CD8 T cells contain a distinct subset of CD8+ CD28- cells. These cells are not present at birth and their frequency increases with age. They frequently contain expanded clones using various TCRalphabeta receptors and these clones can represent >50% of all CD8 cells, specially in old subjects or patients with chronic viral infections such as HIV-1. Herein, it is shown that a large fraction of CD8+ CD28- cells expresses intracellular perforin by three-color flow cytometry, in particular when this subset is expanded. Together with their known ability to exert potent re-directed cytotoxicity, this indicates that CD8+ CD28- T cells comprise cytotoxic effector cells. With BrdU labeling, we show that CD8+ CD28- cells derive from CD8+ CD28+ precursors in vitro. In addition, sorted CD8+ CD28+ cells gave rise to a population of CD8+ CD28- cells after allo-stimulation. Moreover, ex vivo CD8+ CD28+ cells contain the majority of CD8 blasts, supporting the notion that they contain the proliferative precursors of CD8+ CD28- cells. CD95 (Fas) expression was lower in CD8+ CD28- cells, and this subset was less prone to spontaneous apoptosis in ex vivo samples and more resistant to activation-induced cell death induced by a superantigen in vitro. Thus, the persistence of expanded clones in vivo in the CD8+ CD28- subset may be explained by antigen-driven differentiation from CD8+ CD28+ memory precursors, with relative resistance to apoptosis as the clones become perforin(+) effector cells.

CD4 + CD25 + Foxp3 + Tregs play a major role in prevention of autoimmune diseases. The suppressive effect of Tregs on effector T cells (Teffs), the cells that can mediate autoimmunity, has been extensively studied. However, the in vivo impact of Teff activation on Tregs during autoimmunity has not been explored. In this study, we have shown that CD4 + Teff activation strongly boosts the expansion and suppressive activity of Tregs. This helper function of CD4 + T cells, which we believe to be novel, was observed in the pancreas and draining lymph nodes in mouse recipients of islet-specific Teffs and Tregs. Its physiological impact was assessed in autoimmune diabetes. When islet-specific Teffs were transferred alone, they induced diabetes. Paradoxically, when the same Teffs were cotransferred with islet-specific Tregs, they induced disease protection by boosting Treg expansion and suppressive function. RNA microarray analyses suggested that TNF family members were involved in the Teff-mediated Treg boost. In vivo experiments showed that this Treg boost was partially dependent on TNF but not on IL-2. This feedback regulatory loop between Teffs and Tregs may be critical to preventing or limiting the development of autoimmune diseases.

Little is known about the ontogeny of naturally occurring CD4 ؉ CD25 ؉ regulatory/suppressor T cells that play a major role in maintaining self-tolerance in mice and humans. In rodents, thymectomy on day 3 of life leads to multiple organspecific autoimmune diseases that can be prevented by adoptive transfer of regulatory T cells, suggesting their neonatal development. We investigated regulatory T-cell ontogeny in 11 human fetuses. Together with the first mature T cells, thymic CD4 ؉ CD25 ؉ cells were detected as early as 13 weeks of gestation. Thymic CD25 ؉ cells appeared to be positively selected at the CD4 ؉ CD8 ؉ CD3 hi differentiation stage, as assessed by CD1a and CD69 expression. The proportion of thymic CD4 ؉ CD25 ؉ cells appeared quite stable with age, around 6% to 7%, similar to the proportion observed in infant thymi. Extrathymic CD4 ؉ CD25 ؉ T cells could hardly be detected at 13 weeks of gestation but were present from week 14 onwards. As adult regulatory T cells, purified CD4 ؉ CD25 ؉ fetal cells were anergic and suppressed T-cell proliferative responses; they expressed intracellular cytotoxic T-lymphocyte-associated antigen 4 (CTLA- 4 IntroductionT cells capable of suppressing immune responses have been hypothesized for a long time. 1 Functional assays have supported their existence 2,3 but the inability to phenotypically identify them has hampered their further study. However, there is now clear evidence that CD4 ϩ cells expressing the interleukin-2 receptor ␣ chain (CD25) contain a population of naturally and ubiquitously present cells endowed with suppressive activities. [4][5][6][7] The lack of a known specific surface marker makes it hard to distinguish CD4 ϩ CD25 ϩ regulatory T cells (Treg's) from CD4 ϩ activated effector T cells, which may transitorily express CD25. 8 As of today, Treg's are best identified by the expression of (1) high levels of CD25 (CD25 hi ), [8][9][10] (2) the forkhead/winged helix transcription factor Foxp3, 11 (3) high levels of intracytoplasmic cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), 12,13 and (4) the glucocorticoid-induced tumor necrosis factor receptor (GITR) surface marker. 14 Since these individual markers are neither specific nor always simultaneously expressed, the authentication of bona fide Treg's relies on the demonstration that (1) they are anergic in vitro in the absence of exogenous IL-2 and (2) they indeed suppress effector T-cell responses.It is now well established that Treg's play a major role in maintenance of self-tolerance and control of autoimmune diseases. 7,15 They are also involved in regulation of T-cell homeostasis [16][17][18] and modulation of immune responses to alloantigens, tumor cells, and various pathogens. [19][20][21][22][23][24][25][26] In the periphery, at steady-state, Treg's represent a stable proportion of the CD4 ϩ T cells, typically 5% to 12% in mice and humans. Although Treg's are anergic in vitro, in vivo studies have shown that some Treg's are quiescent with a long lifespan whereas others divide extensively du...

