T-cell Receptor Sequencing Research Articles

Host immune signatures as predictors of response to immunotherapy-based regimens in patients with metastatic renal cell carcinoma (mRCC)

Abstract Background Treatment options for mRCC have evolved to include VEGF targeted therapies (VEGF-TT), immune checkpoint inhibitors (ICIs), or combinations of both. However, clinical responses to systemic therapies in mRCC remain largely unpredictable and robust biomarkers are still lacking. The interaction between the tumor and its immune microenvironment has been shown to influence clinical outcomes in patients treated with ICI-based regimens. The aim of this study was to characterize the T-cell and B-cell immune repertoires in patients with mRCC treated with VEGF-TT, ICI or a combination of both, and evaluate their associations with clinical outcomes. Methods We identified patients with mRCC at Dana-Farber Cancer Institute treated with VEGF-TT, ICI or both, and for whom tumor and/or blood samples were available. T-cell receptor sequencing (TCR-seq) was performed on peripheral blood mononuclear cells (PBMCs), collected before and during therapy. Bulk RNA-sequencing (RNA-seq) was performed on available pre-treatment PBMCs and primary tumor samples. Immunoglobulin heavy chain (IgH) isotypes were inferred from bulk RNA-seq data using TRUST4. Parameters of the T-cell and B-cell repertoires were evaluated in responders vs. non-responders to systemic therapies, and between pre- and on-treatment samples within patient subgroups. Results In total, blood (PBMC) samples from 386 patients were available across all treatment cohorts (186 VEGF-TT, 126 ICI and 74 ICI+VEGF-TT). Following quality-control, TCR-seq data were available for 367 patients (228 pre-treatment and 139 on-treatment), while RNA-seq data were available for 105 PBMC and 17 tumor-derived pre-treatment samples. In the TCR-seq analysis, responders to ICI-based regimens (ICI or VEGF-TT+ICI) presented a trend towards an increased baseline (pre-treatment) TCR clonality as compared to non-responders (p=0.06) (Fig. 1A), corresponding to a less polyclonal T cell repertoire in responders at baseline. No significant changes in clonality were seen between pre- and on-treatment samples among responders to ICI regimens (p=0.14), as opposed to non-responders where a significant increase was identified (p=0.001). Therefore, responders to ICI-based regimens seem to have a more oligoclonal TCR repertoire (increased clonality) at baseline with no treatment-induced changes, whereas non-responders to ICI-based regimens seem to evolve from a more polyclonal to a more oligoclonal TCR repertoire in response to treatment. The analysis of IgH isotypes in baseline blood samples showed higher fraction of IgG1 in responders (vs. non-responders) to ICI regimens (p=0.01) (Fig. 1B). This was further confirmed in the analysis of IgH isotypes inferred from tumor samples (p=0.04). No significant differences in IgH isotype fractions were identified between responders and non-responders to VEGF-TT. Furthermore, while shared clonotypes were detected between blood and tumor samples, there were no differences in the Jaccard similarity index between responders and non-responders to ICI-based or VEGF-TT regimens. Figure 1: (A) Evaluation of pre-treatment and post-treatment TCR clonality in responders vs. non-responders across treatment cohorts. (B) Differences in the fraction of IGH isotypes between responders and non-responders across treatment cohorts. Conclusions We were successfully able to characterize T-cell receptor and B-cell IgH repertoires in a large cohort of patients with mRCC, and evaluate their associations with ICI response. Our results show that baseline TCR clonality and IgG1 antibody fraction are associated with the response to ICI regimens, suggesting a potential role for immune biomarker development. CDMRP DOD Funding: yes

A modified IL-18 drug in combination with CTLA-4 blockade enhances anti-tumor efficacy in preclinical models of renal cell carcinoma

