An antagonist IL-15/Fc protein prevents costimulation blockade-resistant rejection.

IL-15 is a powerful T cell growth factor (TCGF) with particular importance for the maintenance of CD8(+) T cells. Because costimulation blockade does not result in universal tolerance, we hypothesized that "escape" from costimulation blockade might represent a CD8(+) and IL-15/IL-15R(+)-dependent process. For this analysis, we have used an IL-15 mutant/Fcgamma2a protein, a potentially cytolytic protein that is also a high-affinity receptor site specific antagonist for the IL-15Ralpha receptor protein, as a therapeutic agent. The IL-15-related fusion protein was used as monotherapy or in combination with CTLA4/Fc in murine islet allograft models. As monotherapies, CTLA4/Fc and an IL-15 mutant/Fcgamma2a were comparably effective in a semiallogeneic model system, and combined treatment with IL-15 mutant/Fcgamma2a plus CTLA4/Fc produced universal permanent engraftment. In a fully MHC-mismatched strain combination known to be refractory to costimulation blockade treatment, combined treatment with both fusion proteins proved to be highly effective; >70% of recipients were tolerized. The analysis revealed that the IL-15 mutant/Fc treatment confers partial protection from both CD4(+) and CD8(+) T cell graft infiltration. In rejections occurring despite CTLA4/Fc treatment, concomitant treatment with the IL-15 mutant/Fcgamma2a protein blocked a CD8(+) T cell-dominated rejection processes. This protection was linked to a blunted proliferative response of alloreactive T cells as well silencing of CTL-related gene expression events. Hence, we have demonstrated that targeting the IL-15/IL-15R pathway represents a new and potent strategy to prevent costimulation blockade-resistant CD8(+) T cell-driven rejection.     

The role of H-2 and non-H-2 antigens and genes in the rejection of murine cardiac allografts

Genes of the major histocompatibility complex (MHC) in the mouse (H-2 complex) have been shown to be an important factor in determining the immune responsiveness of various strains of mice to isolated antigens (e. g., lysozyme). The role of MHC genes in controlling the responsiveness of mice to multiple alloantigens is less well-defined, and although non-MHC genes have been shown to be important in determining responsiveness in some systems (e. g., haptens), they have not been demonstrated as yet to influence the rejection of vascularized organ allografts. In this study, the responsiveness of mice to vascularized cardiac allografts transplanted across well-defined major (H-2) and minor (non-H-2) histocompatibility barriers was investigated using congenic mice in 32 different donor/recipient combinations. The results show that both H-2 and non-H-2 gene products can act as target alloantigens for rejection. At the responder level, they may interact to effect responsiveness of a recipient strain to multiple alloantigens. In no case in this study has any one gene or group of genes been found to confer universal high or low responder status.     

IL-5 mediates eosinophilic rejection of MHC class II-disparate skin allografts in mice.

CD4 T cells play a crucial role in the acute rejection of MHC class II-disparate skin allografts, mainly by Fas/Fas ligand-mediated cytotoxicity. Because recent observations indicate that eosinophils may be found within allografts rejected by CD4 T cells, we evaluated the role played by IL-5, the main eosinophil growth factor, and by eosinophils in the rejection of MHC class II-disparate skin grafts. C57BL/6 mice rapidly rejected MHC class II-disparate bm12 skin grafts. Rejected skins contained a dense, aggressive eosinophil infiltrate. Lymphocytes isolated from lymph nodes draining rejected bm12 skin were primed for IL-5 secretion, and IL-5 mRNA was present within rejected grafts. The IL-5/eosinophil pathway played an effector role in allograft destruction, because the rejection of bm12 skin was significantly delayed in IL-5-deficient mice as compared with wild-type animals. The role of the IL-5/eosinophil pathway was further investigated in MHC class II-disparate donor-recipient strains unable to establish Fas/Fas ligand interactions. Fas ligand-deficient gld/gld mice rejected bm12 skins, and bm12 mice rejected Fas-deficient lpr/lpr C57BL/6 skins. Neutralization of IL-5 prevented acute rejection in both combinations. We conclude that MHC class II-disparate skin allografts trigger an IL-5-dependent infiltration of eosinophils that is sufficient to result in acute graft destruction.     

