PPARγ marks splenic precursors of multiple nonlymphoid-tissue Treg compartments

Foxp3+CD4+ regulatory T cells (Tregs) regulate most types of immune response as well as several processes important for tissue homeostasis, for example, metabolism and repair. Dedicated Treg compartments—with distinct transcriptomes, T cell receptor repertoires, and growth/survival factor dependencies—have been identified in several nonlymphoid tissues. These Tregs are specifically adapted to function and operate in their home tissue—When, where, and how do they take on their specialized characteristics? We recently reported that a splenic Treg population expressing low levels of the transcription factor PPARγ (peroxisome proliferator-activated receptor gamma) contains precursors of Tregs residing in visceral adipose tissue. This finding made sense given that PPARγ, the “master regulator” of adipocyte differentiation, is required for the accumulation and function of Tregs in visceral adipose tissue but not in lymphoid tissues. Here we use single-cell RNA sequencing, single-cell Tcra and Tcrb sequencing, and adoptive-transfer experiments to show that, unexpectedly, the splenic PPARγlo Treg population is transcriptionally heterogeneous and engenders Tregs in multiple nonlymphoid tissues beyond visceral adipose tissue, such as skin and liver. The existence of a general pool of splenic precursors for nonlymphoid-tissue Tregs opens possibilities for regulating their emergence experimentally or therapeutically.

Prion strain-dependent tropism is maintained between spleen and granuloma and relies on lymphofollicular structures

In peripherally acquired prion diseases, prions move through several tissues of the infected host, notably in the lymphoid tissue, long before the occurrence of neuroinvasion. Accumulation can even be restricted to the lymphoid tissue without neuroinvasion and clinical disease. Several experimental observations indicated that the presence of differentiated follicular dendritic cells (FDCs) in the lymphoid structures and the strain type are critical determinants of prion extraneural replication. In this context, the report that granulomatous structures apparently devoid of FDCs could support prion replication raised the question of the requirements for prion lymphotropism. The report also raised the possibility that nonlymphoid tissue-tropic prions could actually target these inflammatory structures. To investigate these issues, we examined the capacity of closely related prions, albeit with opposite lymphotropism (or FDC dependency), for establishment in experimentally-induced granuloma in ovine PrP transgenic mice. We found a positive correlation between the prion capacity to accumulate in the lymphoid tissue and granuloma, regardless of the prion detection method used. Surprisingly, we also revealed that the accumulation of prions in granulomas involved lymphoid-like structures associated with the granulomas and containing cells that stain positive for PrP, Mfge-8 but not CD45 that strongly suggest FDCs. These results suggest that the FDC requirement for prion replication in lymphoid/inflammatory tissues may be strain-dependent.

Molecular diversification of regulatory T cells in nonlymphoid tissues

Foxp3+CD4+regulatory T cells (Tregs) accumulate in certain nonlymphoid tissues, where they control diverse aspects of organ homeostasis. Populations of tissue Tregs, as they have been termed, have transcriptomes distinct from those of their counterparts in lymphoid organs and other nonlymphoid tissues. We examined the diversification of Tregsin visceral adipose tissue, skeletal muscle, and the colon vis-à-vis lymphoid organs from the same individuals. The unique transcriptomes of the various tissue Tregpopulations resulted from layering of tissue-restricted open chromatin regions over regions already open in the spleen, the latter tagged by super-enhancers and particular histone marks. The binding motifs for a small number of transcription factor (TF) families were repeatedly enriched within the accessible chromatin stretches of Tregsin the three nonlymphoid tissues. However, a bioinformatically and experimentally validated transcriptional network, constructed by integrating chromatin accessibility and single-cell transcriptomic data, predicted reliance on different TF families in the different tissues. The network analysis also revealed that tissue-restricted and broadly acting TFs were integrated into feed-forward loops to enforce tissue-specific gene expression in nonlymphoid-tissue Tregs. Overall, this study provides a framework for understanding the epigenetic dynamics of T cells operating in nonlymphoid tissues, which should inform strategies for specifically targeting them.

The Rho regulator Myosin IXb enables nonlymphoid tissue seeding of protective CD8+ T cells

T cells are actively scanning pMHC-presenting cells in lymphoid organs and nonlymphoid tissues (NLTs) with divergent topologies and confinement. How the T cell actomyosin cytoskeleton facilitates this task in distinct environments is incompletely understood. Here, we show that lack of Myosin IXb (Myo9b), a negative regulator of the small GTPase Rho, led to increased Rho-GTP levels and cell surface stiffness in primary T cells. Nonetheless, intravital imaging revealed robust motility of Myo9b−/− CD8+ T cells in lymphoid tissue and similar expansion and differentiation during immune responses. In contrast, accumulation of Myo9b−/− CD8+ T cells in NLTs was strongly impaired. Specifically, Myo9b was required for T cell crossing of basement membranes, such as those which are present between dermis and epidermis. As consequence, Myo9b−/− CD8+ T cells showed impaired control of skin infections. In sum, we show that Myo9b is critical for the CD8+ T cell adaptation from lymphoid to NLT surveillance and the establishment of protective tissue–resident T cell populations.

Local antigen in nonlymphoid tissue promotes resident memory CD8+ T cell formation during viral infection

Tissue-resident memory (Trm) CD8+ T cells are functionally distinct from their circulating counterparts and are potent mediators of host protection against reinfection. Whether local recognition of antigen in nonlymphoid tissues during infection can impact the formation of Trm populations remains unresolved. Using skin infections with vaccinia virus (VacV)–expressing model antigens, we found that local antigen recognition had a profound impact on Trm formation. Activated CD8+ T cells trafficked to VacV-infected skin in an inflammation-dependent, but antigen-independent, manner. However, after viral clearance, there was a subsequent ∼50-fold increase in Trm formation when antigen was present in the tissue microenvironment. Secondary antigen stimulation in nonlymphoid tissue caused CD8+ T cells to rapidly express CD69 and be retained at the site of infection. Finally, Trm CD8+ T cells that formed during VacV infection in an antigen-dependent manner became potent stimulators of localized antigen-specific inflammatory responses in the skin. Thus, our studies indicate that the presence of antigen in the nonlymphoid tissue microenvironment plays a critical role in the formation of functional Trm CD8+ T cell populations, a finding with relevance for both vaccine design and prevention of inflammatory disorders.

The origin and development of nonlymphoid tissue CD103+ DCs

CD103+ dendritic cells (DCs) in nonlymphoid tissues are specialized in the cross-presentation of cell-associated antigens. However, little is known about the mechanisms that regulate the development of these cells. We show that two populations of CD11c+MHCII+ cells separated on the basis of CD103 and CD11b expression coexist in most nonlymphoid tissues with the exception of the lamina propria. CD103+ DCs are related to lymphoid organ CD8+ DCs in that they are derived exclusively from pre-DCs under the control of fms-like tyrosine kinase 3 (Flt3) ligand, inhibitor of DNA protein 2 (Id2), and IFN regulatory protein 8 (IRF8). In contrast, lamina propria CD103+ DCs express CD11b and develop independently of Id2 and IRF8. The other population of CD11c+MHCII+ cells in tissues, which is CD103−CD11b+, is heterogenous and depends on both Flt3 and MCSF-R. Our results reveal that nonlymphoid tissue CD103+ DCs and lymphoid organ CD8+ DCs derive from the same precursor and follow a related differentiation program.