Abstract Background Cytokine-based drugs are currently being explored as alternative cancer immunotherapies. While the cytokine interleukin-18 (IL-18) has immunostimulatory effects, it is negatively regulated by a secreted high-affinity binding protein, IL-18BP, that functions as an immune checkpoint that limits IL-18’s efficacy as a cancer therapeutic. A modified version of IL-18, termed “decoy-resistant” or DR-18, that can avoid trapping by IL-18BP while still maintaining its immune signaling potential, has recently been developed. DR-18 has shown promising preclinical activity in melanoma and colorectal murine models, including potential synergy with anti-PD-1 therapy, and is currently in Phase I trials. In this study, we aim to test the efficacy and determine the cellular mechanism of action of DR-18 in combination with immune checkpoint inhibitors (ICIs) in immunocompetent preclinical models of renal cell carcinoma (RCC), with the goal of establishing the basis for testing these combinations in early phase clinical trials. Methods We engrafted tumors subcutaneously using two different syngeneic, immunocompetent murine RCC models: Renca and RAG. Mice were treated with single-agent DR-18 and combinations of DR-18 with single- and dual-agent anti-PD-1 and anti-CTLA-4. Tumor growth and survival were monitored. In the Renca model, plasma was collected at early time-points and cytokine/chemokine levels were profiled using a 31-plex discovery assay. Single-cell RNA and TCR sequencing was also performed on Renca tumors. Additionally, immune cell depletion studies were conducted in the Renca model with antibodies targeting CD8, CD4, NK cells, and interferon-gamma. Results In the Renca model, DR-18 monotherapy modestly inhibited tumor growth and prolonged survival. The effects were comparable to single- and dual-agent ICIs. Adding PD-1 blockade to DR-18 did not enhance efficacy whereas the addition of anti-CTLA-4 to DR-18 significantly increased anti-tumor effects. Triple-therapy (DR-18 plus anti-PD-1 plus anti-CTLA-4) did not further inhibit tumor growth or prolong survival compared to the doublet (DR-18 plus anti-CTLA-4). The RAG model was more sensitive to ICIs but produced similar results, again showing modest anti-tumor activity of single-agent DR-18 and enhanced benefit of combining with anti-CTLA-4 but not anti-PD-1. Cytokine/chemokine profiling revealed significantly elevated levels of IP-10 (CXCL10) and MIG (CXCL9) after one cycle of DR-18 plus anti-CTLA-4 compared to control and single-agent treatments, suggesting that these chemokines may be key early mediators of the anti-tumor immune response. Single-cell transcriptomic analysis demonstrated changes in intra-tumoral T cell, macrophage, and granulocyte populations with DR-18 plus anti-CTLA-4 relative to other regimens, including enrichment of CD8+ precursor and terminally exhausted T cells and a neutrophil population associated with interferon signaling. Additionally, single-cell TCR analysis showed a reduction in intra-tumoral clonotype diversity with DR-18 plus anti-CTLA-4 compared to other treatments. Immune cell depletion studies identified CD8+ T cells, NK cells, and interferon-gamma, but not CD4+ T cells, as equally required for efficacy of DR-18 plus anti-CTLA-4. Conclusions In this study, we identify DR-18, an IL-18-based drug engineered with resistance to a secreted decoy-receptor protein, in combination with anti-CTLA-4 as having enhanced anti-tumor activity in preclinical models of RCC. This regimen was associated with a more pro-inflammatory immune microenvironment. Further investigation is ongoing to elucidate the cellular mechanism of action of this regimen more fully and lay the groundwork for clinical testing of DR-18-based combination therapy in RCC. In the future, testing novel partner agents outside of anti-PD-1/CTLA-4 and using RCC models of ICI-resistance could be particularly informative and clinically relevant.

Therapeutic cancer vaccination against mutant calreticulin in myeloproliferative neoplasms induces expansion of specific T cells in the periphery but specific T cells fail to enrich in the bone marrow.

Therapeutic cancer vaccination against mutant calreticulin (CALR) in patients with CALR-mutant (CALRmut) myeloproliferative neoplasms (MPN) induces strong T-cell responses against mutant CALR yet fails to demonstrate clinical activity. Infiltration of tumor specific T cells into the tumor microenvironment is needed to attain a clinical response to therapeutic cancer vaccination. Determine if CALRmut specific T cells isolated from vaccinated patients enrich in the bone marrow upon completion of vaccination and explore possible explanations for the lack of enrichment. CALRmut specific T cells from four of ten vaccinated patients were expanded, enriched, and analyzed by T-cell receptor sequencing (TCRSeq). The TCRs identified were used as fingerprints of CALRmut specific T cells. Bone marrow aspirations from the four patients were acquired at baseline and at the end of trial. T cells were enriched from the bone marrow aspirations and analyzed by TCRSeq to identify the presence and fraction of CALRmut specific T cells at the two different time points. In silico calculations were performed to calculate the ratio between transformed cells and effector cells in patients with CALRmut MPN. The fraction of CALRmut specific T cells in the bone marrow did not increase upon completion of the vaccination trial. In general, the T cell repertoire in the bone marrow remains relatively constant through the vaccination trial. The enriched and expanded CALRmut specific T cells recognize peripheral blood autologous CALRmut cells. In silico analyses demonstrate a high imbalance in the fraction of CALRmut cells and CALRmut specific effector T-cells in peripheral blood. CALRmut specific T cells do not enrich in the bone marrow after therapeutic cancer peptide vaccination against mutant CALR. The specific T cells recognize autologous peripheral blood derived CALRmut cells. In silico analyses demonstrate a high imbalance between the number of transformed cells and CALRmut specific effector T-cells in the periphery. We suggest that the high burden of transformed cells in the periphery compared to the number of effector cells could impact the ability of specific T cells to enrich in the bone marrow.