Role of costimulation in the induction of the IL-12/IL-12 receptor pathway and the development of autoimmunity

Costimulation mediated by the interactions of the B7 Ags (CD80/CD86) on APC with CD28 on the responding T cell regulates the magnitude of the immune response and may influence Th1/Th2 development. The IL-12Rbeta2 subunit plays a critical role in maintaining IL-12 responsiveness and controlling Th1 lineage commitment. We demonstrate that IL-2 and IL-12 resulting from CD28/B7 interactions both play a critical role in the induction of expression of the IL-12Rbeta2 subunit and as a result the differentiation of pathogenic autoreactive effector cells. These findings suggest that targeting IL-2 and IL-12 simultaneously may be effective in the treatment of Th1-mediated autoimmunity.     

The role of B7 costimulation in T-cell immunity.

CD4+ T cells are considered to be the major controlling element of the adaptive immune response. They recognize foreign peptides by interaction of the T cell receptor (TCR) with peptide complexed to major histocompatibility complex (MHC) class II molecules on the surface of antigen presenting cells (APC). Once activated, CD4+ T cells orchestrate the various phases of the immune response. They are responsible for the production of numerous cytokines, which activate specific immune effector cell populations including B cells, eosinophils, mast cells and macrophages. Not surprisingly, the activation of CD4+ T cells needs to be tightly regulated and is subject to finely tuned control mechanisms. The requirement for a second or 'costimulatory' signal, in addition to the antigenic signal, provides a key element for the exquisite control of T cell activation. One of the major signalling pathways responsible for delivery of this costimulatory signal is induced by interaction of CD28 on T cells with B7 molecules found only on APC. The present review outlines our current understanding of the physiological role of B7 costimulatory signals in regulating CD4+ T cell responses.     

Rejection of mouse cardiac allografts by costimulation in trans

The activation of T cells by B7 costimulation in trans has been demonstrated in vitro, but the in vivo relevance is unknown. To study costimulation in trans of CD4(+) T cells in vivo, we performed cardiac transplants from B7-1/B7-2-deficient mice to recipients that do not express MHC class II molecules on peripheral APCs, but do have functional CD4(+) T cells (II(-)/4(+) mice). This model restricts the B7-dependent activation of CD4(+) T cells to costimulation in trans and excludes any contribution from indirect Ag presentation. We find that II(-)/4(+) recipients reject B7-deficient grafts as rapidly as wild-type grafts, suggesting that costimulation in trans can mediate rejection as potently as costimulation in cis. Treatment of II(-)/4(+) recipients of B7-deficient grafts with depleting Abs to CD4 or CD8 demonstrates that indirect Ag presentation to CD8(+) cells does not significantly contribute to rejection. This is the first demonstration that costimulation in trans can mediate an immune response in vivo and has important therapeutic implications.     

The IL-2A receptor pathway and its role in lymphocyte differentiation and function

