CD4+ T regulatory cell induction and function in transplant recipients after CD154 blockade is TLR4 independent

Although the role of CD4(+) T regulatory cells (Treg) in transplantation tolerance has been established, putative mechanisms of Treg induction and function in vivo remain unclear. TLR4 signaling has been implicated in the regulation of CD4(+)CD25(+) Treg functions recently. In this study, we first examined the role of recipient TLR4 in the acquisition of operational CD4(+) Treg following CD154 blockade in a murine cardiac transplant model. Then, we determined whether TLR4 activation in allograft tolerant recipients would reverse alloimmune suppression mediated by CD4(+) Treg. We document that donor-specific immune tolerance was readily induced in TLR4-deficient recipients by a single dose of anti-CD154 mAb, similar to wild-type counterparts. The function and phenotype of CD4(+) Treg in both wild-type and TLR4 knockout long-term hosts was demonstrated by a series of depletion experiments examining their ability to suppress the rejection of secondary donor-type test skin grafts and to inhibit alloreactive CD8(+) T cell activation in vivo. Furthermore, TLR4 activation in tolerant recipients following exogenous LPS infusion in conjunction with donor-type skin graft challenge, failed to break Treg-mediated immune suppression. In conclusion, our data reveals a distinctive property of CD4(+) Treg in tolerant allograft recipients, whose induction and function are independent of TLR4 signaling.     

The CD154-CD40 T cell costimulation pathway is required for host sensitization of CD8(+) T cells by skin grafts via direct antigen presentation

Although the CD154-CD40 T cell costimulation pathway has been shown to mediate alloimmune responses in normal recipients, little is known about its role in sensitized hosts. In this work, by using novel models of cardiac allograft rejection in skin-sensitized CD154- and CD40-deficient mice, we reaffirm the key role of CD154-CD40 signaling in host sensitization to alloantigen in vivo. First, we identified CD8(+) T cells as principal effectors in executing accelerated rejection in our model. Disruption of CD154-CD40 signaling in recipients at the T cell side (CD154-deficient) but not at the APC side (CD40-deficient) abrogated accelerated (<2 days) rejection and resulted in long-term (>100 days) graft survival. This suggests that the CD154-dependent mechanism in host CD8(+) T cell sensitization operates via the direct Ag presentation. Then, in comparative studies of alloimmune responses in CD154-deficient and wild-type recipients, we showed that, although alloreactive B cell responses were inhibited, alloreactive T cell responses were down-regulated selectively in the CD8(+) T cell compartment, leaving CD4(+) T cells largely unaffected. This unique alteration in host alloreactivity, seen not only in peripheral lymphocytes but also in allograft infiltrate, may represent the key mechanism by which disruption of CD154-CD40 signaling prevents sensitization to alloantigen in vivo and leads to long-term allograft survival.     

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.     

IL-10- and IL-12-independent down-regulation of allergic sensitization by stimulation of CD40 signaling

Interaction between CD154 (CD40 ligand) on activated T lymphocytes and its receptor CD40 has been shown to be critically involved in the generation of cell-mediated as well as humoral immunity. CD40 triggering activates dendritic cells (DC), enhances their cytokine production, up-regulates the expression of costimulatory molecules, and induces their maturation. It is unknown how stimulation of CD40 during sensitization to an airborne allergen may affect the outcome of allergic airway inflammation. We took advantage of a mouse model of allergic asthma and a stimulatory mAb to CD40 (FGK45) to study the effects of CD40-mediated DC activation on sensitization to OVA and subsequent development of OVA-induced airway inflammation. Agonistic anti-CD40 mAb (FGK45) injected during sensitization with OVA abrogated the development of allergic airway inflammation upon repeated airway challenges with OVA. Inhibition of bronchial eosinophilia corresponded with reduced Th2 cytokine production and was independent of IL-12, as evidenced by a similar down-regulatory effect of anti-CD40 mAb in IL-12 p40-deficient mice. In addition, FGK45 equally down-regulated allergic airway inflammation in IL-10-deficient mice, indicating an IL-10-independent mechanism of action of FGK45. In conclusion, our results show that CD40 signaling during sensitization shifts the immune response away from Th2 cytokine production and suppresses allergic airway inflammation in an IL-12- and IL-10-independent way, presumably resulting from enhanced DC activation during sensitization.     

