Gulcin Demirci

IL-15 and IL-2: A matter of life and death for T cells in vivo

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 α-chain, which defines the binding specificity for IL-2 or IL-15, and shared IL-2 receptor β- and γ-chains. The γ-chain is also a critical signaling component of IL-4, IL-7 and IL-9 receptors. Thus, the γ-chain is called the common γ or γ-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 γ-c expression. Decreased γ-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.

Stimulating PD-1-negative signals concurrent with blocking CD154 co-stimulation induces long-term islet allograft survival

Background. A balanced network of positive and negative T-cell co-stimulatory signals is important in regulating T-cell activation. Blocking CD28, CD154 (CD40L), or both co-stimulatory molecules has been efficacious in preventing acute allograft rejection in certain but not all transplantation models. In the present study, the authors tested the hypothesis that stimulating programmed death 1 (PD-1)–triggered negative signals concurrent with blocking CD154 co-stimulatory signals would facilitate islet allograft tolerance. Methods. The authors used a dimeric PD-L1 immunoglobulin (Ig) fusion protein to stimulate the inhibitory receptor PD-1, and a monoclonal antibody to block CD154. The effects of PD-1 engagement and CD154 blockade on lymphocyte activation were determined by cell proliferation, flow cytometry, and a model of islet transplantation. Results. PD-L1Ig inhibited the proliferation of both CD4+ and CD8+ T cells stimulated by anti-CD3. The inhibitory effect of PD-L1Ig was enhanced by concurrent blockade of CD154 co-stimulatory signals, as demonstrated by T-cell proliferation and expression of cell surface activation markers. PD-L1Ig and anti-CD154 also synergistically blocked the activation and maturation of antigen-presenting cells. In an islet transplantation model, treatment of recipient C57BL/6 (H-2b) mice with PD-L1Ig and anti-CD154 induced long-term survival of DBA/2 (H-2d) islet allografts, whereas treatment with each reagent alone failed to prevent islet allograft rejection. Conclusions. 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.

The Innate NK Cells, Allograft Rejection, and a Key Role for IL-15

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.

Critical Role of OX40 in CD28 and CD154-Independent Rejection

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.

New Insights on OX40 in the Control of T Cell Immunity and Immune Tolerance In Vivo

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+ regulatory T cells (Tregs), but not by resting conventional T cells. The exact role of OX40 in Treg homeostasis and function remains incompletely defined. In this study, we demonstrate that OX40 engagement in vivo in naive 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 naive 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 reveal a novel role for OX40 in promoting immune tolerance and may have important clinical implications.

OX40/OX40L Costimulation Affects Induction of Foxp3+ Regulatory T Cells in Part by Expanding Memory T Cells In Vivo

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.

Striking dichotomy of PD-L1 and PD-L2 pathways in regulating alloreactive CD4+ and CD8+ T cells in vivo

Programmed death‐1 (PD‐1) is a recently identified coinhibitory molecule that belongs to the CD28 superfamily. PD‐1 has two ligands PD‐L1 and PD‐L2. There is some evidence that PD‐L1 and PD‐L2 serve distinct functions, but their exact function in alloimmunity remains unclear. In the present study, we used a GVHD‐like model that allows detailed analyses of T‐cell activation at a single cell level in vivo to examine the role of PD‐1/PD‐L1 and PD‐1/PD‐L2 interactions in regulating proliferation of CD4+ and CD8+ T cells in response to alloantigen stimulation. We found that both CD4+ and CD8+ T cells proliferated vigorously in vivo and that PD‐L1 and PD‐L2 exhibit strikingly different effect on T‐cell proliferation. While blocking PD‐L1 did not affect the in vivo proliferation of CD4+ and CD8+ T cells regardless of CD28 costimulation, blocking PD‐L2 resulted in a marked increase in the responder frequency of CD8+ T‐cells in vivo. The effect of PD‐L2 on the CD8+ T‐cell proliferation is regulated by CD28 costimulation and by the CD4+ T cells. We conclude that PD‐L1 and PD‐L2 function differently in regulating alloreactive T‐cell activation in vivo, and PD‐L2 is predominant in this model in limiting alloreactive CD8+ T‐cell proliferation.

Different Costimulatory and Growth Factor Requirements for CD4+ and CD8+ T Cell-Mediated Rejection

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.

Innate NK Cells and Macrophages Recognize and Reject Allogeneic Nonself In Vivo via Different Mechanisms

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 current study, we examined the roles of NK cells and macrophages in recognizing and rejecting allogeneic cells in vivo. We found that in naive mice NK cells are the primary effector cells in the killing of allogeneic cells via “missing self” recognition. However, in alloantigen-presensitized mice, NK cells are dispensable. Instead, macrophages become alloreactive and readily recognize and reject allogeneic nonself. This effect requires help from activated CD4+ T cells and involves CD40/CD40L engagement, because 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 graft-versus-host disease model. Together, our data uncover an important role for macrophages in the alloimmune response and may have important clinical implications.

Negative T cell costimulation and islet tolerance

Activation of self‐reactive T cells that specifically destroy the pancreatic β‐cells is one of the hallmarks in the development of type 1 diabetes. Thus, for prevention and treatment of this autoimmune disease, approaches to induce and maintain T cell tolerance toward the β‐cells, especially in islet transplantation, have been actively pursued. Noticeably, many of the recent protocols for inducing transplant tolerance involve blockade of positive T cell costimulation extrinsically. Though highly effective in prolonging graft survival, these strategies alone might not be universally sufficient to achieve true tolerance. As the mystery of the suppressive and regulatory T cells unfolds, it is becoming appreciated that exploiting the intrinsic molecular and cellular mechanisms that turn off an immune response would perhaps facilitate the current protocols in establishing T cell tolerance. In this perspective, here we summarize the recent findings on the negative costimulation pathways, in particular, the newly identified PD‐1 : PD‐L interactions. On the basis of these observations, we propose a new principle of curtailing pathogenic T cell response in which blockade of positive T cell costimulation is reinforced by concurrent engagement of the negative costimulation machinery. Such a strategy may hold greater hope for therapeutic intervention of transplant rejection and autoimmune diseases. Copyright