Murine myeloid cells are developed from hemopoietic stem/progenitor cells. Different types of progenitor cells have variable differentiation potentials. Among the ten main types of cells differentiated from lymphoid progenitor cells, regulatory T cells (Tregs), an important cell subpopulation regulating immune and inflammatory responses, arise from the hematopoietic stem cells in the bone marrow. Tregs then differentiate into T lymphocytes and migrate to the thymus and finally generate Treg subsets, which are subsequently activated and regulated by inflammatory cytokines in the peripheral blood. Tregs also have different phenotypes and immunomodulatory functions. The cytokine interleukin-2/interleukin-2 receptor (IL-2/IL-2R) pathway is an important regulatory signaling pathway of Tregs. Besides, different types of CD4⁺ and CD8⁺ cells have different immune effects in the absence of IL-2. IL-2R consists of three subunits, α chain (CD25), β chain (CD122), and γ chain (CD132). Different subunit combinations have different effects on the activation of immune cells. Multiple studies have shown that IL2RA deficiency has various effects on the immune function in mice. This article reviews the subunit composition and signaling pathway of IL-2R, the classification of Tregs in a murine myeloid cell line and the regulatory effect of IL-2/IL-2R on them, the regulatory impact and signaling mechanism of IL-2/IL-2R on CD4⁺/CD8⁺ lymphocyte differentiation, the primary manifestations and molecular mechanism of immune dysfunction in IL-2- and IL-2R-deficient mice, soluble IL-2Rα as a biomarker for diagnosis, prognosis and therapeutic efficacy of treatment in immune system disorders, and the development and clinical application of IL-2 mutants.     

CD28 costimulation accelerates IL-4 receptor sensitivity and IL-4-mediated Th2 differentiation.

The development of Th1 and Th2 cells is determined by the type of antigenic stimulation involved in the initial cell activation step. Evidence indicates that costimulatory signals, such as those delivered by CD28, play an important role in Th2 development, but little is known about how CD28 costimulation contributes to Th2 development. In this study, TCR cross-linking was insufficient for Th2 development, while the addition of CD28 costimulation drastically increased Th2 generation through the IL-4-mediated pathway. Th2 generation following CD28 costimulation was not simply explained by the enhancement of IL-4 production in naive T cells. To generate Th2 cells after TCR cross-linking only, it was necessary to add a 20- to 200-fold excess of IL-4 generated after TCR and CD28 stimulation. TCR cross-linking increased the expression level and binding property of the IL-4R, but enhanced the sensitivity to IL-4 only slightly. In contrast, as evidenced by the enhanced phosphorylation of Jak3, the IL-4Ralpha-chain, and STAT6 following IL-4 stimulation, CD28 costimulation increased IL-4R sensitivity without affecting its expression and binding property. This evidence of the enhancement of IL-4R sensitivity increases our understanding of how CD28 costimulation accelerates Th2 development.     

Prevention of chronic rejection in murine cardiac allografts: a comparison of chimerism- and nonchimerism-inducing costimulation blockade-based tolerance induction regimens

We have previously described a nonirradiation-based regimen combining costimulation blockade, busulfan, and donor bone marrow cells that promotes stable, high level chimerism, deletion of donor-reactive T cells, and indefinite survival of skin allografts in mice. The purpose of the current study is to determine the efficacy of this tolerance regimen in preventing acute and chronic rejection in a vascularized heart graft model and to compare this regimen with other putative tolerance protocols. Mice receiving costimulation blockade (CTLA4-Ig and anti-CD40 ligand) alone or in combination with donor cells enjoyed markedly prolonged heart graft survival and initially preserved histological structure. However, tolerance was not achieved, as evidenced by the eventual onset of chronic rejection characterized by obliterative vasculopathy and the rejection of secondary skin grafts. In contrast, following treatment with costimulation blockade, busulfan, and bone marrow, heart grafts survived indefinitely without detectable signs of chronic rejection or structural damage, even 100 days after placement of a secondary donor skin graft. We detected multilineage chimerism in peripheral blood, spleen, lymph nodes, and thymus, and peripheral deletion of donor-reactive cells was complete by day 90. These findings indicate that only the CD40/CD28 blockade chimerism induction regimen prevents both acute and chronic rejection of vascularized organ transplants. Further testing of these strategies in a preclinical large animal model is warranted.     

IL-33／ST2信号通路在疾病中作用的研究进展

IL-33是在2005年发现的一个多功能细胞因子,属于IL-1家族新成员。IL-33通过受体ST2活化NF-KB和MAPK信号通路,促进Th2细胞因子的产生,在多种疾病的发生发展过程中起着重要的作用。     

Critical role for IL-4 in the development of transplant arteriosclerosis in the absence of CD40-CD154 costimulation.