Large-scale template-based structural modeling of T-cell receptors with known antigen specificity reveals complementarity features.

T-cell receptor (TCR) recognition of foreign peptides presented by the major histocompatibility complex (MHC) initiates the adaptive immune response against pathogens. While a large number of TCR sequences specific to different antigenic peptides are known to date, the structural data describing the conformation and contacting residues for TCR-peptide-MHC complexes is relatively limited. In the present study we aim to extend and analyze the set of available structures by performing highly accurate template-based modeling of these complexes using TCR sequences with known specificity. Identification of CDR3 sequences and their further clustering, based on available spatial structures, V- and J-genes of corresponding T-cell receptors, and epitopes, was performed using the VDJdb database. Modeling of the selected CDR3 loops was conducted using a stepwise introduction of single amino acid substitutions to the template PDB structures, followed by optimization of the TCR-peptide-MHC contacting interface using the Rosetta package applications. Statistical analysis and recursive feature elimination procedures were carried out on computed energy values and properties of contacting amino acid residues between CDR3 loops and peptides, using R. Using the set of 29 complex templates (including a template with SARS-CoV-2 antigen) and 732 specificity records, we built a database of 1585 model structures carrying substitutions in either TCRα or TCRβ chains with some models representing the result of different mutation pathways for the same final structure. This database allowed us to analyze features of amino acid contacts in TCR - peptide interfaces that govern antigen recognition preferences and interpret these interactions in terms of physicochemical properties of interacting residues. Our results provide a methodology for creating high-quality TCR-peptide-MHC models for antigens of interest that can be utilized to predict TCR specificity.

Abstract B013: T cell clonotype expansion is common in advanced renal cell carcinoma but is not associated with altered response to PD-1 blockade

Abstract The diversity of T cell receptors (TCRs) becomes increasingly restricted with advancing stage in renal cell carcinoma (RCC), with individual T cell clones becoming dramatically expanded. Further, prior studies have suggested that TCR clonality (i.e. less diversity) could be associated with improved clinical response to anti-PD-1 treatment. To further interrogate the TCR landscape of advanced RCC and its relation to both T cell phenotype and clinical outcomes, single-cell TCR sequencing (scTCR-seq) was performed in parallel with single-cell RNA sequencing (scRNA-seq) for eligible patients with clear cell or non-clear cell metastatic RCC both pre- (baseline) or post- (progression) treatment with front-line nivolumab monotherapy as part of the HCRN GU16-260 trial (NCT03117309). Clonotype assignments and diversity metrics for 19 tumor samples were determined using VDJdive (R/Bioconductor). Overall, the top 10 clonotypes accounted for 12.3% of all T cells (range by sample: 4.2-34.6%). Of note, 5 of 16 (31%) samples with at least 100 T cells displayed substantial clonotype expansion, with the top 10 clonotypes accounting for at least 20% of tumor-infiltrating T cells. Average TCR diversity (normalized Shannon entropy) was 0.55 (range: 0.38-0.91) for all 19 samples. There were no significant differences in T cell diversity both between baseline (n=10) and post-treatment (n=9) samples (p= 0.66) or between clear cell (n=14) and non-clear cell (n=5) histologies (p= 0.75). To assess the relationship between T cell specificity and phenotypic state, we investigated the distribution of the most expanded clonotypes along a trajectory of T cells. Previous trajectory analysis of T cell populations revealed a bifurcating structure with naïve T cells at the root and lineages terminating in either terminally exhausted CD8+ T cells or SLAMF7+ CD8+ T cells (Braun, ASCO GU, 2023). Of the top 20 most expanded clonotypes across all T cells, 95% were distributed almost exclusively in the terminally exhausted lineage. The TCR diversity decreased along each lineage, from naïve T cell cluster at the root (TCR diversity = 0.61) to either the terminally exhausted CD8+ T cell cluster (0.42) or the SLAMF7+ CD8+ T cell cluster (0.56). There were no significant differences in TCR diversity between patients with progressive disease (n=6) versus complete/partial response (n= 6), (p= 0.96). Similarly, there was no association between TCR diversity and either progression-free (p= 0.41) or overall (p=0.39) survival. Altogether, these data reaffirm the presence of clonal expansion in advanced RCC, with the degree varying by sample. The localization of most expanded clonotypes to a single T cell lineage suggests a low degree of phenotypic plasticity. Enrichment of these clonotypes along an exhausted lineage is consistent with prior findings of lower TCR diversity in terminally exhausted CD8+ T cells. Future directions will focus on inferring the tumor specificity of infiltrating T cells along the diverging lineages. Citation Format: Miya B. Hugaboom, Neil Ruthen, Opeyemi Jegede, Nicholas R. Schindler, Lena Wirth, Sasha Kyrysyuk, David F. McDermott, Elizabeth R. Plimack, Jeffrey A. Sosman, Naomi B. Haas, Michael E. Hurwitz, Hans J. Hammers, Sabina Signoretti, Michael B. Atkins, Catherine J. Wu, David A. Braun, Kelly Street. T cell clonotype expansion is common in advanced renal cell carcinoma but is not associated with altered response to PD-1 blockade [abstract]. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr B013.

