The site of transition between tissue-resident memory (TRM) and circulating phenotypes of T cells is unknown. We integrated clonotype, alloreactivity, and gene expression profiles of graft-repopulating recipient T cells in the intestinal mucosa at the single-cell level after human intestinal transplantation. Host-versus-graft (HvG)–reactive T cells were mainly distributed to TRM, effector T (Teff)/TRM, and T follicular helper compartments. RNA velocity analysis demonstrated a trajectory from TRM to Teff/TRM clusters in association with rejection. By integrating pre- and post-transplantation (Tx) mixed lymphocyte reaction–determined alloreactive repertoires, we observed that pre-existing HvG-reactive T cells that demonstrated tolerance in the circulation were dominated by TRM profiles in quiescent allografts. Putative de novo HvG-reactive clones showed a transcriptional profile skewed to cytotoxic effectors in rejecting grafts. Inferred protein regulon network analysis revealed upstream regulators that accounted for the effector and tolerant T cell states. We demonstrate Teff/TRM interchangeability for individual T cell clones with known (allo)recognition in the human gut, providing novel insight into TRM biology.

The field of cardio-immunology has emerged from discoveries that define roles for innate and adaptive immune responses associated with myocardial inflammation and heart failure. Dendritic cells (DCs) comprise an important cellular compartment that contributes to systemic immune surveillance at the junction of innate and adaptive immunity. Once described as a singular immune subset, we now appreciate that DCs consist of a heterogeneous pool of subpopulations, each with distinct effector functions that can uniquely regulate the acute and chronic inflammatory response. Nevertheless, the cardiovascular-specific context involving DCs in negotiating the biological response to myocardial injury is not well understood. Herein, we review our current understanding of the role of DCs in cardiac inflammation and heart failure, including gaps in knowledge and clinical relevance.

Understanding formation of the human tissue resident memory T cell (TRM) repertoire requires longitudinal access to human non-lymphoid tissues. By applying flow cytometry and next generation sequencing to serial blood, lymphoid tissue, and gut samples from 16 intestinal transplantation (ITx) patients, we assessed the origin, distribution, and specificity of human TRMs at phenotypic and clonal levels. Donor age ≥1 year and blood T cell macrochimerism (peak level ≥4%) were associated with delayed establishment of stable recipient TRM repertoires in the transplanted ileum. T cell receptor (TCR) overlap between paired gut and blood repertoires from ITx patients was significantly greater than that in healthy controls, demonstrating increased gut-blood crosstalk after ITx. Crosstalk with the circulating pool remained high for years of follow-up. TCR sequences identifiable in pre-Tx recipient gut but not those in lymphoid tissues alone were more likely to populate post-Tx ileal allografts. Clones detected in both pre-Tx gut and lymphoid tissue had distinct transcriptional profiles from those identifiable in only one tissue. Recipient T cells were distributed widely throughout the gut, including allograft and native colon, which had substantial repertoire overlap. Both alloreactive and microbe-reactive recipient T cells persisted in transplanted ileum, contributing to the TRM repertoire. Our studies reveal human intestinal TRM repertoire establishment from the circulation, preferentially involving lymphoid tissue counterparts of recipient intestinal T cell clones, including TRMs. We have described the temporal and spatial dynamics of this active crosstalk between the circulating pool and the intestinal TRM pool. This study was funded by the National Institute of Allergy and Infectious Diseases (NIAID) P01 grant AI106697.

The role of conventional type 1 DCs (cDC1s) in tolerance induction to solid organ allografts is unknown and important for strategies that seek to prolong allograft viability. Using a murine model deficient in cDC1s, we report cDC1s are required for donor antigen and costimulation blockade (DST + CoB) tolerance induction and survival of cardiac allografts. cDC1 deficiency led to decreases in CD4 ⁺ CD25 ⁺ FoxP3 ⁺ T cells within allograft and spleen tissue of transplant recipients, and this was found to be antigen specific. Donor antigen stimulation induced TGF-β1 expression in both in vivo cDC1s and in vitro Flt3L-derived cDC1s. Genetic deletion of TGF- β 1 in cDC1s prevented induction of antigen-specific CD4 ⁺ CD25 ⁺ FoxP3 ⁺ T cells and was associated with cardiac allograft rejection. In parallel, single-cell RNA sequencing and metabolic analysis revealed upregulation of cDC1 mitochondrial metabolic signatures after in vivo exposure to DST + CoB. Genetic inactivation of cDC1 mitochondrial metabolism reduced expression of cDC1 TGF-β1, decreased antigen-specific Treg populations, and impaired allograft tolerance. Taken together, our findings implicate cDC1s in strategies to preserve solid organ allografts and also implicate mitochondrial metabolism of cDC1s as a molecular mechanism to enhance the generation of antigen-specific CD4 ⁺ CD25 ⁺ FoxP3 ⁺ T cells through TGF-β1.

Recent studies have revealed novel molecular mechanisms by which innate monocytic cells acutely recognize and respond to alloantigen with significance to allograft rejection and tolerance. What remains unclear is the single-cell heterogeneity of the innate alloresponse, particularly the contribution of dendritic cell (DC) subsets. To investigate the response of these cells to exposure of alloantigen, C57BL/6J mice were administered live allogenic BALB/cJ splenic murine cells versus isogenic cells. In parallel, we infused apoptotic allogenic and isogenic cells, which have been reported to modulate immunity. Forty-eight hours after injection, recipient spleens were harvested, enriched for DCs, and subjected to single-cell mRNA sequencing. Injection of live cells induced a greater transcriptional change across DC subsets compared with apoptotic cells. In the setting of live cell infusion, type 2 conventional DCs (cDC2s) were most transcriptionally responsive with a Ccr2+ cDC2 subcluster uniquely responding to the presence of alloantigen compared with the isogenic control. In vitro experimentation confirmed unique activation of CCR2+ cDC2s following alloantigen exposure. Candidate receptors of allorecognition in other innate populations were interrogated and A type paired Ig-like receptors were found to be increased in the cDC2 population following alloexposure. These results illuminate previously unclear distinctions between therapeutic infusions of live versus apoptotic allogenic cells and suggest a role for cDC2s in innate allorecognition. More critically, these studies allow for future interrogation of the transcriptional response of immune cells in the setting of alloantigen exposure in vivo, encouraging assessment of novel pathways and previously unexamined receptors in this setting.