Toshiro Ito

The role of the ICOS-B7h T cell costimulatory pathway in transplantation immunity

Inducible costimulatory molecule (ICOS) plays a pivotal role in T cell activation and Th1/Th2 differentiation. ICOS blockade has disparate effects on immune responses depending on the timing of blockade. Its role in transplantation immunity, however, remains incompletely defined. We used a vascularized mouse cardiac allograft model to explore the role of ICOS signaling at different time points after transplantation, targeting immune initiation (early blockade) or the immune effector phase (delayed blockade). In major histocompatibility-mismatched recipients, ICOS blockade prolonged allograft survival using both protocols but did so more effectively in the delayed-treatment group. By contrast, in minor histocompatibility-mismatched recipients, early blockade accelerated rejection and delayed blockade prolonged graft survival. Alloreactive CD4+ T cell expansion and alloantibody production were suppressed in both treatment groups, whereas only delayed blockade resulted in suppression of effector CD8+ T cell generation. After delayed ICOS blockade, there was a diminished frequency of allospecific IL-10-producing cells and an increased frequency of both IFN-gamma- and IL-4-producing cells. The beneficial effects of ICOS blockade in regulating allograft rejection were seen in the absence of CD28 costimulation but required CD8+ cells, cytotoxic T lymphocyte antigen-4, and an intact signal transducer and activator of transcription-6 pathway. These data define the complex functions of the ICOS-B7h pathway in regulating alloimmune responses in vivo.

Analysis of the Role of Negative T Cell Costimulatory Pathways in CD4 and CD8 T Cell-Mediated Alloimmune Responses In Vivo

Negative costimulatory signals mediated via cell surface molecules such as CTLA-4 and programmed death 1 (PD-1) play a critical role in down-modulating immune responses and maintaining peripheral tolerance. However, their role in alloimmune responses remains unclear. This study examined the role of these inhibitory pathways in regulating CD28-dependent and CD28-independent CD4 and CD8 alloreactive T cells in vivo. CTLA-4 blockade accelerated graft rejection in C57BL/6 wild-type recipients and in a proportion of CD4−/− but not CD8−/− recipients of BALB/c hearts. The same treatment led to prompt rejection in CD28−/− and a smaller proportion of CD4−/−CD28−/− mice with no effect in CD8−/−CD28−/− recipients. These results indicate that the CTLA-4:B7 pathway provides a negative signal to alloreactive CD8+ T cells, particularly in the presence of CD28 costimulation. In contrast, PD-1 blockade led to accelerated rejection of heart allografts only in CD28−/− and CD8−/−CD28−/− recipients. Interestingly, PD-1 ligand (PD-L1) blockade led to accelerated rejection in wild-type mice and in all recipients lacking CD28 costimulation. This effect was accompanied by expansion of IFN-γ-producing alloreactive T cells and enhanced generation of effector T cells in rejecting allograft recipients. Thus, the PD-1:PD-L1 pathway down-regulates alloreactive CD4 T cells, particularly in the absence of CD28 costimulation. The differential effects of PD-1 vs PD-L1 blockade support the possible existence of a new receptor other than PD-1 for negative signaling through PD-L1. Furthermore, PD-1:PD-L1 pathway can regulate alloimmune responses independent of an intact CD28/CTLA-4:B7 pathway. Harnessing physiological mechanisms that regulate alloimmunity should lead to development of novel strategies to induce durable and reproducible transplantation tolerance.

CD70 Signaling Is Critical for CD28-Independent CD8+ T Cell-Mediated Alloimmune Responses In Vivo

The inability to reproducibly induce robust and durable transplant tolerance using CD28-B7 pathway blockade is in part related to the persistence of alloreactive effector/memory CD8+ T cells that are less dependent on this pathway for their cellular activation. We studied the role of the novel T cell costimulatory pathway, CD27-CD70, in alloimmunity in the presence and absence of CD28-B7 signaling. CD70 blockade prolonged survival of fully mismatched vascularized cardiac allografts in wild-type murine recipients, and in CD28-deficient mice induced long-term survival while significantly preventing the development of chronic allograft vasculopathy. CD70 blockade had little effect on CD4+ T cell function but prevented CD8+ T cell-mediated rejection, inhibited the proliferation and activation of effector CD8+ T cells, and diminished the expansion of effector and memory CD8+ T cells in vivo. Thus, the CD27-CD70 pathway is critical for CD28-independent effector/memory CD8+ alloreactive T cell activation in vivo. These novel findings have important implications for the development of transplantation tolerance-inducing strategies in primates and humans, in which CD8+ T cell depletion is currently mandatory.