Evolution of T cell receptor beta loci in salmonids.

T-cell mediated immunity relies on a vast array of antigen specific T cell receptors (TR). Characterizing the structure of TR loci is essential to study the diversity and composition of T cell responses in vertebrate species. The lack of good-quality genome assemblies, and the difficulty to perform a reliably mapping of multiple highly similar TR sequences, have hindered the study of these loci in non-model organisms. High-quality genome assemblies are now available for the two main genera of Salmonids, Salmo and Oncorhynchus. We present here a full description and annotation of the TRB loci located on chromosomes 19 and 25 of rainbow trout (Oncorhynchus mykiss). To get insight about variations of the structure and composition of TRB locus across salmonids, we compared rainbow trout TRB loci with other salmonid species and confirmed that the basic structure of salmonid TRB locus is a double set of two TRBV-D-J-C loci in opposite orientation on two different chromosomes. Our data shed light on the evolution of TRB loci in Salmonids after their whole genome duplication (WGD). We established a coherent nomenclature of salmonid TRB loci based on comprehensive annotation. Our work provides a fundamental basis for monitoring salmonid T cell responses by TRB repertoire sequencing.

Abstract B019: Clinical and molecular characterization of chromophobe renal cell carcinoma: A focus on immunotherapy based regimens and the tumor immune microenvironment

Abstract Background: Chromophobe renal cell carcinoma (ChRCC) represents 5% of all kidney cancers. In contrast to clear cell RCC (ccRCC), the immune landscape of ChRCC and its response to immunotherapy remain poorly characterized. We sought to evaluate the clinical outcomes of patients with ChRCC treated with immuno-oncology (IO)-based regimens, and assess the immune cell composition, phenotypic state, and T cell specificity in the tumor microenvironment of ChRCC. Methods: Using real-world data from the International Metastatic RCC Database Consortium, we analyzed the survival outcomes and objective responses of patients with advanced ChRCC to currently adopted IO-based regimens (i.e. dual IO therapy or IO + vascular endothelial growth factor targeted therapy [VEGF-TT]) in the first-line setting, as compared to patients with ccRCC. Single-cell RNA sequencing (scRNA-seq) and single-cell T-cell receptor sequencing (scTCR-seq) were performed on ChRCC and related oncocytic neoplasms (i.e. renal oncocytoma [RO] and low-grade oncocytic tumor [LOT]) samples with matched normal kidney specimens. The infiltration of CD45+ immune cells in renal oncocytic tumors and ccRCC samples was quantified using immunohistochemistry (IHC). Results: Compared to patients with ccRCC (n=856) treated with first-line IO-based regimens, patients with ChRCC (n=31) had a lower overall survival (median: 24.7 vs. 50.5 months, p<0.001) and lower time to treatment failure (median: 4.5 vs. 11.0 months, p<0.001). Similarly, patients with ChRCC had a significantly lower overall response rate than those with ccRCC (12.0 vs. 47.1%, respectively; p<0.001). When evaluating immune cell infiltration, renal oncocytic tumors (ChRCC, RO, and LOT) exhibited a low density of CD45+ cells (mean: 739 ± 114 cells/mm2; n=5) compared to ccRCC (mean: 3,420 ± 1,979 cells/mm2; n=5) (p<0.05). Single-cell analysis was performed on 46,817 cells from 5 tumors (ChRCC: n=3, RO: n=1 and LOT: n=1) and 4 samples from adjacent normal kidney. Across all tumors, CD8+ T cell clusters displayed a lower expression of immune checkpoints (i.e. PDCD1, CTLA4, LAG3, HAVCR2, and TIGIT) as compared to CD8+ T-cells from ccRCC. This was further validated in the analysis of bulk RNA-seq data from the TCGA, with a significantly lower expression of all immune checkpoints in ChRCC compared to both ccRCC (p<0.01) and papillary RCC (pRCC; p<0.01). Analysis of the T cell receptor repertoire (scTCR-seq) of ChRCC, RO and LOT samples did not show any pattern of clonal expansion, and a higher proportion of T cells in ChRCC were inferred to have a viral specificity, compared to ccRCC (0.79 vs. 0.1%, respectively). Conclusions: Patients with metastatic ChRCC appear to display poor clinical outcomes when treated with IO-based regimens, compared to ccRCC. Renal oncocytic tumors, including ChRCC, exhibit a low infiltration of immune cells, and a non-exhausted immune phenotype. Citation Format: Michel Alchoueiry, Chris Labaki, Long Zhang, Yue Hou, Kevin Bi, Charbel Hobeika, J. Connor Wells, Kosuke Takemura, Ziad Bakouny, Sabrina Camp, Carmen Priolo, Damir Khabibullin, Nicholas Schindler, Renee Maria Saliby, Eddy Saad, Samer Salem, Melissa Daou, Rana McKay, Sumanta Pal, Daniel Heng, Eliezer Van Allen, Sachet Shukla, Toni Choueiri, David Braun, Elizabeth Henske. Clinical and molecular characterization of chromophobe renal cell carcinoma: A focus on immunotherapy based regimens and the tumor immune microenvironment [abstract]. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr B019.

