Biology subjects

Kadivar, M.

Publications and source records attributed to Kadivar, M..

7 recordsLinked to original sources

bioRxiv2026

KIR Expression Defines a Transcriptionally Distinct CD8+ T-cell Population that Confounds Antigen-Specific T-cell Detection

HLA-C-restricted CD8+ T-cell responses remain poorly defined compared with HLA-A and HLA-B responses, despite the central role of HLA-C as both a peptide-presenting molecule and a ligand for killer-cell immunoglobulin-like receptors (KIRs). This dual function creates a major challenge for pHLA multimer-based antigen-specific T-cell analysis, as HLA-C multimers may bind CD8+ T-cells through either antigen-specific TCRs or KIRs. Here, we show that KIR expression on CD8+ T-cells drives TCR-independent binding to pHLA-C multimers, leading to overestimation of HLA-C-restricted antigen-specific T-cell responses. Using HLA-mismatched donor settings and cancer neoantigen pHLA-C multimers, we demonstrate that apparent multimer-positive CD8+ T-cells can arise from KIR-mediated recognition rather than cognate TCR specificity. We further show that KIR blockade before pHLA-C multimer staining removes non-TCR-specific binding, and applying this strategy to large-scale analysis of CMV, EBV, and SARS-CoV-2 antigens, we identify TCR-specific HLA-C-restricted CD8+ T-cell populations. Importantly, KIR-mediated pHLA-C binding is peptide-selective rather than uniform across all peptide-HLA-C complexes. Single-cell transcriptomic, phenotypic, and clonotypic analyses further demonstrate that KIR-mediated and TCR-mediated pHLA-C multimer binding define transcriptionally and clonally distinct CD8+ T-cell populations. Together, these findings establish KIR-mediated pHLA-C recognition as a major confounder in HLA-C multimer-based T-cell analysis. By separating KIR- from TCR-mediated binding, our approach provides a practical framework for accurate discovery and characterization of HLA-C-restricted antigen-specific CD8+ T-cells across viral infection and cancer.

immunology↗

bioRxiv2026

Impaired HPV driven CD8 T cells recognition and Immune suppression in HPV-Induced Cervical Cancer

Human papillomavirus (HPV) remains the leading cause of cervical cancer, yet the mechanisms underlying its immune evasion remain poorly defined. We performed an integrated analysis of HPV-specific CD8 T cell responses and the immune microenvironment in cervical cancer, high-grade intraepithelial neoplasia (CIN3), and healthy controls using flow cytometry, transcriptomics, and DNA-barcoded peptide-MHC multimer screening across cervical biopsies, peripheral blood, and liquid-based cytology (LBC). Cervical cancer tissues exhibited a profoundly immunosuppressive milieu, with enrichment of exhausted CD8 and CD4 T cells, increased regulatory T cells, and PD-L1-expressing myeloid subsets. Conventional dendritic cells and macrophages showed reduced frequencies, while plasmacytoid dendritic cells and intermediate monocytes were elevated. Interestingly, LBC samples reliably reflected T cell exhaustion signatures observed in biopsies, supporting their use as a minimally invasive tool for T cell immune monitoring; however, they were not suitable for detailed myeloid profiling. Transcriptomic analysis revealed distinct gene expression profiles in tumor tissues with elevated signatures of immune checkpoints and regulatory immune cell infiltration. Importantly, HPV-specific CD8 T cell responses were significantly reduced in cancer patients compared to CIN3 and controls, with decreased breadth and frequency of HPV peptides recognition. HPV peptides screening identified HPV-specific CD8 T cell responses toward 109 unique peptide-MHC complexes, including 37 novel HPV-derived peptide from E2, E6, and E7 proteins. These findings reveal impaired HPV immune recognition and a suppressive tumor microenvironment in cervical cancer, underscoring the need to enhance HPV-specific T cell responses and target immune suppression in therapeutic strategies.

immunology↗

bioRxiv2025

ITRAP2, a flexible and robust strategy to assign antigen recognition of T-cells in a coupled single-cell TCR-pMHC assay