Blockade of the CD40-CD154 pathway can inhibit CD4(+) T cell activation but is unable to prevent immune responses mediated by CD8(+) T cells. However, even in the absence of CD8(+) T cells, inhibition of the CD40-CD154 pathway is insufficient to prevent the development of transplant arteriosclerosis. This study investigated the mechanisms of transplant arteriosclerosis in the absence of the CD40 pathway. C57BL/6 CD40(-/-) (H2(b)) recipients were transplanted with MHC-mismatched BALB/c (H2(d)) aortas. Transplant arteriosclerosis was evident in both CD40(-/-) and CD40(+/-) mice (intimal proliferation was 59 +/- 5% for CD40(-/-) mice vs 58 +/- 4% for CD40(+/-) mice) in the presence or absence of CD8(+) T cells (intimal proliferation was 46 +/- 7% for CD40(-/-) anti-CD8-treated mice vs 50 +/- 10% for CD40(+/-) anti-CD8-treated mice), confirming that CD8(+) T cells are not essential effector cells for the development of this disease. In CD40(-/-) recipients depleted of CD8(+) T cells, the number of eosinophils infiltrating the graft was markedly increased (109 +/- 24 eosinophils/grid for CD40(-/-) anti-CD8-treated mice vs 28 +/- 7 for CD40(+/-) anti-CD8-treated mice). The increased presence of eosinophils correlated with augmented intragraft production of IL-4. To test the hypothesis that IL-4 was responsible for the intimal proliferation, CD8 T cell-depleted CD40(-/-) recipients were treated with anti-IL-4 mAb. This resulted in significantly reduced eosinophil infiltration into the graft (12 +/- 5 eosinophils/grid for CD40(-/-) anti-CD8(+), anti-IL-4-treated mice vs 109 +/- 24 for CD40(-/-) anti-CD8-treated mice), intragraft eotaxin, CCR3 mRNA production, and the level of intimal proliferation (18 +/- 5% for CD40(-/-) anti-CD8(+)-, anti-IL-4-treated mice vs 46 +/- 7% for CD40(-/-) anti-CD8-treated mice). In conclusion, elevated intragraft IL-4 production results in an eosinophil infiltrate and is an important mechanism for CD8(+) T cell-independent transplant arteriosclerosis in the absence of CD40-CD154 costimulation.     

TLR agonists abrogate costimulation blockade-induced prolongation of skin allografts

Costimulation blockade protocols are effective in prolonging allograft survival in animal models and are entering clinical trials, but how environmental perturbants affect graft survival remains largely unstudied. We used a costimulation blockade protocol consisting of a donor-specific transfusion and anti-CD154 mAb to address this question. We observed that lymphocytic choriomeningitis virus infection at the time of donor-specific transfusion and anti-CD154 mAb shortens allograft survival. Lymphocytic choriomeningitis virus 1) activates innate immunity, 2) induces allo-cross-reactive T cells, and 3) generates virus-specific responses, all of which may adversely affect allograft survival. To investigate the role of innate immunity, mice given costimulation blockade and skin allografts were coinjected with TLR2 (Pam3Cys), TLR3 (polyinosinic:polycytidylic acid), TLR4 (LPS), or TLR9 (CpG) agonists. Costimulation blockade prolonged skin allograft survival that was shortened after coinjection by TLR agonists. To investigate underlying mechanisms, we used "synchimeric" mice which circulate trace populations of anti-H2b transgenic alloreactive CD8+ T cells. In synchimeric mice treated with costimulation blockade, coadministration of all four TLR agonists prevented deletion of alloreactive CD8+ T cells and shortened skin allograft survival. These alloreactive CD8+ T cells 1) expressed the proliferation marker Ki-67, 2) up-regulated CD44, and 3) failed to undergo apoptosis. B6.TNFR2-/- and B6.IL-12R-/- mice treated with costimulation blockade plus LPS also exhibited short skin allograft survival whereas similarly treated B6.CD8alpha-/- and TLR4-/- mice exhibited prolonged allograft survival. We conclude that TLR signaling abrogates the effects of costimulation blockade by preventing alloreactive CD8+ T cell apoptosis through a mechanism not dependent on TNFR2 or IL-12R signaling.     