BSBM-19 CROSS-PRESENTATION BY TUMOR-ASSOCIATED MACROPHAGES MEDIATES THE TRANSITION FROM PROGENITOR TO TERMINAL T CELL EXHAUSTION IN PRIMARY AND METASTATIC BRAIN TUMORS

Abstract Glioblastoma (GBM) and brain metastases remain largely resistant to immunotherapeutic intervention. T cell exhaustion, a differentiation state characterized by loss of function and persistence, contributes to this resistance. Two exhaustion subsets, progenitor (Tex_prog) and terminal (Tex_term), have been identified, where only Tex_prog remain responsive to immunotherapy. To date, the dynamics and characteristics of these exhausted populations in GBM and brain metastases remain unclear. Herein, we identify a striking loss of Tex_prog in a murine model of GBM throughout tumor progression. We elucidate the requirements for Tex_prog to Tex_term transition through characterization of T cell exhaustion progression at the RNA (paired scRNA and TCR sequencing) and protein level (flow-cytometry). Tex_prog are enriched for pathways of migration, cell-cell adhesion, and differentiation, whereas Tex_term primarily upregulate cytotoxic pathways. TCRseq revealed that clonal expansion is concentrated to the Tex_term cluster, suggesting a link between Tex_prog transition and proliferation in response to antigen. Likewise, we defined the necessity of hematopoietic antigen presentation, but not tumor-derived antigen presentation for the Tex_prog to Tex_term transition. Of the antigen presenting cells in the TME, tumor-associated macrophages (TAM) had the greatest expression of tumor-antigen loaded MHC I, illustrating their capacity to cross-present. By depleting TAM, we observed disruption of the Tex_prog transition. Similar results were seen in subcutaneous and intracranial melanoma, highlighting the role of TAM in this process across tumor histology and location. Lastly, we identified additional inflammatory cell-cell interactions between T cells and TAM in mice and humans that may contribute to this phenomenon. Taken together, we define T cell exhaustion dynamics in models of primary and metastatic brain tumors, where tumor-antigen presentation by TAM is a critical mediator of the loss of Tex_prog. We extend these findings to human data to better define these complex interactions, which we aim to leverage to overcome immunotherapy resistance.

Sézary syndrome originates from heavily mutated hematopoietic progenitors

AbstractThe pathogenesis of cutaneous T-cell lymphoma (CTCL) remains unclear. Using single-cell RNA or T-cell receptor (TCR) sequencing of 32 619 CD3+CD4+ and CD26+/CD7+ and 29 932 CD3+CD4+ and CD26−/CD7− lymphocytes from the peripheral blood of 7 patients with CTCL, coupled to single-cell ATAC-sequencing of 26,411 CD3+CD4+ and CD26+/CD7+ and 33 841 CD3+CD4+ and CD26−/CD7− lymphocytes, we show that tumor cells in Sézary syndrome and mycosis fungoides (MF) exhibit different phenotypes and trajectories of differentiation. When compared to MF, Sézary cells exhibit narrower repertoires of TCRs and exhibit clonal enrichment. Surprisingly, we identified ≥200 mutations in hematopoietic stem cells from multiple patients with Sézary syndrome. Mutations in key oncogenes were also present in peripheral Sézary cells, which also showed the hallmarks of recent thymic egression. Together our data suggest that CTCL arises from mutated lymphocyte progenitors that acquire TCRs in the thymus, which complete their malignant transformation in the periphery.