Determining T-cell specificity forms a crucial step toward understanding T-cell involvement in health and disease. Single-cell sequencing technologies allow for co-capture of TCR alpha and beta chains, and their antigen specificity can be determined through peptide-MHC (pMHC) multimer binding and capture of a co-attached barcode oligo. However, SC sequencing often includes a high level of dropouts and risk of cross-contamination. Similarly, barcoded pMHC readouts often suffer from significant background noise. These issues complicate the automatic assignment of pMHC recognition to TCR clonotypes. To overcome these challenges, we developed a method for data denoising - Improved T-cell Receptor Antigen Paring 2 (ITRAP2). This approach significantly reduces noise in single-cell pMHC readouts and allows for accurate identification of TCR specificity. ITRAP2 incorporates statistical tests and confidence metrics for each TCR-pMHC pairing, offering user flexibility in pairing rigor, and allows multiple pMHC assignments to the same T-cell clone in the event of cross-binding within the pMHC multimer library. We tested this method on an in-house generated dataset of 8141 single cells, screened for CD8 T-cell binding using a panel of 100 different barcode-labelled pMHC multimers holding virus-derived peptides, and on a larger public dataset from 10x Genomics with 208,589 T-cells evaluated for recognition using a panel of 50 different pMHCs. In both datasets, ITRAP2 was able to recover TCR-pMHC hits that were missed either when investigating the raw data or analyzing the data using alternative tools. Importantly, we demonstrate that the size of the pMHC multimer library is crucial for accurate pMHC-TCR pairing and that a minimum of 25 pMHC multimer should be included to optimally determine background characteristic, and assigning true positive events.

bioinformatics↗

bioRxiv2025

Antigen-scaffolds loaded with hyper-stable Neoleukin-2/15 expand antigen-specific T cells with a favorable phenotype for adoptive cell therapy

Adoptive cell therapy (ACT) has shown promising results in cancer treatment, however, achieving effective ex vivo expansion of potent, functionally active, and cytotoxic T cells remains challenging. To overcome this, we loaded the engineered cytokine Neoleukin-2/15 (Neo2/15) on our recently established artificial antigen-presenting scaffolds (Ag-scaffolds) to expand antigen-specific T cells. Neo2/15 selectively binds to IL-2R{beta}/{gamma} receptors, enhancing CD8+ T cell proliferation while limiting regulatory T cell expansion. Our study assessed the efficacy of Neo2/15-loaded Ag-scaffolds (Ag-Neo2/15 scaffolds) in expanding antigen-specific T cells from peripheral blood mononuclear cells (PBMCs) of healthy donors. We optimized Ag-scaffold configurations by varying the number of Neo2/15 molecules loaded on Ag-scaffolds and evaluated their impact on T-cell expansion and functionality. We showed that Ag-Neo2/15 scaffolds promoted significant T-cell expansion, with a comparable frequency of antigen-specific CD8+ T cells compared to IL-2/IL-21-loaded Ag-scaffolds (Ag-IL2/21 scaffolds). The CD8+ T cells expanded with Ag-Neo2/15 scaffolds exhibited potent TNF and IFN{gamma} production and expressed high levels of 4{beta}7 integrin, a homing molecule which is important for directing T cells to specific tissues, potentially enhancing their therapeutic potential. T cells expanded with Ag-Neo2/15 scaffolds had superior and durable cytotoxicity against tumor target cells compared to T cells expanded with Ag-IL2/21 scaffolds. These findings were further supported by our single-cell analysis revealing that T cells expanded with Ag-Neo2/15 scaffolds had higher cytotoxic scores and lower dysfunctionality scores compared to T cells expanded with Ag-IL2/21 scaffolds. The single-cell analysis also indicated increased expression of genes linked to cell division and enhanced proliferative capacity in Ag-Neo2/15 expanded T cells. Furthermore, TCR clonality analysis demonstrated that Ag-Neo2/15 scaffolds promoted the expansion of functionally superior T-cell clones. The top clones of CD8+ T cells expanded with Ag-Neo2/15 scaffolds exhibited a favorable phenotype, essential for effective antigen recognition and sustained T-cell mediated cytotoxicity. Our findings suggest that Ag-Neo2/15 scaffolds represent an advancement in ACT by producing high-quality, functional antigen-specific T cells. This method has the potential to improve clinical outcomes in cancer therapy by generating large numbers of highly functional T cells, thereby optimizing the balance between cytotoxicity and proliferation capacity with less exhausted T-cells in expansion protocols.

immunology↗

bioRxiv2025

Comprehensive longitudinal profiling of SARS-CoV-2-specific CD8+ T-cells reveal strong functional impairment and recognition bias as markers for disease severity