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-gamma-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.     

Intestinal lamina propria retaining CD4+CD25+ regulatory T cells is a suppressive site of intestinal inflammation

It is well known that immune responses in the intestine remain in a state of controlled inflammation, suggesting that not only does active suppression by regulatory T (T(REG)) cells play an important role in the normal intestinal homeostasis, but also that its dysregulation of immune response leads to the development of inflammatory bowel disease. In this study, we demonstrate that murine CD4(+)CD25(+) T cells residing in the intestinal lamina propria (LP) constitutively express CTLA-4, glucocorticoid-induced TNFR, and Foxp3 and suppress proliferation of responder CD4(+) T cells in vitro. Furthermore, cotransfer of intestinal LP CD4(+)CD25(+) T cells prevents the development of chronic colitis induced by adoptive transfer of CD4(+)CD45RB(high) T cells into SCID mice. When lymphotoxin (LT)alpha-deficient intercrossed Rag2 double knockout mice (LTalpha(-/-) x Rag2(-/-)), which lack mesenteric lymph nodes and Peyer's patches, are transferred with CD4(+)CD45RB(high) T cells, they develop severe wasting disease and chronic colitis despite the delayed kinetics as compared with the control LTalpha(+/+) x Rag2(-/-) mice transferred with CD4(+)CD45RB(high) T cells. Of note, when a mixture of splenic CD4(+)CD25(+) T(REG) cells and CD4(+)CD45RB(high) T cells are transferred into LTalpha(-/-) x Rag2(-/-) recipients, CD4(+)CD25(+) T(REG) cells migrate into the colon and prevent the development of colitis in LTalpha(-/-) x Rag2(-/-) recipients as well as in the control LTalpha(+/+) x Rag2(-/-) recipients. These results suggest that the intestinal LP harboring CD4(+)CD25(+) T(REG) cells contributes to the intestinal immune suppression.     

CTLA-4 engagement and regulatory CD4+CD25+ T cells independently control CD8+-mediated responses under costimulation blockade

Blockade of costimulatory signals is a promising therapeutic target to prevent allograft rejection. In this study, we sought to characterize to what extent CTLA-4 engagement contributes to the development of transplantation tolerance under the cover of CD40/CD40L and CD28/CD86 blockade. In vitro, we found that inhibition of the primary alloresponse and induction of alloantigen hyporesponsiveness by costimulation blockade was abrogated by anti-CTLA-4 mAb. In addition, regulatory CD4(+)CD25(+) T cells (T(REG)) were confirmed to play a critical role in the induction of hyporesponsiveness by anti-CD40L and anti-CD86 mAb. Our data indicated that CTLA-4 engagement is not required for activation or suppressor function of T(REG). Instead, in the absence of either CTLA-4 signaling or T(REG), CD8(+) T cell division was enhanced, whereas the inhibition of CD4(+) T cell division by costimulation blockade remained largely unaffected. In vivo, the administration of additional anti-CTLA-4 mAb abrogated anti-CD40L- and anti-CD86 mAb-induced cardiac allograft survival. Correspondingly, rejection was accompanied by enhanced allograft infiltration of CD8(+) cells. We conclude that CTLA-4 signaling and T(REG) independently cooperate in the inhibition of CD8(+) T cell expansion under costimulation blockade.     

The role of the CD134-CD134 ligand costimulatory pathway in alloimmune responses in vivo

The CD134-CD134 ligand (CD134L) costimulatory pathway has been shown to be critical for both T and B cell activation; however, its role in regulating the alloimmune response remains unexplored. Furthermore, its interactions with other costimulatory pathways and immunosuppressive agents are unclear. We investigated the effect of CD134-CD134L pathway blockade on allograft rejection in fully MHC-mismatched rat cardiac and skin transplantation models. CD134L blockade alone did not prolong graft survival compared with that of untreated recipients, and in combination with donor-specific transfusion, cyclosporine, or rapamycin, was less effective than B7 blockade in prolonging allograft survival. However, in combination with B7 blockade, long-term allograft survival was achieved in all recipients (>200 days). Moreover, this was synergistic in reducing the frequency of IFN-gamma-producing alloreactive lymphocytes and inhibiting the generation of activated/effector lymphocytes. Most impressively, this combination prevented rejection in a presensitized model using adoptive transfer of primed lymphocytes into athymic heart transplant recipients. In comparison to untreated recipients (mean survival time (MST): 5.3 +/- 0.5 days), anti-CD134L mAb alone modestly prolonged allograft survival (MST: 14 +/- 2.8 days) as did CTLA4Ig (MST: 21.5 +/- 1.7 days), but all grafts were rejected within 24 days. Importantly, combined blockade further and significantly prolonged allograft survival (MST: 75.3 +/- 12.7 days) and prevented the expansion and/or persistence of primed/effector alloreactive T cells. Our data suggest that CD134-CD134L is a critical pathway in alloimmune responses, especially recall/primed responses, and is synergistic with CD28-B7 in mediating T cell effector responses during allograft rejection. Understanding the mechanisms of collaboration between these different pathways is important for the development of novel strategies to promote long-term allograft survival.     