Abstract P3069: ScRNA-seq And Flow Cytometry Based Analysis Reveals That Chronic Heart Failure Associates With Enhanced Activation And Clonal Expansion Of T Cells With Predictive Autoreactive Capacity

Background: Heart failure (HF) is marked by adverse remodelling and chronic inflammation causing impaired function of the heart and poor prognosis. While much is known about the immune response immediately after MI, little is known of the role of the immune system in chronic HF. Our aim is to elucidate the response of T cells to HF. Methods and Results: In a pilot study assessing healthy controls (HC) and HF patients (n=16), scRNA-seq showed an enhanced T cell activation profile in HF patients. A confirmation cohort of n=180 HC and HF patients using flow cytometry of immune cells confirmed an increase of T cell memory/effector cells (HF=65%, HC=53% activated T cells). Correspondingly, circulating antigen presenting cells showed augmented inflammatory profiles (ICAM-1, TREM-1). Given that T cells showed strong activation profiles in HF patients, we investigated whether T cells are preferentially clonally expanded in the same patients. Bulk T cell receptor (TCR) sequencing revealed that TCR diversity was significantly reduced (p=0.03) along with clonal expansion of T cells in HF patients. To assess what T cells might be targeting, we utilized epitope prediction tools to identify 22 unique human-derived epitopes found only in HF patients. ScRNA-seq of CD45+ sorted cardiac-derived and circulating cells of the same HF patient (N=1) versus healthy controls (n=4) showed clonal expansion of TCRs in the cardiac tissue and blood in HF and epitope prediction confirmed epitopes found in bulk TCR sequencing. When modelling this in mice, a scRNA-seq post-MI time course also found an accumulation of specifically pro-inflammatory (Th17) T cells in the heart at days 14, 28 and 48 post-MI. To assess the impact of immune dysregulation in HF on cardiac cells, we cultured the secretome of HF or HC immune cells with endothelial cells and found decreased viability of endothelial cells and increased monocyte adhesion to stimulated endothelial cells (p=0.005). Conclusion: This study suggests that chronic HF may drive persistent T cell activation and clonal expansion, with potential cardiac-relevant autoimmune implications causing or promoting adverse remodelling. These data also provide additional insights for therapeutic cardio-immunological interventions.

CD39 inhibition and VISTA blockade may overcome radiotherapy resistance by targeting exhausted CD8+ T cells and immunosuppressive myeloid cells

Although radiotherapy (RT) has achieved great success in the treatment of non-small cell lung cancer (NSCLC), local relapses still occur and abscopal effects are rarely seen even when it is combined with immune checkpoint blockers (ICBs). Here, we characterize the dynamic changes of tumor-infiltrating immune cells after RT in a therapy-resistant murine tumor model using single-cell transcriptomes and Tcell receptor sequencing. Atthe early stage, the innate and adaptive immune systems are activated. At the late stage, however, the tumorimmune microenvironment (TIME) shifts into immunosuppressive properties. Our study reveals that inhibition of CD39 combined with RT preferentially decreases the percentage of exhausted CD8+ Tcells. Moreover, we find that the combination of V-domain immunoglobulin suppressor of T cell activation (VISTA) blockade and RT synergistically reduces immunosuppressive myeloid cells. Clinically, high VISTA expression is associated with poor prognosis in patients with NSCLC. Altogether, our data provide deep insight into acquired resistance to RT from an immune perspective and present rational combination strategies.

BERTrand-peptide:TCR binding prediction using Bidirectional Encoder Representations from Transformers augmented with random TCR pairing.

The advent of T-cell receptor (TCR) sequencing experiments allowed for a significant increase in the amount of peptide:TCR binding data available and a number of machine-learning models appeared in recent years. High-quality prediction models for a fixed epitope sequence are feasible, provided enough known binding TCR sequences are available. However, their performance drops significantly for previously unseen peptides. We prepare the dataset of known peptide:TCR binders and augment it with negative decoys created using healthy donors' T-cell repertoires. We employ deep learning methods commonly applied in Natural Language Processing to train part a peptide:TCR binding model with a degree of cross-peptide generalization (0.69 AUROC). We demonstrate that BERTrand outperforms the published methods when evaluated on peptide sequences not used during model training. The datasets and the code for model training are available at https://github.com/SFGLab/bertrand.