Differential Role of CCR2 in Islet and Heart Allograft Rejection: Tissue Specificity of Chemokine/Chemokine Receptor Function In Vivo

Chemokines have a pivotal role in the mobilization and activation of specific leukocyte subsets in acute allograft rejection. However, the role of specific chemokines and chemokine receptors in islet allograft rejection has not been fully elucidated. We now show that islet allograft rejection is associated with a steady increase in intragraft expression of the chemokines CCL8 (monocyte chemoattractant protein-2), CCL9 (monocyte chemoattractant protein-5), CCL5 (RANTES), CXCL-10 (IFN-γ-inducible protein-10), and CXCL9 (monokine induced by IFN-γ) and their corresponding chemokine receptors CCR2, CCR5, CCR1, and CXCR3. Because CCR2 was found to be highly induced, we tested the specific role of CCR2 in islet allograft rejection by transplanting fully MHC mismatched islets from BALB/c mice into C57BL/6 wild-type (WT) and CCR2-deficient mice (CCR2−/−). A significant prolongation of islet allograft survival was noted in CCR2−/− recipients, with median survival time of 24 and 12 days for CCR2−/− and WT recipients, respectively (p < 0.0001). This was associated with reduction in the generation of CD8+, but not CD4+ effector alloreactive T cells (CD62LlowCD44high) in CCR2−/− compared with WT recipients. In addition, CCR2−/− recipients had a reduced Th1 and increased Th2 alloresponse in the periphery (by ELISPOT analysis) as well as in the grafts (by RT-PCR). However, these changes were only transient in CCR2−/− recipients that ultimately rejected their grafts. Furthermore, in contrast to the islet transplants, CCR2 deficiency offered only marginal prolongation of heart allograft survival. This study demonstrates the important role for CCR2 in early islet allograft rejection and highlights the tissue specificity of the chemokine/chemokine receptor system in vivo in regulating allograft rejection.

Allorecognition and Effector Pathways of Islet Allograft Rejection in Normal versus Nonobese Diabetic Mice

Islet transplantation is becoming an accepted therapy to cure type I diabetes mellitus. The exact mechanisms of islet allograft rejection remain unclear, however. In vivo CD4(+) and CD8(+) T cell-depleting strategies and genetically altered mice that did not express MHC class I or class II antigens were used to study the allorecognition and effector pathways of islet allograft rejection in different strains of mice, including autoimmunity-prone nonobese diabetic (NOD) mice. In BALB/c mice, islet rejection depended on both CD4(+) and CD8(+) T cells. In C57BL/6 mice, CD8(+) T cells could eventually mediate islet rejection by themselves, but they produced rejection more efficiently with help from CD4(+) T cells stimulated through either the direct or indirect pathway. In C57BL/6 mice, CD4(+) T cells alone caused islet rejection when only the direct pathway was available but not when only the indirect pathway was available. In contrast, in NOD mice, CD4(+) T cells alone, with only the indirect pathway, could mediate islet and cardiac allograft rejection. These findings indicate that different mouse strains can make use of different pathways for T cell-mediated rejection of islet allografts. In addition, they demonstrate that NOD mice, which develop autoimmunity and are known to be resistant to tolerance induction, have an unusually powerful CD4(+) cell indirect mechanism that can cause rejection of both islet and cardiac allografts. These data shed light on the mechanisms of islet allograft rejection in different responder strains, including those with autoimmunity.