CD8+ T-cells are essential for controlling and resolving SARS-CoV-2 infection, yet their antigen-specific resolution in relation to disease severity, functional dynamics during acute infection, and long-term memory formation remain incompletely understood. Using comprehensive longitudinal profiling of 553 SARS-CoV-2 immunogenic antigens across globally prevalent HLAs, we identified antigen-specific CD8+ T-cell responses that were either critical for early viral clearance or associated with severe disease outcomes. During acute infection, patients with severe COVID-19 exhibited a broader and more robust CD8+ T-cell response than those with mild disease. Notably, we identified HLA-A1-restricted immunodominant antigen-specific T-cells strongly associated with severe disease. These T-cells were present at extremely high frequencies but showed significantly reduced expression of cytotoxic molecules at both the transcriptomic (PRF1, GZMB, GZMH, GNLY) and protein levels (IFN-{gamma}, TNF-, IL-2), as revealed by multidimensional single-cell and cytokine profiling. In contrast, patients with mild disease had T-cells that recognized a more restricted set of antigens, showed only partial overlap with those in severe cases, and showed enhanced cytotoxicity, along with enrichment in gene sets associated with cytotoxic function, hypoxia, and glycolysis. Furthermore, the long-term memory CD8+ T-cells were maintained for a limited subset of immunodominant antigens, with their persistence correlating with their initial frequency during infection. Importantly, SARS-CoV-2 vaccination following infection expanded the long-term T-cell repertoire by enhancing pre-existing responses and generating de novo responses, regardless of prior disease severity. These findings resolve the antigen-specific kinetics and durability of CD8+ T-cells in SARS-CoV-2 infection and provide key insights into their functional landscape. This knowledge could inform future vaccine strategies and therapeutic interventions to enhance protective immunity against emerging viral threats.

immunology↗

bioRxiv2024

Identification and characterization of neoantigen-reactive CD8+ T cells following checkpoint blockade therapy in a pan-cancer setting

BackgroundImmune checkpoint blockade (ICB) has been approved as first-line or second-line therapies for an expanding list of malignancies. T cells recognizing mutation-derived neoantigens are hypothesized to play a major role in tumor elimination. However, the dynamics and characteristics of such neoantigen-reactive T cells (NARTs) in the context of ICB are still limitedly understood. MethodsTo explore this, tumor biopsies and peripheral blood were obtained pre- and post-treatment from 20 patients with solid metastatic tumors, in a Phase I basket trial. From whole-exome sequencing and RNA-seq data, patient-specific libraries of neopeptides were predicted and screened with DNA barcode-labeled MHC multimers for CD8+ T cell reactivity, in conjunction with the evaluation of T cell phenotype. ResultsWe were able to detect NARTs in the peripheral blood and tumor biopsies for the majority of the patients; however, we did not observe any significant difference between the disease control and progressive disease patient groups, in terms of the breadth and magnitude of the detected NARTs. We also observed that the hydrophobicity of the peptide played a role in defining neopeptides resulting in NARTs response. A trend towards a treatment-induced phenotype signature was observed in the NARTs post-treatment, with the appearance of Ki67+ CD27+ PD-1+ subsets in the PBMCs and CD39+ Ki67+ TCF-1+ subsets in the TILs. Finally, the estimation of T cells from RNAseq was increasing post versus pre-treatment for disease control patients. ConclusionOur data demonstrates the possibility of monitoring the characteristics of NARTs from tumor biopsies and peripheral blood, and that such characteristics could potentially be incorporated with other immune predictors to understand further the complexity governing clinical success for ICB therapy.

immunology↗

bioRxiv2022

ATRAP - Accurate T cell Receptor Antigen Pairing through data-driven filtering of sequencing information from single-cells

Novel single-cell based technologies hold the promise of matching T cell receptor (TCR) sequences with their cognate peptide-MHC recognition motif in a high-throughput manner. Parallel capture of TCR transcripts and peptide-MHC is enabled through the use of reagents labeled with DNA barcodes. However, analysis and annotation of such single-cell sequencing (SCseq) data is challenged by dropout, random noise, and other technical artifacts that must be carefully handled in the downstream processing steps. We here propose a rational, data-driven method termed ATRAP (Accurate T cell Receptor Antigen Paring) to deal with these challenges, filtering away likely artifacts, and enable the generation of large sets of TCR-pMHC sequence data with a high degree of specificity and sensitivity, thus outputting the most likely pMHC target per T cell. We have validated this approach across 10 different virus-specific T cell responses in 16 healthy donors. Across these samples we have identified up to 1494 high-confident TCR-pMHC pairs derived from 4135 single-cells.

immunology↗