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 TCRαβ 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 organ-specific 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) and Foxp3 mRNA. Altogether, our results indicate that the generation of regulatory/suppressor T cells is consubstantial to the generation of a functional and self-tolerant immune system. (Blood. 2005;105:4715-4721)

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.

Little is known about the in vivo conditions in which CD4⁺CD25⁺ regulatory T cells (T_reg) exert their suppressive effect in nonlymphopenic mice. To this end, we analyzed T_reg-mediated suppression of expansion and cytokine production at different levels of Ag-specific CD4⁺CD25⁻ T cell activation. Using Ab-mediated depletion of endogenous T_reg, we show that basal immunosuppression is dependent on effector T cell activation. These polyclonal T_reg, 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 T_reg. Those efficiently inhibited T cell proliferation and cytokine production even in strong T cell activation context. Interestingly, T_reg 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 T_reg depletion exacerbated diabetes of NOD mice only at the early stage of the disease. Overall, our study suggests that T_reg-mediated suppression depends on the relative activation of T_reg and effector T cells in vivo. This balance may be a critical factor in the regulation of immune responses.

Achieving cell-specific expression of a therapeutic transgene by gene transfer vectors represents a major goal for gene therapy. To achieve specific expression of a transgene in CD4⁺ cells, we have generated lentiviral vectors expressing the enhanced green fluorescent protein (eGFP) reporter gene under the control of regulatory sequences derived from theCD4 gene—a minimal promoter and the proximal enhancer, with or without the silencer. Both lentiviral vectors could be produced at high titers (more than 10⁷ infectious particles per milliliter) and were used to transduce healthy murine hematopoietic stem cells (HSCs). On reconstitution of RAG-2–deficient mice with transduced HSCs, the specific vectors were efficiently expressed in T cells, minimally expressed in B cells, and not expressed in immature cells of the bone marrow. Addition of the CD4gene-silencing element in the vector regulatory sequences led to further restriction of eGFP expression into CD4⁺ T cells in reconstituted mice and in ex vivo–transduced human T cells. Non–T CD4⁺ dendritic and macrophage cells derived from human CD34⁺ cells in vitro expressed the transgene of the specific vectors, albeit at lower levels than CD4⁺ T cells. Altogether, we have generated lentiviral vectors that allow specific targeting of transgene expression to CD4⁺ cells after differentiation of transduced mice HSCs and human mature T cells. Ultimately, these vectors may prove useful for in situ injections for in vivo gene therapy of HIV infection or genetic immunodeficiencies.