Importance of Donor- and Recipient-Derived Selectins in Cardiac Allograft Rejection

The selectins expressed on activated endothelial cells (E- and P-selectin), leukocytes (L-selectin), and platelets (P-selectin) play crucial roles in the rolling and tethering of leukocytes. We explored the importance of donor and recipient selectins in acute and chronic cardiac allograft rejection using mice deficient in all three selectins (ELP-/-). In BALB/c recipients, survival of fully allomismatched hearts from ELP-/- C57BL/6 donors was almost double that of wild-type grafts. In ELP-/- cardiac allografts, mononuclear cell infiltration and vasculitis of intramyocardial coronary arteries were significantly reduced. Interestingly, ELP-/- grafts were rejected similarly in both the presence and the absence of recipient selectins, and both wild-type and ELP-/- recipients promptly rejected wild-type hearts. Alternative adhesive molecules such as alpha4beta7 integrin may compensate for the lack of selectins and may mediate rejection in ELP-/- recipients. Chronic rejection was evaluated in a major histocompatibility complex (MHC) class II mismatch model using C57BL/6.C-H2(bm12) mice. While lack of selectins in recipients did not offer protection against chronic rejection, luminal stenosis of coronary arteries in ELP-/- grafts was markedly diminished. In conclusion, donor-derived selectins contribute to the development of both acute and chronic cardiac allograft rejection, and targeting donor selectins may open novel therapeutic approaches in clinical transplantation.

Mg-ATPase and Ca2+ activated myosin ATPase activity in ventricular myofibrils from non-failing and diseased human hearts: effects of calcium sensitizing agents MCI-154, DPI 201-106, and caffeine

We investigated the effects of two purported calcium sensitizing agents, MCI-154 and DPI 201–106, and a known calcium sensitizer caffeine on Mg-ATPase (myofibrillar ATPase) and myosin ATPase activity of left ventricular myofibrils isolated from non-failing, idiopathic (IDCM) and ischemic cardiomyopathic (ISCM) human hearts (i.e. failing hearts). The myofibrillar ATPase activity of non-failing myofibrils was higher than that of diseased myofibrils. MCI-154 increased myofibrillar ATPase Ca2+ sensitivity in myofibrils from non-failing and failing human hearts. Effects of caffeine similarly increased Ca2+ sensitivity. Effects of DPI 201–106 were, however, different. Only at the 10−6 M concentration was a significant increase in myofibrillar ATPase calcium sensitivity seen in myofibrils from non-failing human hearts. In contrast, in myofibrils from failing hearts, DPI 201–106 caused a concentration-dependent increase in myofibrillar ATPase Ca2+ sensitivity. Myosin ATPase activity in failing myocardium was also decreased. In the presence of MCI-154, myosin ATPase activity increased by 11, 19, and 24% for non-failing, IDCM, and ISCM hearts, respectively. DPI 201–106 caused an increase in the enzymatic activity of less than 5% for all preparations, and caffeine induced an increase of 4, 11, and 10% in non-failing, IDCM and ISCM hearts, respectively. The mechanism of restoring the myofibrillar Ca2+ sensitivity and myosin enzymatic activity in diseased human hearts is most likely due to enhancement of the Ca2+ activation of the contractile apparatus induced by these agents. We propose that myosin light chain-related regulation may play a complementary role to the troponin-related regulation of myocardial contractility.

Role of nuclear factor of activated T cell (NFAT) transcription factors in skin and vascularized cardiac allograft rejection.

The fungal metabolites cyclosporine A and tacrolimus (FK506) are among the most potent immunosuppressive drugs available today (1). As inhibitors of the calcium-dependent serine/threonine phosphatase calcineurin, these drugs inhibit the dephosphorylation of the transcription factor nuclear factor of activated T cells (NFAT), a process that is essential for the nuclear localization of the cytoplasmic components of the NFAT transcription complex. Currently, five different NFAT family members have been identified: NFAT1 (also known as NFATp/NFATc2), NFAT2 (NFATc1), NFAT3 (NFATc4), NFAT4 (NFATx/NFATc3), and NFAT5 (2). Our collaborator has previously demonstrated a preferential T helper 2 (Th2) differentiation and modulated T-cell receptor responsiveness of naive T cells in mice deficient in NFATc2 and NFATc3 (NFATc2/c3 double deficient [DKO] mice: BALB/c (H-2) background) (3). Moreover, several studies of one or two NFAT family members have attempted to address the role of NFAT in regulation of CD4 Th1/Th2 differentiation (4 – 8). Although NFAT family members play crucial roles in the development and function of the immune system, little work seems to have addressed their direct role in transplantation. In this study, we sought to explore the role of these molecules in both skin and cardiac transplantation models using allografts from BALB.B (H-2) donors. Allograft survival was slightly but significantly prolonged in mice deficient in NFATc2 and NFATc3 (DKO) recipients of skin grafts (18 days vs. 12.5 days, P 0.0001) (Fig. 1b) and in recipients of heart allografts (14 days vs. 8 days, P 0.0004) (Fig. 1a). Our in vitro studies showed that alloantigen proliferative responses are unaffected in DKO recipients by mixed lymphocyte reaction (data not shown). Enzyme-linked immunosorbent spot (ELISPOT) showed that interFIGURE 1. Allograft survival in DKO recipients; (a and b) Allograft survival was significantly prolonged in DKO recipients of skin grafts (b) and somewhat less prolonged in recipients of heart allografts (a). Th2 cytokine generation was enhanced by NFATc2/c3 deficiency; (c) production of the Th1 cytokine IFNwas significantly lower in the NFATc2and NFATc3-deficient (DKO) mice. DKO mice enhanced their production of IL-4. Data are representative of three independent experiments using at least n 3 mice per group. Allograft survival in TKO recipients; (d and e) TKO mice rejected both skin (e) and cardiac allografts (d) as promptly as wild-type BALB/c controls. Data from the figure (a and b): allograft survival in control and DKO is also used in the figure (d and e). **P less than 0.001; ***P less than 0.0001. DKO, NFATc2/c3 double-deficient; NFAT, nuclear factor of activated T cells; IL, interleukin; TKO, NFATc2/c3 and IL-4 triple-deficient.