Critical role of CD4 help in CD154 blockade-resistant memory CD8 T cell activation and allograft rejection in sensitized recipients

Allograft rejection in sensitized recipients remains the major problem in clinical organ transplantation. We have developed a donor-type skin-sensitized mouse cardiac allograft model (BALB/c-->C57BL/6) in which both rejection (<5 days) and alloreactive CD8 activation are resistant to CD154 blockade. First, we attempted to elucidate why CD154 blockade fails to protect cardiac grafts in sensitized recipients. The gene array analysis has revealed that treatment with anti-CD154 mAb (MR1) had distinctive impact on host immunity in naive vs sensitized animals. Unlike in naive counterparts, host sensitization mitigated the impact of CD154 blockade on critical immune signaling pathways. Indeed, we identified 3234 genes in cardiac grafts that were down-regulated by MR1 in naive (at least 5-fold), but remained unaffected in sensitized hosts. Moreover, MR1 treatment failed to prevent accumulation of CD4 T cells in cardiac allografts of sensitized recipients. Then, to determine the role of CD4 help in CD154 blockade-resistant immune response, we used CD4-depleting and CD4-blocking Ab, in conjunction with MR1 treatment. Our data revealed that CD154 blockade-resistant CD8 activation in sensitized mice was dependent on CD4 T cells. In the absence of CD4 help, CD154 blockade prevented differentiation of alloreactive CD8 T cells into CTL effector/memory cells and abrogated acute rejection (cardiac graft survival for >30 days), paralleled by selective target gene depression at the graft site. These results provide the rationale to probe potential synergy of adjunctive therapy targeting CD4 and CD154 to overcome graft rejection in sensitized recipients.     

The role of mononuclear phagocytes in cardiac allograft rejection in the rat: II. Characterization of mononuclear phagocytes extracted from rat cardiac allografts

A method was developed for the extraction of leukocytes infiltrating rat cardiac allografts. Mononuclear phagocytes (MNP) comprised 52.4 ± 5.5% of the cells extracted from allografts at the time of rejection (Day 7). Day 4 allografts and Day 7 syngeneic grafts yielded considerably fewer MNP although numbers of lymphoid cells were similar in all three groups. Allograft MNP were phagocytic for latex particles but only very low numbers were adherent to a variety of surfaces. About 50% were positive for attachment and internalization of opsonized sheep red cells via the Fc receptor. However, fewer cells were able to internalize sheep red cells than were able to bind them when complement receptor-mediated phagocytosis was investigated. Large amounts of plasminogen activator were secreted by allograft MNP while cells from syngeneic grafts produced very little. The possible participation of MNP in the effector phase of a mechanism for allograft rejection similar to delayed-type hypersensitivity is discussed.     

The role of the ubiquitin-proteasome pathway in apoptosis.

Coordinated intracellular protein degradation mediated by the ubiquitin-proteasome pathway is crucial to a vast array of cellular processes including orderly progression through the mitotic cycle. Similarly important to both the fates of individual cells, as well as to the normal function of multicellular organisms, is the process of apoptosis, or programmed cell death. Execution of this latter process has been known for some time to be intimately associated with the activity of caspases, a family of proteases related to interleukin-1-beta-converting enzyme. Evidence is now accumulating, however, that the ubiquitin-proteasome system itself plays an important role in apoptosis, and some of the cellular pathways that are impacted upon by the proteasome, and may lead to apoptosis, are beginning to be dissected. This review provides a summary of the experimental basis by which components of the ubiquitin-proteasome pathway have been linked to apoptosis, and attempts are made to formulate a hypothesis about its role in this process.