IL-6 promotes cardiac graft rejection mediated by CD4+ cells

IL-6 mediates numerous immunologic effects relevant to transplant rejection; however, its specific contributions to these processes are not fully understood. To this end, we neutralized IL-6 in settings of acute cardiac allograft rejection associated with either CD8(+) or CD4(+) cell-dominant responses. In a setting of CD8(+) cell-dominant graft rejection, IL-6 neutralization delayed the onset of acute rejection while decreasing graft infiltrate and inverting anti-graft Th1/Th2 priming dominance in recipients. IL-6 neutralization markedly prolonged graft survival in the setting of CD4(+) cell-mediated acute rejection and was associated with decreased graft infiltrate, altered Th1 responses, and reduced serum alloantibody. Furthermore, in CD4(+) cell-dominated rejection, IL-6 neutralization was effective when anti-IL-6 administration was delayed by as many as 6 d posttransplant. Finally, IL-6-deficient graft recipients were protected from CD4(+) cell-dominant responses, suggesting that IL-6 production by graft recipients, rather than grafts, is necessary for this type of rejection. Collectively, these observations define IL-6 as a critical promoter of graft infiltration and a shaper of T cell lineage development in cardiac graft rejection. In light of these findings, the utility of therapeutics targeting IL-6 should be considered for preventing cardiac allograft rejection.     

T cell subsets and in vitro immune regulation in "infectious" transplantation tolerance.

CD4-targeted mAb therapy results in permanent acceptance of cardiac allografts in rat recipients, in conjunction with features of the infectious tolerance pathway. Although CD4(+) T cells play a central role, the actual cellular and molecular tolerogenic mechanisms remain elusive. This study was designed to analyze in vitro alloimmune responses of T lymphocytes from CD4 mAb-treated engrafted hosts. Spleen, but not lymph node, cells lost proliferative response against donor alloantigen in MLR and suppressed test allograft rejection in adoptive transfer studies, suggesting compartmentalization of tolerogenic T cells in transplant recipients. A high dose of exogenous IL-2 restored the allogeneic response of tolerogenic T cells, indicating anergy as a putative mechanism. Vigorous proliferation of the tolerogenic T cells in in vivo MLR supports the existence of alloreactive lymphocytes in tolerogenic T cell repertoire and implies an active operational suppression mechanism. The tolerogenic splenocytes suppressed proliferation of naive splenocytes in vitro, consistent with their in vivo property of dominant immune regulation. Finally, CD45RC(+) but not CD45RC(-) T cells from tolerant hosts were hyporesponsive to alloantigen and suppressed the proliferation of normal T cells in the coculture assay. Thus, nondeletional, anergy-like regulatory mechanisms may operate via CD4(+)CD45RC(+) T cells in the infectious tolerance pathway in transplant recipients.     

Abrogation of antibody-mediated allograft rejection by regulatory CD4 T cells with indirect allospecificity

