Abstract
Checkpoint blockade immunotherapy has shown great success in certain types of cancers, but are ineffective in most cancers. We do not understand why these therapies fail.
Here, we seek to ...better understand functional changes in T cells in responsive tumors through single cell TCR sequencing and high dimensional phenotypic analysis within single tumor-infiltrating T cells. Through surgical biopsy, we analyze the same tumor longitudinally before and after treatment, enabling us to track the fate of individual T cell clones.
Our data show that the CD4/CD8 ratio decreases upon treatment. T cells expressing inhibitory markers such as PD1, CTLA4, diminish post treatment. We see distinct clusters of effector CD4 and CD8 T cells in treated tumors, including effector CD4 populations with Tfh, Th1 and Th2 phenotypes. CD4 Tregs are greatly diminished upon successful treatment, and diminish through clonal contraction. Furthermore, TCR acts as a unique marker for T cell ancestry, which links T cell function to T cell specificity. Based on TCR clonality, we observe that expanded T cell clones before treatment do not share the same TCRs with expanded T cell clones after treatment. This implies new CD8 T cell clones are recruited into the tumor upon treatment.
From our data, we will be able to identify T cell clones that are phenotypically distinct and expanded in responsive tumors which is critical in understanding what makes for a successful T cell response after immunotherapy.
Expressed on epidermal Langerhans cells, CD1a presents a range of self-lipid antigens found within the skin; however, the extent to which CD1a presents microbial ligands from bacteria colonizing the ...skin is unclear. Here we identified CD1a-dependent T cell responses to phosphatidylglycerol (PG), a ubiquitous bacterial membrane phospholipid, as well as to lysylPG, a modified PG, present in several Gram-positive bacteria and highly abundant in Staphylococcus aureus. The crystal structure of the CD1a-PG complex showed that the acyl chains were buried within the A'- and F'-pockets of CD1a, while the phosphoglycerol headgroup remained solvent exposed in the F'-portal and was available for T cell receptor contact. Using lysylPG and PG-loaded CD1a tetramers, we identified T cells in peripheral blood and in skin that respond to these lipids in a dose-dependent manner. Tetramer
CD4
T cell lines secreted type 2 helper T cell cytokines in response to phosphatidylglycerols as well as to co-cultures of CD1a
dendritic cells and Staphylococcus bacteria. The expansion in patients with atopic dermatitis of CD4
CD1a-(lysyl)PG tetramer
T cells suggests a response to lipids made by bacteria associated with atopic dermatitis and provides a link supporting involvement of PG-based lipid-activated T cells in atopic dermatitis pathogenesis.
T cells are implicated in the pathogenesis of cardiac allograft vasculopathy (CAV), yet their clonality, specificity, and function are incompletely defined. Here we used T cell receptor β chain ...(TCRB) sequencing to study the T cell repertoire in the coronary artery, endomyocardium, and peripheral blood at the time of retransplant in four cases of CAV and compared it to the immunoglobulin heavy chain variable region (IGHV) repertoire from the same samples. High‐dimensional flow cytometry coupled with single‐cell PCR was also used to define the T cell phenotype. Extensive overlap was observed between intragraft and blood TCRBs in all cases, a finding supported by robust quantitative diversity metrics. In contrast, blood and graft IGHV repertoires from the same samples showed minimal overlap. Coronary infiltrates included CD4+ and CD8+ memory T cells expressing inflammatory (IFNγ, TNFα) and profibrotic (TGFβ) cytokines. These were distinguishable from the peripheral blood based on memory, activation, and tissue residency markers (CD45RO, CTLA‐4, and CD69). Importantly, high‐frequency rearrangements were traced back to endomyocardial biopsies (2–6 years prior). Comparison with four HLA‐mismatched blood donors revealed a repertoire of shared TCRBs, including a subset of recently described cross‐reactive sequences. These findings provide supportive evidence for an active local intragraft bystander T cell response in late‐stage CAV.
Repertoire analysis and single‐cell phenotypic characterization of T cell infiltrates during cardiac allograft vasculopathy suggests a predominance of bystander T cells within graft‐infiltrating T cells.
