SUMMARY Knowledge of human T cells derives chiefly from studies of peripheral blood, whereas their distribution and function in tissues remains largely unknown. Here, we present a unique analysis of human T cells in lymphoid and mucosal tissues obtained from individual organ donors, revealing tissue-intrinsic compartmentalization of naive, effector and memory subsets conserved between diverse individuals. Effector-memory CD4+ T cells producing IL-2 predominated in mucosal tissues and accumulated as central-memory subsets in lymphoid tissue, whereas CD8+ T cells were maintained as naïve subsets in lymphoid tissues and IFN-γ-producing effector-memory CD8+ T cells in mucosal sites. The T cell activation marker, CD69, was constitutively expressed by memory T cells in all tissues, distinguishing them from circulating subsets, with mucosal memory T cells exhibiting additional distinct phenotypic and functional properties. Our results provide an assessment of human T cell compartmentalization as a new baseline for understanding human adaptive immunity.

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.

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The Epstein-Barr virus (EBV)-encoded nuclear antigen EBNA1 is critical for the persistence of the viral episome in replicating EBV-transformed human B cells. Therefore, all EBV-induced tumors express this foreign antigen. However, EBNA1 is invisible to CD8+ cytotoxic T lymphocytes because its Gly/Ala repeat domain prevents proteasome-dependent processing for presentation on major histocompatibility complex (MHC) class I. We now describe that CD4+ T cells from healthy adults are primed to EBNA1. In fact, among latent EBV antigens that stimulate CD4+ T cells, EBNA1 is preferentially recognized. We present evidence that the CD4+ response may provide a protective role, including interferon γ secretion and direct cytolysis after encounter of transformed B lymphocyte cell lines (B-LCLs). Dendritic cells (DCs) process EBNA1 from purified protein and from MHC class II–mismatched, EBNA1-expressing cells including B-LCLs. In contrast, B-LCLs and Burkitt's lymphoma lines likely present EBNA1 after endogenous processing, as their capacity to cross-present from exogenous sources is weak or undetectable. By limiting dilution, there is a tight correlation between the capacity of CD4+ T cell lines to recognize autologous B-LCL–expressing EBNA1 and DCs that have captured EBNA1. Therefore, CD4+ T cells can respond to the EBNA1 protein that is crucial for EBV persistence. We suggest that this immune response is initiated in vivo by DCs that present EBV-infected B cells, and that EBNA1-specific CD4+ T cell immunity be enhanced to prevent and treat EBV-associated malignancies.

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The γ-herpesvirus, EBV, is reliably found in a latent state in endemic Burkitt’s lymphoma. A single EBV gene product, Epstein-Barr nuclear Ag 1 (EBNA1), is expressed at the protein level. Several mechanisms prevent immune recognition of these tumor cells, including a block in EBNA1 presentation to CD8+ killer T cells. Therefore, no EBV-specific immune response has yet been found to target Burkitt’s lymphoma. We now find that EBNA1-specific, Th1 CD4+ cytotoxic T cells recognize Burkitt’s lymphoma lines. CD4+ T cell epitopes of EBNA1 are predominantly found in the C-terminal, episome-binding domain of EBNA1, and ∼0.5% of peripheral blood CD4+ T cells are specific for EBNA1. Therefore, adaptive immunity can be directed against Burkitt’s lymphoma, and perhaps this role for CD4+ Th1 cells extends to other tumors that escape MHC class I presentation.

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Dendritic cells (DC) utilize at least two pathways to process viral antigens onto MHC class I molecules. The conventional endogenous route is used to acquire antigens from both infectious and non‐replicating virions. Exogenous pathways are used by DC to acquire and "cross‐present" antigens derived from virus‐infected donor cells that by themselves lack the ability to activate T cells directly. We analyzed the role of this pathway for antigens derived from vaccinia, a virus which inhibits DC maturation and causes extensive apoptosis of infected cells, yet is highly immunogenic. Using recombinant vaccinia virus encoding the influenza matrix protein as model vector, DC were shown to cross‐present vaccinia‐derived antigens from both apoptotic and necrotic infected cells to antigen‐specific CD8+ T cells. Efficient cross presentation required uptake of dead cells by immature DC and exposure to maturation stimuli, especially CD40 ligand. The responding CD8+ Tcells secreted IL‐2 and IFN‐γ, proliferated and developed into cytotoxic effectors. Quantification of the cross presentation of vaccinia‐derived antigens showed this pathway to be highly efficient, corresponding to a peptide pulse of 10–100 nM. While monocytes also phagocytosed apoptotic and necrotic cells, they were far less efficient at cross‐presenting vaccinia‐derived antigens to CD8+ T cells. The ability of DC to cross‐present vaccinia‐derived antigens from infected apoptotic cells or necrotic cell lysates, bypasses the deleterious effects of direct infection of DC and provides one explanation for this pathogen's immunogenicity.