Alloantibody is an important effector mechanism for allograft rejection. In this study, we tested the hypothesis that regulatory T cells with indirect allospecificity can prevent humoral rejection by using a rat transplant model in which acute rejection of MHC class I-disparate PVG.R8 heart grafts by PVG.RT1(u) recipients is mediated by alloantibody and is dependent upon help from CD4 T cells that can recognize the disparate MHC alloantigen only via the indirect pathway. Pretransplant treatment of PVG.RT1(u) recipients with anti-CD4 mAb plus donor-specific transfusion abrogated alloantibody production and prolonged PVG.R8 graft survival indefinitely. Naive syngeneic splenocytes injected into tolerant animals did not effect heart graft rejection, suggesting the presence of regulatory mechanisms. Adoptive transfer experiments into CD4 T cell-reconstituted, congenitally athymic recipients confirmed that regulation was mediated by CD4 T cells and was alloantigen-specific. CD4 T cell regulation could be broken in tolerant animals either by immunizing with an immunodominant linear allopeptide or by depleting tolerant CD4 T cells, but surprisingly this resulted in neither alloantibody generation nor graft rejection. These findings demonstrate that anti-CD4 plus donor-specific transfusion treatment results in the development of CD4 regulatory T cells that recognize alloantigens via the indirect pathway and act in an Ag-specific manner to prevent alloantibody-mediated rejection. Their development is associated with intrinsic tolerance within the alloantigen-specific B cell compartment that persists after T cell help is made available.     

CD28, TNF receptor,and IL-12 are critical for CD4-independent cross-priming of therapeutic antitumor CD8+ T cells

Previously, we have shown that priming of therapeutic CD8(+) T cells in tumor vaccine-draining lymph nodes of mice vaccinated with GM-CSF secreting B16BL6 melanoma cells occurs independent of CD4 T cell help. In this study, we examined the contribution of the major costimulatory molecules, CD40 ligand (CD40L), CD80, and CD86, in the priming of CD8(+) T cells. Priming of therapeutic CD8(+) T cells by a GM-CSF-transduced tumor vaccine did not require CD40 and CD40L interactions, as therapeutic T cells could be generated from mice injected with anti-CD40L Ab and from CD40L knockout mice. However, costimulation via either CD80 or CD86 was required, as therapeutic T cells could be generated from mice injected with either anti-CD80 or anti-CD86 Ab alone, but administration of both Abs completely inhibited the priming of therapeutic T cells. Blocking experiments also identified that priming of therapeutic T cells in MHC class II-deficient mice required TNFR and IL-12 signaling, but signaling through CD40, lymphotoxin-betaR, or receptor activator of NF-kappaB was not essential. Thus, cross-priming of therapeutic CD8(+) T cells by a tumor vaccine transduced with GM-CSF requires TNFR, IL-12, and CD28 signaling.     

A critical precursor frequency of donor-reactive CD4+ T cell help is required for CD8+ T cell-mediated CD28/CD154-independent rejection

Ag-specific precursor frequency is increasingly being appreciated as an important factor in determining the kinetics, magnitude, and degree of differentiation of T cell responses, and recently was found to play a critical role in determining the relative requirement of CD8(+) T cells for CD28- and CD154-mediated costimulatory signals during transplantation. We addressed the possibility that variations in CD4(+) T cell precursor frequency following transplantation might affect CD4(+) T cell proliferation, effector function, and provision of help for donor-reactive B cell and CD8(+) T cell responses. Using a transgenic model system wherein increasing frequencies of donor-reactive CD4(+) T cells were transferred into skin graft recipients, we observed that a critical CD4(+) T cell threshold precursor frequency was necessary to provide help following blockade of the CD28 and CD154 costimulatory pathways, as measured by increased B cell and CD8(+) T cell responses and precipitation of graft rejection. In contrast to high-frequency CD8(+) T cell responses, this effect was observed even though the proliferative and cytokine responses of Ag-specific CD4(+) T cells were inhibited. Thus, we conclude that an initial high frequency of donor-reactive CD4(+) T cells uncouples T cell proliferative and effector cytokine production from the provision of T cell help.     

IDO and regulatory T cell support are critical for cytotoxic T lymphocyte-associated Ag-4 Ig-mediated long-term solid organ allograft survival

