Mucosal T cell memory to pathogens

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PROJECT SUMMARY Iterative T cell stimulation can result in senescence, exhaustion, or death (SED) as observed during chronic viral infections, certain heterologous prime boost (HPB) vaccination studies, or cancer. Knowing whether there are axiomatic limits to T cell clonal expansion is a critical gap in knowledge relevant for immunotherapy of diverse diseases as well as fundamental understanding of immunobiology. In pilot experiments that began >8y ago, we identified a stimulation strategy revealing that memory CD8 T populations are essentially infinitely expansible, our oldest population having gone through 38 booster immunizations over 3200 days (longer than any mouse lives) and effectively producing >1030 progeny. We hypothesize that this proof-of-principle, extreme-of-nature experiment reveals fundamental T cell biology highly relevant to our understanding of the immune system and provides unexpected observations that could be exploited for medically relevant purposes. We will leverage this unique resource (ISTCs, iteratively stimulated T cells) to explore the consequences of iterative stimulation on T cell biology. We will address the rules for avoiding SED despite repeated stimulation, the relevance of ISTCs expressing exhaustion-associated genes without appearing functionally exhausted, and the mechanisms by which ISTCs durably retain the unique effector-like property of circulation between blood and mucosal tissues. Aim 1. To define the regulation and evolution of the ISTC differentiation program. We will 1) define the evolution of ISTCs at the single cell level using bioinformatic and computational biology approaches, 2) define rules for avoiding T cell SED by modifying the parameters of our stimulation strategy, and 3) contrast ISTCs from exhausted T cells (Tex) on a molecular level by comparing gene expression patterns of Tex and consecutive generations of ISTCs. We will test the hypotheses that 1) everlasting proliferative capacity depends on avoiding terminal differentiation of a ‘stem’ population, 2) excessive division over a short period of time promotes terminal differentiation, and 3) despite sharing key features with exhausted cells, functional ISTCs will reveal discrete patterns of gene expression thus refining the molecular definition of exhaustion. Aim 2. To define ISTC recirculation properties, mechanisms of immunosurveillance, and antimicrobial functions. ISTCs represent a unique opportunity to interrogate mechanisms of nonlymphoid tissue recirculation. We will 1) define the migration properties of ISTCs using parabiosis surgeries, photoactivatable mouse lines, and perturbing homing molecules, 2) assess the protective potential of ISTCs in a LM-N challenge model, and 3) extend these findings to ‘dirty’ mice (mice which have been iteratively exposed to natural pathogens) and humans. We will test the hypotheses that 1) ISTCs recirculate through NLTs, 2) ISTCs can protect against pathogen challenge, and 3) ISTCs can be identified in ‘dirty’ mice and humans.