Rationale: Pulmonary arterial hypertension is characterized by vascular remodeling and neomuscularization. PW1 + progenitor cells can differentiate into smooth muscle cells (SMCs) in vitro. Objective: To determine the role of pulmonary PW1 + progenitor cells in vascular remodeling characteristic of pulmonary arterial hypertension. Methods and Results: We investigated their contribution during chronic hypoxia–induced vascular remodeling in Pw1 nLacZ+/− mouse expressing β-galactosidase in PW1 + cells and in differentiated cells derived from PW1 + cells. PW1 + progenitor cells are present in the perivascular zone in rodent and human control lungs. Using progenitor markers, 3 distinct myogenic PW1 + cell populations were isolated from the mouse lung of which 2 were significantly increased after 4 days of chronic hypoxia. The number of proliferating pulmonary PW1 + cells and the proportion of β-gal + vascular SMC were increased, indicating a recruitment of PW1 + cells and their differentiation into vascular SMC during early chronic hypoxia–induced neomuscularization. CXCR4 inhibition using AMD3100 prevented PW1 + cells differentiation into SMC but did not inhibit their proliferation. Bone marrow transplantation experiments showed that the newly formed β-gal + SMC were not derived from circulating bone marrow–derived PW1 + progenitor cells, confirming a resident origin of the recruited PW1 + cells. The number of pulmonary PW1 + cells was also increased in rats after monocrotaline injection. In lung from pulmonary arterial hypertension patients, PW1-expressing cells were observed in large numbers in remodeled vascular structures. Conclusions: These results demonstrate the existence of a novel population of resident SMC progenitor cells expressing PW1 and participating in pulmonary hypertension–associated vascular remodeling.

Several drugs targeting members of the TNF superfamily or TNF receptor superfamily (TNFRSF) are widely used in medicine or are currently being tested in therapeutic trials. However, their mechanism of action remains poorly understood. Here, we explored the effects of TNFRSF co-stimulation on murine Foxp3 + regulatory T cell (Treg) biology, as they are pivotal modulators of immune responses. We show that engagement of TNFR2, 4-1BB, GITR, and DR3, but not OX40, increases Treg proliferation and survival. Triggering these TNFRSF in Tregs induces similar changes in gene expression patterns, suggesting that they engage common signal transduction pathways. Among them, we identified a major role of canonical NF-κB. Importantly, TNFRSF co-stimulation improves the ability of Tregs to suppress colitis. Our data demonstrate that stimulation of discrete TNFRSF members enhances Treg activation and function through a shared mechanism. Consequently, therapeutic effects of drugs targeting TNFRSF or their ligands may be mediated by their effect on Tregs.

The diversity of the human immune repertoire and how it relates to a functional immune response has not yet been studied in detail in humanized NOD.SCID.cc À/À immunodeficient mice. Here, we used a multiplex PCR on genomic DNA to quantify the combinatorial diversity of all possible V-J rearrangements at the TCR-b chain and heavy chain Ig locus. We first show that the combinatorial diversity of the TCR-b chain generated in the thymus was well preserved in the periphery, suggesting that human T cells were not vastly activated in mice, in agreement with phenotypic studies. We then show that the combinatorial diversity in NOD.SCID.cc À/À mice reached 100% of human reference samples for both the TCR and the heavy chain of Ig. To document the functionality of this repertoire, we show that a detectable but weak HLA-restricted cellular immune response could be elicited in reconstituted mice after immunization with an adenoviral vector expressing HCV envelope glycoproteins. Altogether, our results suggest that humanized mice express a diversified repertoire and are able to mount antigen-specific immune responses.

Little is known about the in vivo conditions in which CD4+CD25+ regulatory T cells (Treg) exert their suppressive effect in nonlymphopenic mice. To this end, we analyzed Treg-mediated suppression of expansion and cytokine production at different levels of Ag-specific CD4+CD25− T cell activation. Using Ab-mediated depletion of endogenous Treg, we show that basal immunosuppression is dependent on effector T cell activation. These polyclonal Treg, which were poorly activated in our immunization conditions, were effective in weak but not high T cell activation context. In contrast, the same immunization conditions led to proliferation of cotransferred Ag-specific Treg. Those efficiently inhibited T cell proliferation and cytokine production even in strong T cell activation context. Interestingly, Treg selectively suppressed expansion or cytokine production depending on the experimental approach. The importance of the immune context for efficient suppression is further supported by the observation that Treg depletion exacerbated diabetes of NOD mice only at the early stage of the disease. Overall, our study suggests that Treg-mediated suppression depends on the relative activation of Treg and effector T cells in vivo. This balance may be a critical factor in the regulation of immune responses.

CD4+Foxp3+ regulatory T (Treg) cells are central modulators of autoimmune diseases. However, the timing and location of Treg cell–mediated suppression of tissue-specific autoimmunity remain undefined. Here, we addressed these questions by investigating the role of tumor necrosis factor (TNF) receptor 2 (TNFR2) signaling in Treg cells during experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis. We found that TNFR2-expressing Treg cells were critical to suppress EAE at peak disease in the central nervous system but had no impact on T cell priming in lymphoid tissues at disease onset. Mechanistically, TNFR2 signaling maintained functional Treg cells with sustained expression of CTLA-4 and Blimp-1, allowing active suppression of pathogenic T cells in the inflamed central nervous system. This late effect of Treg cells was further confirmed by treating mice with TNF and TNFR2 agonists and antagonists. Our findings show that endogenous Treg cells specifically suppress an autoimmune disease by acting in the target tissue during overt inflammation. Moreover, they bring a mechanistic insight to some of the adverse effects of anti-TNF therapy in patients.