Costimulatory blockade of CD28-B7 interaction with CTLA4Ig is a well-established strategy to induce transplantation tolerance. Although previous in vitro studies suggest that CTLA4Ig upregulates expression of the immunoregulatory enzyme IDO in dendritic cells, the relationship of CTLA4Ig and IDO in in vivo organ transplantation remains unclear. In this study, we studied whether concerted immunomodulation in vivo by CTLA4Ig depends on IDO. C57BL/6 recipients receiving a fully MHC-mismatched BALB/c heart graft treated with CTLA4Ig + donor-specific transfusion showed indefinite graft survival (>100 d) without signs of chronic rejection or donor specific Ab formation. Recipients with long-term surviving grafts had significantly higher systemic IDO activity as compared with rejectors, which markedly correlated with intragraft IDO and Foxp3 levels. IDO inhibition with 1-methyl-dl-tryptophan, either at transplant or at postoperative day 50, abrogated CTLA4Ig + DST-induced long-term graft survival. Importantly, IDO1 knockout recipients experienced acute rejection and graft survival comparable to controls. In addition, αCD25 mAb-mediated depletion of regulatory T cells (Tregs) resulted in decreased IDO activity and again prevented CTLA4Ig + DST induced indefinite graft survival. Our results suggest that CTLA4Ig-induced tolerance to murine cardiac allografts is critically dependent on synergistic cross-linked interplay of IDO and Tregs. These results have important implications for the clinical development of this costimulatory blocker.     

Altered distribution of H60 minor H antigen-specific CD8 T cells and attenuated chronic vasculopathy in minor histocompatibility antigen mismatched heart transplantation in Cxcr3-/- mouse recipients

Chemokine-chemokine receptor interactions and the subsequent recruitment of T lymphocytes to the graft are believed to be among the initial events in the development of acute and chronic rejection of heart transplants. We sought to determine the role of chemokine receptor Cxcr3 on the development of acute and chronic rejection in a multiple minor Ag mismatched mouse heart transplant model. The frequencies and kinetics of immunodominant H60 (LTFNYRNL) miHA-specific CD8 T cells in wild-type or Cxcr3-/- C57BL/6 recipients were monitored using MHC class I tetramer after BALB/b donor hearts were transplanted. Acceptance of grafts, severity of rejection, and infiltration of T cells were not altered in Cxcr3-/- recipients. However, graft survival was moderately prolonged in Cxcr3-/- recipient mice undergoing acute rejection. Analyses of splenocytes, PBLs, and graft-infiltrating cells revealed increased alloreactive T cells (H60-specific CD8 T cells) in the peripheral blood and spleen but not in the graft. Adoptively transferred Cxcr3-/- CD8 T cells in the BALB/b heart-bearing B6 scid mice showed retention of alloreactive CD8 T cells in the blood but less infiltration into the graft. Cxcr3-/- recipients with long-term graft survival also showed a marked decrease of CD8+ T cell infiltration and reduced neo-intimal hyperplasia. These data indicate that Cxcr3 plays a critical role in the trafficking as well as activation of alloreactive T cells. This role is most eminent in a transplant model when a less complex inflammatory milieu is involved such as a well-matched graft and chronic rejection.     

Prolonged blockade of CD40-CD40 ligand interactions by gene transfer of CD40Ig results in long-term heart allograft survival and donor-specific hyporesponsiveness, but does not prevent chronic rejection

Previous work on blockade of CD40-CD40 ligand interaction in mice and primates with anti-CD40 ligand mAbs has resulted in a moderate prolongation of allograft survival without the development of true allograft tolerance. In this study, we show in rats that adenovirus-mediated gene transfer of CD40Ig sequences into the graft resulted in prolonged (>200 days) expression of CD40Ig and in long-term (>300 days) survival. Recipients expressing CD40Ig displayed strongly (>90%) inhibited mixed leukocyte reactions and alloantibody production at early (days 5 and 17) and late time points (>100 day) after transplantation, but showed limited inhibition of leukocyte infiltration and cytokine production as evaluated by immunohistology at early time points (day 5). Recipients of long-surviving hearts showed donor-specific hyporesponsiveness since acceptance of second cardiac allografts was donor specific. Nevertheless, long-term allografts (>100 days) displayed signs of chronic rejection vasculopathy. Occluded vessels showed leukocyte infiltration, mainly composed of CD4(+) and CD8(+) cells, macrophages, and mast cells. These recipients also showed antidonor CTL activity. Recipients expressing CD40Ig did not show nonspecific immunosuppression, as they were able to mount anticognate immune responses that were partially inhibited at early time points and were normal thereafter. We conclude that gene transfer-mediated expression of CD40Ig resulted in a highly efficient inhibition of acute heart allograft rejection in rats. This treatment induced donor-specific inhibition of certain alloreactive mechanisms in the short-, but not the long-term, which resulted in long-term survival of allografts concomitant with the development of chronic rejection.     

