Interleukin (IL)-2 and IL-15 are redundant in stimulating T-cell proliferation in vitro. Their precise role in vivo in governing T-cell expansion and T-cell homeostasis is less clear. Each may have distinct functions and regulate distinct aspects of T-cell activation. The functional receptors for IL-2 and IL-15 consist of a private alpha-chain, which defines the binding specificity for IL-2 or IL-15, and shared IL-2 receptor beta- and gamma-chains. The gamma-chain is also a critical signaling component of IL-4, IL-7 and IL-9 receptors. Thus, the gamma-chain is called the common gamma or gamma-c. As these receptor subunits can be expressed individually or in various combinations resulting in the formation of receptors with different affinities, distinct signaling capabilities or both, we hypothesized that differential expression of IL-2 and IL-15 receptor subunits on cycling T cells in vivo may direct activated T cells to respond to IL-2 or IL-15, thereby regulating the homeostasis of T-cell response in vivo. By observing in vivo T-cell divisions and expression of IL-2 and IL-15 receptor subunits, we demonstrate that IL-15 is a critical growth factor in initiating T cell divisions in vivo, whereas IL-2 limits continued T-cell expansion via downregulation of the gamma-c expression. Decreased gamma-c expression on cycling T cells reduced sustained Bcl-2 expression and rendered cells susceptible to apoptotic cell death. Our study provides data that IL-2 and IL-15 regulate distinct aspects of primary T-cell expansion in vivo.

These results suggest that engaging the negative receptor PD-1 exhibits critical immunoregulatory effects in the allograft response, and blocking positive co-stimulatory molecules with active delivery of inhibitory signals may represent a novel therapeutic strategy in transplantation.

Transplant rejection is mediated primarily by adaptive immune cells such as T cells and B cells. The T and B cells are also responsible for the specificity and memory of the rejection response. However, destruction of allografts involves many other cell types including cells in the innate immune system. As the innate immune cells do not express germline-encoded cell surface receptors that directly recognize foreign Ags, these cells are thought to be recruited by T cells to participate in the rejection response. In this study, we examined the alloreactivity of the innate NK cells in Rag−/− mice using a stringent skin transplant model and found that NK cells at a resting state readily reject allogeneic cells, but not the skin allografts. We also found that IL-15, when preconjugated to its high affinity IL-15Rα-chain, is remarkably potent in stimulating NK cells in vivo, and NK cells stimulated by IL-15 express an activated phenotype and are surprisingly potent in mediating acute skin allograft rejection in the absence of any adaptive immune cells. Furthermore, NK cell-mediated graft rejection does not show features of memory responses. Our data demonstrate that NK cells are potent alloreactive cells when fully activated and differentiated under certain conditions. This finding may have important clinical implications in models of transplantation and autoimmunity.

Blocking both CD28 and CD154 costimulatory pathways can induce transplant tolerance in some, but not all, transplant models. Under stringent conditions, however, this protocol often completely fails to block allograft rejection. The precise nature of such CD28/CD154 blockade-resistant rejection is largely unknown. In the present study we developed a new model in which both CD28 and CD154, two conventional T cell costimulatory molecules, are genetically knocked out (i.e., CD28/CD154 double-knockout (DKO) mice) and used this model to examine the role of novel costimulatory molecule-inducible costimulator (ICOS), OX40, 4-1BB, and CD27 in mediating CD28/CD154-independent rejection. We found that CD28/CD154 DKO mice vigorously rejected fully MHC-mismatched DBA/2 skin allografts (mean survival time, 12 days; n = 6) compared with the wild-type controls (mean survival time, 8 days; n = 7). OX40 costimulation is critically important in skin allograft rejection in this model, as blocking the OX40/OX40 ligand pathway, but not the ICOS/ICOS ligand, 4-1BB/4-1BBL, or CD27/CD70 pathway, markedly prolonged skin allograft survival in CD28/CD154 DKO mice. The critical role of OX40 costimulation in CD28/CD154-independent rejection is further confirmed in wild-type C57BL/6 mice, as blocking the OX40/OX40 ligand pathway in combination with CD28/CD154 blockade induced long term skin allograft survival (>100 days; n = 5). Our study revealed a key cellular mechanism of rejection and identified OX40 as a critical alternative costimulatory molecule in CD28/CD154-independent rejection.

