Biology subjects

Tamhane, T.

Publications and source records attributed to Tamhane, T..

7 recordsLinked to original sources

bioRxiv2026

DNA-barcode labelled MHCII multimers for detection of antigen-specific CD4 T cells across large libraries of epitopes

The role of antigen-specific T cells responding to antigen is a topic of intense studies, and critical for mechanistic insight of diseases and development of therapeutic strategies. Methods for broad-scale detection of antigen-specific CD4 T cells are lacking, while such methods have demonstrated great value in exploring CD8 T cell response in health and disease. Furthermore, major histocompatibility complex II (MHCII) assays are technically challenging due to high HLA diversity, lower binding affinities, low frequencies of ex vivo antigen-specific CD4 T cells and several bottlenecks in production and peptide exchange of MHCII monomers. Here we use peptide-loaded MHCII (pMHCII) proteins multimerized on a barcode- and fluorophore-labelled dextran backbone to provide a method for the detection of peptide-specific CD4 T cells by using a large display of MHCII-associated peptides. We have established a protocol for MHCII production and peptide-exchange suitable for the generation of large libraries of peptide-MHCII complexes. We validate the use of such pMHCII complexes in the form of barcode-labelled MHCII multimers to detect antigen-specific CD4 T cells. We demonstrate that we can identify antigen specific CD4 T cells, using these DNA barcoded peptide-MHCII multimer. The multimer bound CD4 T cells were selected based on the fluorochrome signal, and the co-attached DNA barcodes were hereafter amplified and used to identify the peptide-MHCII response/binding. In cases where the peptide-specific CD4 T cells frequencies are very low, we expanded the cell population with peptide-pools and in the presence of IL2. The given CD4 T cell populations hereby reach a cell number allowing for the DNA-barcoded pMHCII multimers to detect responses otherwise missed out. Applying this technology, we utilized a panel of 150 peptides derived from human cytomegalo virus (CMV), Epstein barr virus (EBV), Influenza (Flu), SARS CoV 2 and SARS CoV1, Hepatitis B virus (HBV), and Hepatitis C virus (HCV) loaded onto HLA-DRB1*01:01 and DRB1*04:01 to screen peripheral blood mononuclear cells (PBMC). We assessed ex vivo responses in 16 participants with HCV infection, and successfully detected naturally occurring viral-specific CD4 T cells at frequencies as low as 0.004% of total CD4 T cells. The low-frequency responses, identified via the barcode screen, were rigorously validated using individual fluorophore-labelled tetramer staining after a peptide-driven expansion in 15 participants. Furthermore, we assessed the recognition of novel HCV epitopes in 11 additional participants. Through this, we identified a total of 12 distinct HCV epitopes, including 9 that have not been previously utilized in assays to detect CD4 T cells. Overall, this barcoded-multimer platform provides a powerful tool for the large-scale discovery of class II epitopes and the broad profiling of CD4 T cell specificities. This method will allow for in-depth analyses of immune interactions, provide a better understanding of the antigen-driven associations between CD4 and CD8 T cell responses, and help dissect the complexities of CD4 T cell protection in HCV infection.

immunology↗

bioRxiv2025

Mapping of CD8 T-cell recognition to latent EBV infection and neuroantigens reveals HLA-specific depletion of T-cell responses in multiple sclerosis

Multiple sclerosis (MS) is a complex autoimmune disease with an unclear contribution from antigen-specific CD8 T cells. To enumerate adaptive immune responses associated with MS and to identify MS-related epitopes recognized by CD8 T cells, we evaluated T-cell recognition of 1,158 predicted major histocompatibility complex (MHC)-binding peptides derived from latent EBV antigens (laEBVs) and selected neuroantigens (NAgs) using DNA barcode-labelled pMHC multimers. HLA-B*07:02+ individuals living with relapsing-remitting MS (RRMS) and progressive MS (PMS) were characterized by a relative reduction in EBNA6-specific and MAG-specific CD8 T cells compared to healthy donors. Furthermore, both RRMS and PMS patients exhibited a delayed expansion of CMV- and laEBV-specific CD8 T cells compared to healthy donors indicating an age-specific interaction with disease. Female RRMS patients were moreover characterized by a specific reduction of laEBV-specific CD8 T cells compared to male RRMS patients. NAg-specific CD8 T cells recognized heterogeneous peptides, were of low cellular frequency in peripheral blood, and exhibited a circulating, naive-like, anergic phenotype. In contrast, virus-specific CD8 T cells were characterized by high-frequency cellular responses with an effector-like, tissue-homing phenotype. These immunophenotypes were largely shared between MS and healthy donors. Collectively, our data provide novel insights into CD8 T-cell recognition of both NAg and laEBV antigens in MS and constitute a key reference map of epitopes for further immunomonitoring of EBV- and self-specific CD8 T cells in health and disease.

immunology↗

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↗

bioRxiv2023

Simultaneous analysis of pMHC binding and reactivity unveils virus-specific CD8 T cell immunity to a concise epitope set