The Limits of Linked Suppression for Regulatory T Cells

Background We have previously found that CD4+CD25+ regulatory T cells (Tregs) can adoptively transfer tolerance after its induction with costimulatory blockade in a mouse model of murine cardiac allograft transplantation. In these experiments, we tested an hypothesis with three components: (1) the Tregs that transfer tolerance have the capacity for linked suppression, (2) the determinants that stimulate the Tregs are expressed by the indirect pathway, and (3) the donor peptides contributing to these indirect determinants are derived from donor major histocompatibility complex (MHC) antigens (Ags). Methods First heart transplants were performed from the indicated donor strain to B10.D2 recipients along with costimulatory blockade treatment (250 μg i.p. injection of MR1 on day 0 and 250 μg i.p. injection of CTLA-4 Ig on day 2). At least 8 weeks later, a second heart transplant was performed to a new B10.D2 recipient who had been irradiated with 450 cGy. This recipient was given 40 × 106 naive B10.D2 spleen cells + 40 × 106 B10.D2 spleen cells from the first (tolerant) recipient. We performed three different types of heart transplants using various donors. Results (1) Tregs suppress the graft rejection in an Ag-specific manner. (2) Tregs generated in the face of MHC disparities suppress the rejection of grafts expressing third party MHC along with tolerant MHC. Conclusion The limits of linkage appear to be quantitative and not universally determined by either the indirect pathway or by peptides of donor MHC Ags.

Myofibrillar responsiveness to cAMP, PKA, and caffeine in an animal model of heart failure

We investigated whether an alteration of myofilament calcium responsiveness and contractile activation may in part contribute to heart failure. A control group of Broad Breasted White turkey poults was given regular feed without additive, whereas the experimental group was given the control ration with 700 ppm of furazolidone at 1 week of age for 3 weeks (DCM). At 4 weeks of age, left ventricular trabeculae carneae were isolated from hearts and calcium-force relationships studied. No differences in calcium-activation between fibers from control or failing hearts were noted under standard experimental conditions. Also failing hearts demonstrated no significant shift in the population of troponin T isoforms but we did observe a significant 4-fold decrease in TnT content in failing hearts compared to non-failing hearts. Addition of caffeine, however, resulted in a greater leftward shift on the calcium axis in fibers from failing hearts. At pCa 6, caffeine increased force by 26+/-2.1% in control fibers and 44.5+/-8.7% in myopathic fibers. Cyclic AMP resulted in a greater rightward shift on the calcium axis in failing myocardium. In control muscles, the frequency of minimum stiffness (f(min)) was higher than in muscles from failing hearts. cAMP and caffeine both shifted f(min) to higher frequencies in control fibers whereas in fibers from failing hearts both caused a greater shift. These results lead us to conclude that heart failure exerts differential effects on cAMP and caffeine responsiveness. Our data suggest that changes at the level of the thin myofilaments may alter myofilament calcium responsiveness and contribute to the contractile dysfunction seen in heart failure.