Control of CD4+ T Cell Function by CD4+CD25+ Regulatory T Cells

Thesis (Ph.D.)--University of Rochester. School of Medicine and Dentistry. Dept. of Microbiology and Immunology, 2009.In the periphery thymus derived, naturally occurring CD4+CD25+Foxp3+ (Treg(s)) have the potential to suppress an extensive array of immune responses that exceed their original identification of preventing autoimmune disease. In recent years Treg mediated beneficial and harmful suppressive activities have been attributed to limiting responses to infectious agents and tumor antigens, respectively. In various immune settings Tregs have been implicated in controlling initial T cell activation, proliferation, differentiation, effector function, and migration. Although Tregs have an extensive suppressive agenda, their mechanisms of action are not well understood. Costimulatory signals from CD28 and CTLA-4 have been identified to regulate both target T cells and Tregs and can either help the cell escape suppression or mediate the suppression. This thesis will focus on the CD4+ effector functions that are modified by Tregs in the presence of CD28 signaling as well as the role of CTLA-4 in Treg function. To get a better understanding of Treg action we first investigated the kinetics of murine Treg activity in vitro. Tregs were suppressive within a surprisingly narrow kinetic window: necessary and sufficient only in the first 6-10h of culture. Importantly, the timing of suppression was dictated by the kinetics of target T cell activation suggesting that early target T cell signals may alter susceptibility to suppression. CD28 signals enabled target T cells to resist the early Treg-induced downregulation of IL-2. Disabling one mode of Treg suppressive activity, IL-2 secretion and proliferation, by provision of CD28 allowed us to test other arms of Treg suppressive activities. Here we define specific requirements for suppression of IL-2 secretion and proliferation than that for suppression of IFN- secretion. CD28 signaling abrogates Treg action through rendering the target T cell resistant to Treg suppression. We reveal that while CD28 signals do bypass the Treg blockade of IL-2 secretion and proliferation, the target cells remain susceptible to Treg suppression of IFN- secretion. Early during priming, secretion of IFN- by proliferating CD4+ T cells is suppressed by Tregs. This acute shutoff of effector cytokine production is IL-10 dependent but independent of TGF-. The Treg source of IL-10 suppresses IFN- secretion but plays no role in suppressing IL-2 secretion and proliferation. In addition, Tregs from p35 KO and p40 KO mice fail to suppress IFN- secretion but do not lose their ability to suppress IL-2 secretion and proliferation. Thus, Tregs use a distinct set of suppressive factors for the regulation of IL-2 secretion and proliferation than for effector cytokine production. The loss of Treg function in the absence of IL-10, p35 and p40 suggests that either these molecules are part of the Treg suppressive arsenal, or perhaps more interestingly, that they are required to ‘condition’ Tregs to gain suppressive function for IFN-. In a separate set of in vivo experiments we set out to test the contributions of a constitutively expressed molecule on Tregs, CTLA-4, and the role that it may play in the suppressive functions of Tregs. Although blockade of CTLA-4 interferes with Treg function, studies using CTLA-4 deficient Tregs have failed to reveal an essential requirement for CTLA-4 in Treg suppression in vivo. We demonstrate that Treg expression of CTLA-4 is essential for Treg control of lymphopenia-induced CD4+ T cell expansion. Despite IL-10 expression, CTLA-4 deficient Tregs were unable to control the expansion of CD4+ target cells in a lymphopenic environment. Moreover, unlike their WT counterparts, CTLA-4-deficient Tregs failed to inhibit cytokine production associated with homeostatic expansion and were unable to prevent colitis. Thus, while Tregs developing in the absence of CTLA-4 appear to acquire some compensatory suppressive mechanisms in vitro, we identify a non-redundant role for CTLA-4 in Treg function in vivo. Our data reveal that Tregs have the ability to act early and suppress IL-2 secretion of CD4+ target T cells prior to the first cell division. While CD28 signals bypass the Treg blockade of IL-2 secretion and proliferation, the target T cells remain susceptible to Treg suppression of IFN- secretion. We identified specific molecules that are required by the Tregs to suppress IFN- secretion but not proliferation. Therefore Tregs disable their targets through multiple mechanisms; each suppressive tool controlling a highly specific aspect of immune function