Identification and classification of distinct surface markers of T regulatory cells.

Background: Regulatory T (Treg) cells have emerged as key players in the maintenance of immune homeostasis. Although significant progress has been made in recent years to define the Treg surface markers involved with or identifying their suppressive function, there remains much to be elucidated, and many questions persist. This study determined the expression of surface markers on human peripheral Treg cells and conventional T (Tconv) cells in a steady state and after activation to gain insight into their mechanism of action and more precisely characterize this regulatory population in humans. Methods: To screen Treg and Tconv cells, peripheral blood mononuclear cells (PBMCs) were isolated from volunteers, stained with a commercially available lyophilized antibody array comprising 371 surface antigens, and analyzed by flow cytometry. To compare Treg cells with activated Tconv cells, PBMCs were stimulated with PMA and further stained similar to freshly isolated cells. Results: Treg and Tconv cells were positive for 135 and 168 of the 371 antigens, respectively. Based on the frequency distribution, all of the most highly expressed markers identified were shared by both Treg and Tconv cells and participate in T cell activation, act as costimulatory and signaling molecules, or exhibit adhesion and migratory functions. Additionally, we identified several differences in marker expression between Treg and Tconv cells, with most found in the expression of co-stimulatory (ICOS, GITR, 4-1BB) and co-inhibitory (TIGIT, CTLA-4) molecules, as well as chemokine receptors (CXCR4, CXCR5, CCR4, CCR5, CCR7, CCR8, and CXCR7). Furthermore, post-activation expression of surface molecules identified molecules capable of discriminating Treg cells from activated Tconv cells (GITR, 4-1BB, TIGIT, CD120b, and CD39); however, almost all of these markers were also expressed in a small fraction of activated Tconv cells. Conclusions: These results offer insight into the biology of Tregs and contribute to their accurate identification and characterization in variety of immunological diseases as well as physiological processes.

The effect of Toll-like receptor stimulation on the motility of regulatory T cells.

Regulatory T cells (Tregs) have suppressive functions and play an important role in controlling inflammation and autoimmunity. The migratory capacity of Tregs determines their location and their location determines whether they inhibit the priming of naïve lymphocytes in lymphoid tissues or the effector phase of immune responses at inflamed sites. Tregs generated or expanded in vitro are currently being tested in clinics for the treatment of autoimmune disorders, however, little is known about the factors controlling their migration towards therapeutically relevant locations. In this study, we have modulated Treg dynamics using Toll-like receptor (TLR) agonists. Dynamic imaging with confocal and two-photon microscopy revealed that Tregs generated in vitro and stimulated with P3C (a TLR2 agonist) but not with R848 (a TLR7 agonist) or LPS (a TLR4 agonist) showed enhanced cell migration within splenic white pulp or draining lymph node when transferred into mice intravenously or into the footpad, respectively. In summary, our data demonstrate that Tregs are more motile in response to direct TLR stimulation in particular towards TLR2 signals. This may have implications for efficient clinical Treg induction protocols.

Impact of tissue enzymatic digestion on analysis of immune cells in mouse reproductive mucosa with a focus on γδ T cells.

Mucosal tissues are enriched in γδ T lymphocytes, which maintain epithelial homeostasis, however, the homeostatic mechanisms are still incompletely understood. To elucidate their role in the tissue integrity governance within the female genital mucosa we employed flow cytometry, which is a powerful tool used for the characterization of tissue-resident immune cells, however, often requiring cell release upon tissue enzymatic disaggregation. Here, we analyzed the impact of various proteolytic enzymes in their ability to effectively isolate viable immune cells from the reproductive system of non-pregnant mice. Murine vaginas and uteri were digested using commercially available enzyme blends (liberases) and single enzymes (dispase II and collagenase IV). Among tested enzymes, liberases released the highest number of cells from digested tissues while dispase II and collagenase IV led to a significant decrease in the number of isolated live cells. Also, liberases had only minor detrimental effects on cell viability and detection of CD45, CD3ε, γδ TCR and CD11c positive cells. We found that a single liberase blend called Liberase TL was the most suited for the analysis of γδ T cells in the reproductive tract. By examining two distinct phases of the estrous cycle - estrus and diestrus, characterized by high and low epithelial stratification, respectively, we showed that higher numbers of γδ T lymphocytes were present in the latter cycle phase in vagina and uterus. Interestingly, the diestrus-associated increase in γδ T lymphocyte number was also observed in reproductive tract draining lumbar lymph nodes but not in more distant, inguinal lymph nodes. Our data indicate that enzymes used for reproductive mucosa digestion have profound effects on the cell viability and isolation efficiency, which consequently influence the phenotypic and quantitative analysis of immune cells.