T cells are implicated in the pathogenesis of cardiac allograft vasculopathy (CAV), yet their clonality, specificity, and function are incompletely defined. Here we used T-cell receptor β chain ...(TCRB) sequencing to study the T-cell repertoire in the coronary artery, endomyocardium, and peripheral blood at the time of retransplant in 4 cases of CAV and compared it to the immunoglobulin heavy chain (IGHV) repertoire from the same samples. High-dimensional flow cytometry coupled with single-cell PCR was used to define the T-cell phenotype. Extensive overlap was observed between intragraft and blood TCRBs in all cases, a finding supported by robust quantitative diversity metrics. In contrast, blood and graft IGHV repertoires from the same samples showed minimal overlap. Coronary infiltrates included CD4+ and CD8+ memory T cells expressing inflammatory (IFNγ, TNFα) and profibrotic (TGFβ) cytokines. These were distinguishable from the peripheral blood based on memory, activation, and tissue residency markers (CD45RO, CTLA-4 and CD69). Importantly, high frequency rearrangements were traced back to endomyocardial biopsies (2–6 years prior). Comparison with 4 HLA mismatched blood donors revealed a repertoire of shared TCRBs, including a subset of recently described crossreactive sequences. These findings provide supportive evidence for an active local intragraft bystander T-cell response in late-stage CAV.
Interleukin (IL)-17 plays a key role in immunity. In acute infections, a rapid IL-17 response must be induced without prior antigen exposure, and γδ T cells are the major initial IL-17 producers. In ...fact, some γδ T cells make IL-17 within hours after an immune challenge. These cells appear to acquire the ability to respond to IL-1 and IL-23 and to make IL-17 naturally in naïve animals. They are known as the natural Tγδ17 (nTγδ17) cells. The rapidity of the nTγδ17 response, and the apparent lack of explicit T cell receptor (TCR) engagement for its induction have led to the view that this is a cytokine (IL-1, IL-23)-mediated response. However, pharmacological inhibition or genetic defects in TCR signaling drastically reduce the nTγδ17 response and/or their presence. To better understand antigen recognition in this rapid IL-17 response, we analyzed the antigen receptor repertoire of IL-1R(+)/IL-23R(+) γδ T cells, a proxy for nTγδ17 cells in naïve animals directly ex vivo, using a barcode-enabled high throughput single-cell TCR sequence analysis. We found that regardless of their anatomical origin, these cells have a highly focused TCR repertoire. In particular, the TCR sequences have limited V gene combinations, little or no junctional diversity and much reduced or no N region diversity. In contrast, IL-23R(-) cells at mucosal sites similar to most of the splenic γδ T cells and small intestine epithelial γδ lymphocytes expressed diverse TCRs. This remarkable commonality and restricted repertoire of IL-1R(+)/IL-23R(+) γδ T cells underscores the importance of antigen recognition in their establishment/function.
Abstract
Analysis of TCR repertoires is useful for monitoring T cell responses. Integrating TCR sequencing with expression of targeted genes at the single cell level allows comprehensive analysis of ...T cell function and specificity. RT-PCR reactions of single T cells were performed using 76 primers from VJC regions and 136 primers for targeted T cell genes. A series of nested PCR reactions incorporated with barcodes and adapters for pooled amplicons sequencing. A bioinformatics pipeline was used data analysis. 2880 single T cells were collected and tested. TCR sequences were obtained in up to 90% of the wells. Based on the CDR3 region, multiple dominant TCR clones were identified. 52 of 68 target genes were validated with sorted target-specific T cells. FOXP3, IL10, PRF1, IL13, and RUNX3 were highly expressed in memory activated cells. The specific CD8+ T cells had high frequency of CCR9, LAG3, CD8, CD62L, and IFNG expression. TGFB, TNFA, IL12, FOXP3, PD1, TBET, CTLA4, MKI67, RUNX1, and GATA3 were major biomarkers in the specific CD4 cells. The gene expression patterns were often associated with TCR repertoire CDR3 variations and/or the TCRβ and TCRα chain usage although the same TCRα/β sequences may have different target genes expressed. We expanded a method enabling sequencing of TCR repertoire and multiple functional genes in single, sorted, T cells through targeted RNA-seq technology. This approach could reveal important insights into T cell functions and clonal development.
Celiac disease is an intestinal autoimmune disease driven by dietary gluten and gluten-specific CD4+ T-cell responses. In celiac patients on a gluten-free diet, exposure to gluten induces the ...appearance of gluten-specific CD4+ T cells with gut-homing potential in the peripheral blood. Here we show that gluten exposure also induces the appearance of activated, gut-homing CD8+ αβ and ... T cells in the peripheral blood. Single-cell T-cell receptor sequence analysis indicates that both of these cell populations have highly focused T-cell receptor repertoires, indicating that their induction is antigen-driven. These results reveal a previously unappreciated role of antigen in the induction of CD8+ αβ and ... T cells in celiac disease and demonstrate a coordinated response by all three of the major types of T cells. More broadly, these responses may parallel adaptive immune responses to viral pathogens and other systemic autoimmune diseases. (ProQuest: ... denotes formulae/symbols omitted.)
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