Blake E. Smith, Winiffer Conce Alberto, Ellen J. Kim, Michael L. Dougan, Harshabad Singh, Michael E. Birnbaum, Stephanie K. Dougan. Identification of KRAS-specific TCRs from human peripheral blood [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C020.

The T-cell repertoire of Spanish patients with COVID-19 as a strategy to link T-cell characteristics to the severity of the disease

BackgroundThe architecture and dynamics of T cell populations are critical in orchestrating the immune response to SARS-CoV-2. In our study, we used T Cell Receptor sequencing (TCRseq) to investigate TCR repertoires in 173 post-infection COVID-19 patients.MethodsThe cohort included 98 mild and 75 severe cases with a median age of 53. We amplified and sequenced the TCR β chain Complementary Determining Region 3 (CDR3b) and performed bioinformatic analyses to assess repertoire diversity, clonality, and V/J allelic usage between age, sex and severity groups. CDR3b amino acid sequence inference was performed by clustering structural motifs and filtering validated reactive CDR3b to COVID-19.ResultsOur results revealed a pronounced decrease in diversity and an increase in clonal expansion in the TCR repertoires of severe COVID-19 patients younger than 55 years old. These results reflect the observed trends in patients older than 55 years old (both mild and severe). In addition, we identified a significant reduction in the usage of key V alleles (TRBV14, TRBV19, TRBV15 and TRBV6-4) associated with disease severity. Notably, severe patients under 55 years old had allelic patterns that resemble those over 55 years old, accompanied by a skewed frequency of COVID-19-related motifs.ConclusionsPresent results suggest that severe patients younger than 55 may have a compromised TCR repertoire contributing to a worse disease outcome.Graphical