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

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

Single-cell T-cell receptor sequencing of paired human atherosclerotic plaques and blood reveals autoimmune-like features of expanded effector T-cells

Single-cell T-cell receptor sequencing of paired human atherosclerotic plaques and blood reveals autoimmune-like features of expanded effector T-cells

The pseudokinase Trib1 regulates the transition of exhausted T cells to a KLR+ CD8+ effector state, and its deletion improves checkpoint blockade

CD8+ Tcell exhaustion (TEX) impairs the ability of Tcells to clear chronic infection or cancer. While TEX are hypofunctional, some TEX retain effector gene signatures, a feature associated with killer lectin-like receptor (KLR) expression. Although KLR+ TEX (TKLR) may improve control of chronic antigen, the signaling molecules regulating this population are poorly understood. Using single-cell RNA sequencing (scRNA-seq), flow cytometry, RNA velocity, and single-cell Tcell receptor sequencing (scTCR-seq), we demonstrate that deleting the pseudokinase Trib1 shifts TEX toward CX3CR1+ intermediates with robust enrichment of TKLR via clonal Tcell expansion. Adoptive transfer studies demonstrate this shift toward CD8+ TKLR in Trib1-deficient cells is CD8 intrinsic, while CD4-depletion studies demonstrate CD4+ Tcells are required for improved viral control in Trib1 conditional knockout mice. Further, Trib1 loss augments anti-programmed death-ligand 1 (PD-L1) blockade to improve viral clearance. These data identify Trib1 as an important regulator of CD8+ TEX whose targeting enhances the TKLR effector state and improves checkpoint inhibitor therapy.

TCR Sequencing in Mouse Models of Allorecognition Unveils the Features of Directly and Indirectly Activated Clonotypes.

Allorecognition is known to involve a large number of lymphocytes carrying diverse T-cell receptor repertoire. Thus, one way to understand allorecognition and rejection mechanisms is via high-throughput sequencing of T-cell receptors. In this study, in order to explore and systematize the properties of the alloreactive T-cell receptor repertoire, we modeled direct and indirect allorecognition pathways using material from inbred mice in vitro and in vivo. Decoding of the obtained T-cell receptor genes using high-throughput sequencing revealed some features of the alloreactive repertoires. Thus, alloreactive T-cell receptor repertoires were characterized by specific V-gene usage patterns, changes in CDR3 loop length, and some amino acid occurrence probabilities in the CDR3 loop. Particularly pronounced changes were observed for directly alloreactive clonotypes. We also revealed a clustering of directly and indirectly alloreactive clonotypes by their ability to bind a single antigen; amino acid patterns of the CDR3 loop of alloreactive clonotypes; and the presence in alloreactive repertoires of clonotypes also associated with infectious, autoimmune, and tumor diseases. The obtained results were determined by the modeling of the simplified allorecognition reaction in inbred mice in which stimulation was performed with a single MHCII molecule. We suppose that the decomposition of the diverse alloreactive TCR repertoire observed in humans with transplants into such simple reactions will help to find alloreactive repertoire features; e.g., a dominant clonotype or V-gene usage pattern, which may be targeted to correct the entire rejection reaction in patients. In this work, we propose several technical ways for such decomposition analysis, including separate modeling of the indirect alloreaction pathway and clustering of alloreactive clonotypes according to their ability to bind a single antigen, among others.

CD4 T cells and toxicity from immune checkpoint blockade.

Immune-related toxicities, otherwise known as immune-related adverse events (irAEs), occur in a substantial fraction of cancer patients treated with immune checkpoint inhibitors (ICIs). Ranging from asymptomatic to life-threatening, ICI-induced irAEs can result in hospital admission, high-dose corticosteroid treatment, ICI discontinuation, and in some cases, death. A deeper understanding of the factors underpinning severe irAE development will be essential for improved irAE prediction and prevention, toward maximizing the benefits and safety profiles of ICIs. In recent work, we applied mass cytometry, single-cell RNA sequencing, single-cell V(D)J sequencing, bulk RNA sequencing, and bulk T-cell receptor (TCR) sequencing to identify pretreatment determinants of severe irAE development in patients with advanced melanoma. Across 71 patients separated into three cohorts, we found that two baseline features in circulation-elevated activated CD4 effector memory T-cell abundance and TCR diversity-are associated with severe irAE development, independent of the affected organ system within 3 months of ICI treatment initiation. Here, we provide an extended perspective on this work, synthesize and discuss related literature, and summarize practical considerations for clinical translation. Collectively, these findings lay a foundation for data-driven and mechanistic insights into irAE development, with the potential to reduce ICI morbidity and mortality in the future.

