The extent to which tissue-specific viral infections generate memory T cells specifically adapted to and maintained within the target infection site is unknown. Here, we show that respiratory virus-specific memory T cells in mice and humans are generated and maintained in compartmentalized niches in lungs, distinct from populations in lymphoid tissue or circulation. Using a polyclonal mouse model of influenza infection combined with an in vivo antibody labeling approach and confocal imaging, we identify a spatially distinct niche in the lung where influenza-specific T cell responses are expanded and maintained long term as tissue resident memory (TRM) CD4 and CD8 T cells. Lung TRM are further distinguished from circulating memory subsets in lung and spleen based on CD69 expression and persistence independent of lymphoid stores. In humans, influenza-specific T cells are enriched within the lung TRM subset, while memory CD8 T cells specific for the systemic virus CMV are distributed in both lung and spleen, suggesting that the site of infection affects TRM generation. Our findings reveal a precise spatial organization to virus-specific T cell memory, determined by the site of the initial infection, with important implications for the development of targeted vaccination and strategies to boost immunity at appropriate tissue sites.

A paradigm in transplantation states that graft-infiltrating T cells are largely non-alloreactive “bystander” cells. However, the origin and specificity of allograft T cells over time has not been investigated in detail in animals or humans. Here, we use polychromatic flow cytometry and high throughput TCR sequencing of serial biopsies to show that gut-resident T cell turnover kinetics in human intestinal allografts are correlated with the balance between intra-graft host-vs-graft (HvG) and graft-vs-host (GvH) reactivities and with clinical outcomes. In the absence of rejection, donor T cells were enriched for GvH-reactive clones that persisted long-term in the graft. Early expansion of GvH clones in the graft correlated with rapid replacement of donor APCs by the recipient. Rejection was associated with transient infiltration by blood-like recipient CD28+ NKG2DHi CD8+ alpha beta T cells, marked predominance of HvG clones, and accelerated T cell turnover in the graft. Ultimately, these recipient T cells acquired a steady state tissue-resident phenotype, but regained CD28 expression during rejections. Increased ratios of GvH to HvG clones were seen in non-rejectors, potentially mitigating the constant threat of rejection posed by HvG clones persisting within the tissue-resident graft T cell population.

The efficacy of memory T cells in protective immunity has been shown in mouse models, but their lifelong compartmentalization and maintenance in humans remains unclear. To systematically probe lifelong memory T cell generation and maintenance, we have analyzed their distribution and function among 9 tissues acquired through from over 40 healthy donors aged 3 to 73 years old. We statistically analyzed data derived from FACS analysis of these donors, and generated a quantitative map of T cell compartmentalization and maintenance over the human lifespan. Our analyses revealed that differentiated effector memory subsets primarily resided in peripheral tissues while central and naïve subsets remained in circulatory sites. Moreover, expression of the resident memory marker CD69 effectively predicts compartmentalization in peripheral tissues, indicating the need for increased activation to enter the periphery. CD127 and CD28 expression revealed differential maintenance of the subsets in distinct sites, with more cytokine and antigen-driven maintenance prevalent in TEM and TEMRA subsets localized to the periphery. We found that the distribution and maintenance patterns of these subsets are established early in life, and vary little with increasing age. These findings suggest that tissue localization is the primary driver of human T cell maintenance and compartmentalization, and remains stable over the lifetime.

Lung-resident T cells are uniquely poised to mediate early in situ responses to respiratory pathogens. We have previously identified a subset of lung-resident CD4 T cells that mediate optimal protective immunity to respiratory infection. Here, we have examined the mechanisms for the establishment of lung-resident memory CD4 T cells and their role in anti-viral responses. Using intravenous (iv) infusion of antibody to differentiate between cells in the lung interstitium and those accessible to the circulation, we discovered that a minor subset of lung αβ CD4 T and most γδ T cells are protected from iv antibody staining (protected subset) while the remaining αβ CD4 T cells are readily stained (accessible subset). The protected αβ CD4 T cell subset and γδ T cells similarly expressed increased levels of CD11a, CD69, CD103 and CCR6 as compared to the accessible subset. The protected population of αβ CD4 T cells is established through respiratory infection as it comprises less than 10% of αβ CD4 T cells in naïve animals but represents approximately 30% of lung cells in influenza immune mice during the memory stage. Once established, the protected memory αβ CD4 T cells along with γδ T cells produces IFN-γ and IL-17 in situ early after influenza rechallenge. Our results show that the lung CD4 T cell population is comprised of a circulating subset and a tissue-resident subset with the tissue resident αβ CD4 T and γδ T cells acting as early responders to respiratory infections.