OX40 is a T cell costimulatory molecule that belongs to the TNFR superfamily. In the absence of immune activation, OX40 is selectively expressed by Foxp3+ Tregs, but not by resting conventional T cells. The exact role of OX40 in Treg homeostasis and function remains incompletely defined. Here, we demonstrate that OX40 engagement in vivo in naïve mice induces initial expansion of Foxp3+ Tregs, but the expanded Tregs have poor suppressive function and exhibit features of exhaustion. We also show that OX40 enables the activation of the Akt and Stat5 pathways in Tregs, resulting in transient proliferation of Tregs and reduced levels of Foxp3 expression. This creates a state of relative IL-2 deficiency in naïve mice that further impacts Tregs. This exhausted Treg phenotype can be prevented by exogenous IL-2, as both OX40 and IL-2 agonists drive further expansion of Tregs in vivo. Importantly, Tregs expanded by both OX40 and IL-2 agonists are potent suppressor cells, and in a heart transplant model, they promote long-term allograft survival. Our data uncover a novel role for OX40 in promoting immune tolerance and may have important clinical implications.

Both innate and adaptive immune cells are involved in the allograft response. But how the innate immune cells respond to allotransplants remains poorly defined. In the present study, we examined the role of NK cells and macrophages in recognizing and rejecting allogeneic cells in vivo. We found that in naïve mice NK cells are the primary effector cells in killing of allogeneic cells via “the missing self” recognition. However, in alloantigen pre-sensitized mice, NK cells are dispensable. Instead, macrophages become alloreactive and readily recognize and reject allogeneic non-self. This effect requires help from activated CD4+ T cells and involves CD40/CD40L engagement, as blocking CD40/CD40L interactions prevents macrophage mediated rejection of allogeneic cells. Conversely, actively stimulating CD40 triggers macrophage-mediated rejection in the absence of CD4+ T cells. Importantly, alloantigen primed and CD4+ T cell-helped macrophages (licensed macrophages) exhibit potent regulatory function in vivo in an acute GVHD model. Together, our data uncover an important role for macrophages in the alloimmune response and may have important clinical implications.

OX40 is a member of the TNFR superfamily and has potent T cell costimulatory activities. OX40 also inhibits the induction of Foxp3+ regulatory T cells (Tregs) from T effector cells, but the precise mechanism of such inhibition remains unknown. In the present study, we found that CD4+ T effector cells from OX40 ligand-transgenic (OX40Ltg) mice are highly resistant to TGF-β mediated induction of Foxp3+ Tregs, whereas wild-type B6 and OX40 knockout CD4+ T effector cells can be readily converted to Foxp3+ T cells. We also found that CD4+ T effector cells from OX40Ltg mice are heterogeneous and contain a large population of CD44highCD62L− memory T cells. Analysis of purified OX40Ltg naive and memory CD4+ T effector cells showed that memory CD4+ T cells not only resist the induction of Foxp3+ T cells but also actively suppress the conversion of naive CD4+ T effector cells to Foxp3+ Tregs. This suppression is mediated by the production of IFN-γ by memory T cells but not by cell-cell contact and also involves the induction of T-bet. Importantly, memory CD4+ T cells have a broad impact on the induction of Foxp3+ Tregs regardless of their origins and Ag specificities. Our data suggest that one of the mechanisms by which OX40 inhibits the induction of Foxp3+ Tregs is by inducing memory T cells in vivo. This finding may have important clinical implications in tolerance induction to transplanted tissues.

Costimulatory signals and growth factor signals play a key role in commanding T cell activation and T cell effector function. However, how costimulatory signals and growth factor signals interact and integrate into the activation program of CD4+ and CD8+ T cells during the allograft response remains poorly defined. In the present study we found that either CD4- or CD8-deficient mice can vigorously reject the skin allografts. Blocking rapamycin-sensitive growth factor signals produced long term skin allograft survival in CD4-deficient mice (mean survival time, >120 days), but not in CD8-deficient mice (mean survival time, 20 days). Analysis of CFSE-labeled cells proliferating in the allogeneic hosts revealed that clonal expansion of CD4+ T cells in vivo was more resistant to growth factor blockade than that of CD8+ T cells. However, blockade or genetic absence of CD28/CD154 costimulatory molecules rendered CD4+ T cell-mediated rejection sensitive to rapamycin, and long term skin allograft survival can be readily induced by rapamycin in the absence of CD28/CD154 signals (>100 days). Furthermore, blocking OX40 costimulation induced long term skin allograft survival in CD4-deficient mice and CD8-deficient mice when both CD28 and CD154 were transiently blocked. We conclude that CD4+ and CD8+ T cells exhibit distinct sensitivity to growth factor blockade in transplant rejection, and CD28/CD154-independent rejection is sensitive to rapamycin and appears to be supported by OX40 costimulation.