On the feasibility of using TCR sequencing to follow a vaccination response - lessons learned.

T cells recognize pathogens by their highly specific T-cell receptor (TCR), which can bind small fragments of an antigen presented on the Major Histocompatibility Complex (MHC). Antigens that are provided through vaccination cause specific T cells to respond by expanding and forming specific memory to combat a future infection. Quantification of this T-cell response could improve vaccine monitoring or identify individuals with a reduced ability to respond to a vaccination. In this proof-of-concept study we use longitudinal sequencing of the TCRβ repertoire to quantify the response in the CD4+ memory T-cell pool upon pneumococcal conjugate vaccination. This comes with several challenges owing to the enormous size and diversity of the T-cell pool, the limited frequency of vaccine-specific TCRs in the total repertoire, and the variation in sample size and quality. We defined quantitative requirements to classify T-cell expansions and identified critical parameters that aid in reliable analysis of the data. In the context of pneumococcal conjugate vaccination, we were able to detect robust T-cell expansions in a minority of the donors, which suggests that the T-cell response against the conjugate in the pneumococcal vaccine is small and/or very broad. These results indicate that there is still a long way to go before TCR sequencing can be reliably used as a personal biomarker for vaccine-induced protection. Nevertheless, this study highlights the importance of having multiple samples containing sufficient T-cell numbers, which will support future studies that characterize T-cell responses using longitudinal TCR sequencing.

Lsd1 safeguards T-cell development via suppressing endogenous retroelements and interferon responses.

The histone demethylase Lsd1 has been shown to play multiple essential roles in mammalian biology. However, its physiological functions in thymocyte development remain elusive. We observed that the specific deletion of Lsd1 in thymocytes caused significant thymic atrophy and reduced peripheral T cell populations with impaired proliferation capacity. Single-cell RNA sequencing combined with strand-specific total RNA-seq and ChIP-seq analysis revealed that ablation of Lsd1 led to the aberrant derepression of endogenous retroelements, which resulted in a viral mimicry state and activated the interferon pathway. Furthermore, the deletion of Lsd1 blocked the programmed sequential down-regulation of CD8 expression at the DP→CD4+CD8lo stage and induced an innate memory phenotype in both thymic and peripheral T cells. Single-cell TCR sequencing revealed the kinetics of TCR recombination in the mouse thymus. However, the preactivation state after Lsd1 deletion neither disturbed the timeline of TCR rearrangement nor reshaped the TCR repertoire of SP cells. Overall, our study provides new insight into the function of Lsd1 as an important maintainer of endogenous retroelement homeostasis in early T-cell development.

LRT: Integrative analysis of scRNA-seq and scTCR-seq data to investigate clonal differentiation heterogeneity.

Single-cell RNA sequencing (scRNA-seq) data has been widely used for cell trajectory inference, with the assumption that cells with similar expression profiles share the same differentiation state. However, the inferred trajectory may not reveal clonal differentiation heterogeneity among T cell clones. Single-cell T cell receptor sequencing (scTCR-seq) data provides invaluable insights into the clonal relationship among cells, yet it lacks functional characteristics. Therefore, scRNA-seq and scTCR-seq data complement each other in improving trajectory inference, where a reliable computational tool is still missing. We developed LRT, a computational framework for the integrative analysis of scTCR-seq and scRNA-seq data to explore clonal differentiation trajectory heterogeneity. Specifically, LRT uses the transcriptomics information from scRNA-seq data to construct overall cell trajectories and then utilizes both the TCR sequence information and phenotype information to identify clonotype clusters with distinct differentiation biasedness. LRT provides a comprehensive analysis workflow, including preprocessing, cell trajectory inference, clonotype clustering, trajectory biasedness evaluation, and clonotype cluster characterization. We illustrated its utility using scRNA-seq and scTCR-seq data of CD8+ T cells and CD4+ T cells with acute lymphocytic choriomeningitis virus infection. These analyses identified several clonotype clusters with distinct skewed distribution along the differentiation path, which cannot be revealed solely based on scRNA-seq data. Clones from different clonotype clusters exhibited diverse expansion capability, V-J gene usage pattern and CDR3 motifs. The LRT framework was implemented as an R package 'LRT', and it is now publicly accessible at https://github.com/JuanXie19/LRT. In addition, it provides two Shiny apps 'shinyClone' and 'shinyClust' that allow users to interactively explore distributions of clonotypes, conduct repertoire analysis, implement clustering of clonotypes, trajectory biasedness evaluation, and clonotype cluster characterization.