Knowledge of widely recognized T-cell epitopes against common virus infections are vital for immune monitoring and characterization of relevant antigen-specific CD8 T cells and their antigen receptors. We therefore aimed to establish a concise and validated epitope panel for monitoring human virus-specific immunity complete with data on both prevalence of recognition and reactivity in humans. To achieve this, we first establish TCR downregulation, and loss of peptide major histocompatibility (pMHC) multimer-binding, as an early and sensitive marker of T cell reactivity after peptide stimulation. We next applied TCR downregulation in a high-throughput assay by monitoring binding, and loss of binding (i.e. reactivity), to libraries of DNA-barcode labelled pMHC multimers in paired unstimulated/stimulated samples. This novel method allowed us to access T-cell responses in 48 donors towards 929 epitopes recorded in the Immune Epitope Database (IEDB) encompassing 29 virus common infections and 25 different HLA alleles. This yielded a concise panel of 137 virus epitopes, many of which were underrepresented in the public domain, recognized by T cells in peripheral blood. 84% of these epitopes exhibited prevalent reactivity to peptide stimulation, which was associated with effector and long-term memory phenotypes. Conversely, non-reactive responses correlated with naive and immunosenescence phenotypes. This study represents the largest effort to unbiasedly assess T-cell recognition and reactivity to common virus infections in healthy individuals providing a minimal epitope panel for monitoring adaptive immune responses in humans. Significance StatementCD8 T-cell epitopes are widely available in public databases yet many are not recognized in the general population. Here we undertook an exhaustive screening process using "state-of-the-art" methods to assess both T-cell recognition and reactivity against common virus infections, which holds significant implications for shaping T-cell immunity and disease protection. We identify 137 commonly recognized epitopes from common virus infections to which T cell responses are expected to occur in human donors. Importantly, several of the verified epitopes were underreported in public databases compared to their observed prevalence of recognition and high cellular frequency making this an important reference dataset and resource for immunologists studying antigen-specific T cells across different immunopathologies and contexts including autoimmunity, infectious disease and cancer immunotherapy.

immunology↗

bioRxiv2022

Three-doses of BNT162b2 COVID-19 mRNA vaccine establishes long-lasting CD8+ T cell immunity in CLL and MDS patients

Patients with hematological malignancies are prioritized for COVID-19 vaccine due to their high risk for severe SARS-CoV-2 infection related disease and mortality. To understand T cell immunity, its long-term persistence, and correlation with antibody response, we evaluated the BNT162b2 COVID-19 mRNA vaccine-specific immune response in chronic lymphocytic leukemia (CLL) and myeloid dysplastic syndrome (MDS) patients. Longitudinal analysis of CD8+ T cells using DNA-barcoded peptide-MHC multimers covering the full SARS-CoV-2 Spike-protein (415 peptides) showed vaccine-specific T cell activation and persistence of memory T cells up to six months post-vaccination. Surprisingly, a higher frequency of vaccine-induced antigen-specific CD8+ T cell was observed in the patient group compared to a healthy donor group. Furthermore, and importantly, immunization with the second booster dose significantly increased the frequency of antigen-specific CD8+ T cells as well as the total number of T cell specificities. Altogether 59 BNT162b2 vaccine-derived immunogenic epitopes were identified, of which 23 established long-term CD8+ T cell memory response with a strong immunodominance for NYNYLYRLF (HLA-A24:02) and YLQPRTFLL (HLA-A02:01) epitopes. In summary, we mapped the vaccine-induced antigen-specific CD8+ T cells and showed a booster-specific activation and enrichment of memory T cells that could be important for long-term disease protection in this patient group. Key PointsO_LICOVID-19 mRNA vaccine induced an early and persistent activation of antigen-specific CD8+ T cells in this patient group. C_LIO_LIVaccination with a booster dose is required to maintain vaccine-specific CD8+ T cells. C_LI

immunology↗

bioRxiv2020

SARS-CoV-2 genome-wide mapping of CD8 T cell recognition reveals strong immunodominance and substantial CD8 T cell activation in COVID-19 patients

To understand the CD8+ T cell immunity related to viral protection and disease severity in COVID-19, we evaluated the complete SARS-CoV-2 genome (3141 MHC-I binding peptides) to identify immunogenic T cell epitopes, and determine the level of CD8+ T cell involvement using DNA-barcoded peptide-major histocompatibility complex (pMHC) multimers. COVID-19 patients showed strong T cell responses, with up to 25% of all CD8+ lymphocytes specific to SARS-CoV-2-derived immunodominant epitopes, derived from ORF1 (open reading frame 1), ORF3, and Nucleocapsid (N) protein. A strong signature of T cell activation was observed in COVID-19 patients, while no T cell activation was seen in the non-exposed and high exposure risk healthy donors. Interestingly, patients with severe disease displayed the largest T cell populations with a strong activation profile. These results will have important implications for understanding the T cell immunity to SARS-CoV-2 infection, and how T cell immunity might influence disease development.

immunology↗