Defining molecular circuits of CD8+ T cell responses in tissues during latent viral infection

Latent viral infections rely on a precise coordination of the immune response to control sporadic viral reactivation. CD8+ T cells play a crucial role in controlling viral latency by generating diverse memory responses in an epitope-specific manner. Among these distinct responses, conventional and inflationary memory responses have been described during herpesvirus infections. Using a newly generated T cell receptor transgenic mouse strain, we investigated the transcriptomic and epigenetic remodeling of distinct epitope-specific CD8+ T cells during cytomegalovirus (CMV) infection across tissues at both population and single-cell levels. Our findings reveal that whereas the transcriptomic and epigenetic landscape of conventional and inflationary memory responses diverge in the spleen and liver, these molecular programs converge in the salivary gland, a site of CMV persistence. Thus, we provide evidence that the dynamics of memory CD8+ T cell responses are nuanced and exquisitely distinct between tissues. For preparation of transgenic cells for single-cell multiome sequencing, congenically marked naïve TCRTg cells (M45Tg and M38Tg) were transferred into congenically distinct naïve recipient mice prior to infection with MCMV. On the day of cell isolation, each mouse was injected with 50µg of anti-ARTC2 nanobody (i.p.) 30 minutes before sacrifice (BioLegend, Catalog No. 149803, Clone S+16a). Intravenous (I.V.) labeling was not performed prior to sorting due to its limited ability to distinguish circulating from bona fide tissue-resident cells in the liver. Notably, I.V. labeling showed minimal signal in the salivary gland, comparable to the lymph nodes, which remain unlabeled (data not shown). Upon cell sorting into 1× PBS + 0.04% BSA, TCRTg cells were stained with mouse TotalSeq-A Hash Tag Oligonucleotides antibodies (BioLegend, tag number A0301-A0310), oligo-tagged anti-rat/mouse CD90.1 (BioLegend, Catalog No. 202547, Clone OX-7), and anti-mouse CD90.2 (BioLegend, Catalog No. 105345, Clone 30-H12). Hash tagged cells were then pooled at approximately equal ratios for multiome sequencing.