Alpha tumor necrosis factor contributes to CD8(+) T cell survival in the transition phase

Cytokine and costimulation signals determine CD8(+) T cell responses in proliferation phase. In this study, we assessed the potential effect of cytokines and costimulations to CD8(+) T cell survival in transition phase by transferring in vitro ovalbumin (OVA)-pulsed dendritic cell-activated CD8(+) T cells derived from OVA-specific T cell receptor transgenic OT I mice into wild-type C57BL/6 mice or mice with designated gene knockout. We found that deficiency of IL-10, IL-12, IFN-gamma, CD28, CD40, CD80, CD40L, and 41BBL in recipients did not affect CD8(+) T cell survival after adoptive transfer. In contrast, TNF-alpha deficiency in both recipients and donor CD8(+) effector T cells significantly reduced CD8(+) T cell survival. Therefore, our data demonstrate that the host- and T cell-derived TNF-alpha signaling contributes to CD8(+) effector T cell survival and their transition to memory T cells in the transition phase, and may be useful information when designing vaccination.     

Ex vivo expansion of CD4+CD25+FoxP3+ T regulatory cells based on synergy between IL-2 and 4-1BB signaling

Naturally occurring CD4(+)CD25(+)FoxP3(+) T regulatory (Treg) cells require three distinct signals transduced via TCR, CD28, and IL-2R for their development and maintenance. These requirements served as the basis for several recently developed ex vivo expansion protocols that relied on the use of solid support-bound Abs to CD3 and CD28 in the presence of high dose IL-2. We report in this study that Treg cells up-regulate the expression of inducible costimulatory receptor 4-1BB in response to IL-2, and stimulation using this receptor via a novel form of 4-1BB ligand (4-1BBL) fused to a modified form of core streptavidin (SA-4-1BBL) was effective in expanding these cells up to 110-fold within 3 wk. Expanded cells up-regulated CD25, 4-1BB, and membranous TGF-beta, suppressed T cell proliferation, and prevented the rejection of allogeneic islets upon adoptive transfer into graft recipients. Importantly, SA-4-1BBL rendered CD4(+)CD25(-) T effector cells refractive to suppression by Treg cells. This dual function of signaling via 4-1BB, vis-à-vis Treg cell expansion and licensing T effector cells resistant to Treg cell suppression, as well as the up-regulation of 4-1BB by IL-2 may serve as important regulatory mechanisms for immune homeostasis following antigenic challenge. Stimulation using a soluble form of SA-4-1BBL represents a novel approach to expand Treg cells with potential therapeutic applications in autoimmunity and transplantation.     

Activation of alloreactive CD8+ T cells operates via CD4-dependent and CD4-independent mechanisms and is CD154 blockade sensitive

CD154, one of the most extensively studied T cell costimulation molecules, represents a promising therapeutic target in organ transplantation. However, the immunological mechanisms of CD154 blockade that result in allograft protection, particularly in the context of alloreactive CD4/CD8 T cell activation, remain to be elucidated. We now report on the profound inhibition of alloreactive CD8(+) T cells by CD154 blockade via both CD4-dependent and CD4-independent activation pathways. Using CD154 KO recipients that are defective in alloreactive CD8(+) T cell activation and unable to reject cardiac allografts, we were able to restore CD8 activation and graft rejection by adoptively transferring CD4(+) or CD8(+) T cells from wild-type syngeneic donor mice. CD4-independent activation of alloreactive CD8(+) T cells was confirmed following treatment of wild-type recipients with CD4-depleting mAb, and by using CD4 KO mice. Comparable levels of alloreactive CD8(+) T cell activation was induced by allogenic skin engraftment in both animal groups. CD154 blockade inhibited CD4-independent alloreactive CD8(+) T cell activation. Furthermore, we analyzed whether disruption of CD154 signaling affects cardiac allograft survival in skin-sensitized CD4 KO and CD8 KO recipients. A better survival rate was observed consistently in CD4 KO, as compared with CD8 KO recipients. Our results document CD4-dependent and CD4-independent activation pathways for alloreactive CD8(+) T cells that are both sensitive to CD154 blockade. Indeed, CD154 blockade was effective in preventing CD8(+) T cell-mediated cardiac allograft rejection.