MAIT cell heterogeneity across paired human tissues reveals specialization of distinct regulatory and enhanced effector profiles.

Mucosal-associated invariant T (MAIT) cells are unconventional T cells that recognize microbial riboflavin pathway metabolites presented by evolutionarily conserved MR1 molecules. We explored the human MAIT cell compartment across organ donor-matched blood, barrier, and lymphoid tissues. MAIT cell population size was donor dependent with distinct tissue compartmentalization patterns and adaptations: Intestinal CD103+ resident MAIT cells presented an immunoregulatory CD39highCD27low profile, whereas MAIT cells expressing NCAM1/CD56 dominated in the liver and exhibited enhanced effector capacity with elevated response magnitude and polyfunctionality. Both intestinal CD39high and hepatic CD56+ adaptations accumulated with donor age. CD56+ MAIT cells displayed limited T cell receptor-repertoire breadth, elevated MR1 binding, and a transcriptional profile skewed toward innate activation pathways. Furthermore, CD56 was dynamically up-regulated to a persistent steady-state equilibrium after exposure to antigen or IL-7. In summary, we demonstrate functional heterogeneity and tissue site adaptation in resident MAIT cells across human barrier tissues with distinct regulatory and effector signatures.

Comparison of Lung-Homing Receptor Expression and Activation Profiles on NK Cell and T Cell Subsets in COVID-19 and Influenza.

Respiratory viral infections with SARS-CoV-2 and influenza viruses commonly induce a strong infiltration of immune cells into the human lung, with potential detrimental effects on the integrity of the lung tissue. Despite comprising the largest fractions of circulating lymphocytes in the lung, rather little is known about how peripheral blood natural killer (NK) cell and T cell subsets are equipped for lung-homing in COVID-19 and influenza. Here, we provide a detailed comparative analysis of NK cells and T cells in patients infected with SARS-CoV-2 or influenza virus, focusing on the protein and gene expression of chemokine receptors known to be involved in recruitment to the lung. For this, we used 28-colour flow cytometry as well as re-analysis of a publicly available single-cell RNA-seq dataset from bronchoalveolar lavage (BAL) fluid. Frequencies of NK cells and T cells expressing CXCR3, CXCR6, and CCR5 were altered in peripheral blood of COVID-19 and influenza patients, in line with increased transcript expression of CXCR3, CXCR6, and CCR5 and their respective ligands in BAL fluid. NK cells and T cells expressing lung-homing receptors displayed stronger phenotypic signs of activation compared to cells lacking lung-homing receptors, and activation was overall stronger in influenza compared to COVID-19. Together, our results indicate a role for CXCR3+, CXCR6+, and/or CCR5+ NK cells and T cells that potentially migrate to the lungs in moderate COVID-19 and influenza patients, identifying common targets for future therapeutic interventions in respiratory viral infections.

Acquisition of murine splenic myeloid cells for protein and gene expression profiling by advanced flow cytometry and CITE-seq.

Here, we outline detailed protocols to isolate and profile murine splenic dendritic cells (DCs) through advanced flow cytometry of the myeloid compartment and single-cell transcriptomic profiling with integrated cell surface protein expression through CITE-seq. This protocol provides a general transferrable road map for different tissues and species. For complete details on the use and execution of this protocol, please refer to Lukowski et al. (2021).

Absence of Batf3 reveals a new dimension of cell state heterogeneity within conventional dendritic cells.

Conventional dendritic cells (cDCs) are traditionally subdivided into cDC1 and cDC2 lineages. Batf3 is a cDC1-required transcription factor, and we observed that Batf3-/- mice harbor a population of cDC1-like cells co-expressing cDC2-associated surface molecules. Using single-cell RNA sequencing with integrated cell surface protein expression (CITE-seq), we found that Batf3-/- mitotic immature cDC1-like cells showed reduced expression of cDC1 features and increased levels of cDC2 features. In wild type, we also observed a proportion of mature cDC1 cells expressing surface features characteristic to cDC2 and found that overall cDC cell state heterogeneity was mainly driven by developmental stage, proliferation, and maturity. We detected population diversity within Sirpa+ cDC2 cells, including a Cd33+ cell state expressing high levels of Sox4 and lineage-mixed features characteristic to cDC1, cDC2, pDCs, and monocytes. In conclusion, these data suggest that multiple cDC cell states can co-express lineage-overlapping features, revealing a level of previously unappreciated cDC plasticity.