Biology subjects

Sant, A. J.

Publications and source records attributed to Sant, A. J..

3 recordsLinked to original sources

bioRxiv2025

A funnel approach to enable analyses of epitope-specific human CD4 T cells specific for influenza and SARS-CoV-2.

Protection from pathogenic organisms relies heavily on the adaptive immune response, for which key regulators are CD4 T cells. CD4 T cells, notable in the complexity of their repertoire and functional potential, can most easily be dissected with the ability to identify, quantify, characterize and isolate epitope-specific cells. In the study reported here, we present a systematic and unbiassed strategy that has enabled identification of highly immunogenic peptide epitopes derived from influenza virus and SARS-CoV-2, presented by human HLA-DR proteins. Coupling the use of HLA-DR transgenic mice with infection and vaccination with highly sensitive epitope specific cytokine ELISpot assays, we have narrowed the potential epitopes from 450-600 peptides to 5-15 peptides, by an iterative process of elimination and selection which we have termed a funnel approach. These epitopes have been validated in HLA-DR typed human CD4 T cells directly ex vivo and enabled derivation and implementation of HLA-DR peptide tetramers. Tetramer staining of human PBMCs enriched CD4 T memory populations from healthy adult subjects highlighting this approach as a sensitive and specific method of identifying novel epitopes and subsequent CD4 T cell responses to human viral infections. ImportanceTracking single epitope-specific CD4 T cells enables sophisticated analyses of the human response to infectious pathogens, vaccines and probing the human CD4 T cell immune memory compartment. The studies presented here provide a unbiased strategy for accomplishing this goal and provide a verified compilation of candidate HLA-DR restricted CD4 T cell peptide epitopes for future studies by researchers in the field of human immunology.

immunology↗

bioRxiv2025

Re-shaping the immune response to influenza vaccination in a host with immune memory from influenza

Although CD4 T cells are critical orchestrators of protective immunity to viral respiratory pathogens, vaccine strategies that optimize generation of these cells have yet to be prioritized. In this manuscript, to mimic the typical human vaccine recipient using a mouse model, we evaluated the impact of previous influenza infection on the adaptive immune response elicited by the recombinant influenza vaccine Flublok, co-delivered with either AddaVax, an MF59 mimetic or a nanolipoparticle innate activator R-DOTAP. In the context of influenza B infection memory, a repolarization of the responding CD4 T cells and dramatic change in the fate of the vaccine-elicited CD4 T cells was discovered. A rapidly evolving CD4 T cell response enriched in TNF- and IFN-{psi} was observed, with the HA-B-specific CD4 T cells also displaying increased expression of chemokine receptors associated with lung homing potential and ultimate accumulation in the lung tissue. Unexpectedly, similar shifts in the features of the H3-specific CD4 T cell and antibody response were also observed, drawn from the naive repertoire. These results are consistent with the view the microenvironment of the vaccine draining lymph node, developed in the context of immune memory, rather than infection-induced CD4 T cell imprinting, plays the decisive role in the functional phenotype, magnitude, and fate of vaccine-elicited CD4 T cells. These results have important implications for both pre-clinical models of vaccination and future vaccine strategies.

immunology↗

bioRxiv2023

copepodTCR: Identification of Antigen-Specific T Cell Receptors with combinatorial peptide pooling

T cell receptor (TCR) repertoire diversity enables the antigen-specific immune responses against the vast space of possible pathogens. Identifying TCR-antigen binding pairs from the large TCR repertoire and antigen space is crucial for biomedical research. Here, we introduce copepodTCR, an open-access tool to design and interpret high-throughput experimental TCR specificity assays. copepodTCR implements a combinatorial peptide pooling scheme for efficient experimental testing of T cell responses against large overlapping peptide libraries, that can be used to identify the specificity of (or "deorphanize") TCRs. The scheme detects experimental errors and, coupled with a hierarchical Bayesian model for unbiased interpretation, identifies the response-eliciting peptide sequence for a TCR of interest out of hundreds of peptides tested using a simple experimental set-up. Using in silico simulations, we demonstrate the varied experimental settings in which copepodTCR yields efficient and interpretable TCR specificity results. We validated our approach on a library of 253 overlapping peptides covering the SARS-CoV-2 spike protein, split across 12 pools. A single stimulation with combinatorial pools identified the correct epitope of two TCRs with known specificity and then deorphanized two SARS-CoV-2 associated TCRs shared among a large cohort of COVID-19 patients. We provide experimental guides to efficiently design larger screens covering thousands of peptides which will be crucial to identify antigen-specific T cells and their targets from limited clinical material.

immunology↗