Evidence for a role for T cell receptors (TCR) in the effector phase of acute bone marrow graft rejection. TCR V beta 5 transgenic mice lack effector cells able to cause graft rejection.

Lethally irradiated mice reject within 24 h certain marrow grafts, a phenomenon called either allogeneic or hybrid resistance. The cells responsible for this rejection (NK1+ CD3+ cells (TNK) express Ag of NK cells as well as the TCR-associated CD3 complex. This raises the question whether TCR participate in the function of these cells during graft rejection. By using flow cytometry it is shown that the majority of TNK cells expresses the TCR-alpha/beta chains and by using adoptive cell transfer assays evidence is presented that it is the TCR-alpha/beta expressing cells that cause rejection. To explore whether any particular TCR chains have to be expressed on these cells, C57L mice were assayed and found to be responders suggesting that the V beta chains deleted in these mice are not obligatory. However, introduction of a specific TCR V beta 5 chain into C57BL/6 mice as a transgene leads to inability to transfer resistance. TNK cells of V beta 5 transgenic mice express the introduced gene suggesting that it is the transgenic TCR that is responsible for the lack of function. In assessing T cell functions in V beta 5 transgenic mice it is shown that although these mice generate CTL specific for H-2d targets there is a deficiency to recognize H-2Dd, i.e., of determinants presumed to be recognized in the acute rejection mechanism. Thus TNK cells and CTL share the inability to recognize H-2Dd epitopes due to expression of the V beta 5 transgene. The notion that TCR on TNK cells play a role in the acute rejection process makes it necessary to postulate a receptor selection mechanism for these cells.     

Activation and migration of allo-peptide specific TCR transgenic T cells in cardiac allograft rejection

T cells from TCR transgenic mice, expressing receptors specific for an allogeneic MHC class I peptide, were used to track T cell activation and migration in normal adoptive recipients that were subsequently transplanted with heterotopic hearts that were syngeneic except for a transgenic MHC class I antigen. T cells rapidly disappeared from the blood into the lymphoid tissues where they were activated within one day after transplantation. T cells initially formed discrete clusters in the spleen and lymph nodes. After proliferating for 2-4 days in lymphoid tissues, T cells reappeared in the blood and migrated to the heart and the intestines. The T cells underwent another round of proliferation in the heart, but not the intestines, and induced cardiac rejection uniformly on 6 day.     

Endothelial induction of fgl2 contributes to thrombosis during acute vascular xenograft rejection

Thrombosis is a prominent feature of acute vascular rejection (AVR), the current barrier to survival of pig-to-primate xenografts. Fibrinogen-like protein 2 (fgl2/fibroleukin) is an inducible prothrombinase that plays an important role in the pathogenesis of fibrin deposition during viral hepatitis and cytokine-induced fetal loss. We hypothesized that induction of fgl2 on the vascular endothelium of xenografts contributes to thrombosis associated with AVR. We first examined fgl2 as a source of procoagulant activity in the pig-to-primate combination. The porcine fgl2 (pfgl2) was cloned and its chromosomal locus was identified. Recombinant pfgl2 protein expressed in vitro was detected on the cell surface and generated thrombin from human prothrombin. Studies of pig-to-baboon kidney xenografts undergoing AVR in vivo revealed induction of pfgl2 expression on graft vascular endothelial cells (ECs). Cultured porcine ECs activated by human TNF-alpha in vitro demonstrated induction of pfgl2 expression and enhanced activation of human prothrombin. The availability of gene-targeted fgl2-deficient mice allowed the contribution of fgl2 to the pathogenesis of AVR to be directly examined in vivo. Hearts heterotopically transplanted from fgl2(+/+) and fgl2(+/-) mice into Lewis rats developed AVR with intravascular thrombosis associated with induction of fgl2 in graft vascular ECs. In contrast, xenografts from fgl2(-/-) mice were devoid of thrombosis. These observations collectively suggest that induction of fgl2 on the vascular endothelium plays a role in the pathogenesis of AVR-associated thrombosis. Manipulation of fgl2, in combination with other interventions, may yield novel strategies by which to overcome AVR and extend xenograft survival.     

Immunological and regenerative aspects of hepatic mast cells in liver allograft rejection and tolerance

The precise roles of mast cells in liver allograft rejection and tolerance are still unknown. This study aimed to explore the roles of mast cells in immune regulation and liver regeneration for tolerance induction by using rat models of orthotopic liver transplantation (OLT). Stem cell factor (SCF) and its receptor c-Kit, which are critical to the migration and development of not only stem cells but also mast cells, significantly increased in the tolerogenic livers as compared with rejected livers. The significant elevation of mast cell tryptase, high-affinity IgE receptor, and histamine suggested the activation of mast cells in liver allografts at the tolerogenic phase after OLT. Immunohistochemical analysis using confocal microscope clearly showed colocalization of mast cells, Foxp3+ Tregs, γδ T cells, and recipient-derived hepatic progenitor cells with higher expression of SCF, IL-9, IL-10, TGF-β1, and IL-17 related to immunoregulation and liver regeneration in the donor grafts of a tolerogenic OLT model. Cross-talk among mast cells and other cells was evaluated by in vitro studies demonstrating that syngeneic bone marrow-derived mast cells (BMMCs) co-cultured with na?ve splenocytes or primary hepatocytes significantly increased the population of splenic γδ T cells by mitogen stimulation or by mast cell degranulation, and also significantly induced the hepatocyte proliferation, respectively. Our results suggested that mast cells in the donor grafts may play important roles in the induction/maintenance of immune tolerance and liver regeneration resulting in the replacement of hepatic cells from donor to recipient.     

The role of TNF-related activation-induced cytokine-receptor activating NF-kappa B interaction in acute allograft rejection and CD40L-independent chronic allograft rejection

We analyzed the role of TNF-related activation-induced cytokine (TRANCE), a member of the TNF family expressed on activated T cells that shares functional properties with CD40L, and its receptor-activating NF-kappaB (RANK) which is mostly expressed on mature dendritic cells, during allogenic responses in vivo using a rodent heart allograft model. TRANCE mRNA was strongly up-regulated in acutely rejected allografts on days 4 and 5 posttransplantation whereas RANK was detected as early as day 1 but did not show further up-regulation during the first week. Immunofluoresence analyses of heart allografts showed that 80 and 100% of TRANCE and RANK-expressing cells were T cells and APCs, respectively. We show for the first time that short-term TRANCE blockade using a mouse RANKIg fusion molecule can significantly prolong heart allograft survival in both rat and mouse models. Similarly, rat heart allografts transduced with a RANKIg encoding recombinant adenovirus exhibited a significant prolongation of survival (14.3 vs 7.6 days, p < 0.0001). However, TRANCE blockade using RANKIg did not appear to inhibit allogeneic T and B cell priming humoral responses against RANKIg. Interestingly, TRANCE blockade induced strong up-regulation of CD40 ligand (CD40L) mRNA in allografts. Combined CD40L and TRANCE blockade resulted in significantly decreased chronic allograft rejection lesions as well as allogeneic humoral responses compared with CD40L blockade alone. We conclude that TRANCE-RANK interactions play an important role during acute allograft rejection and that CD40L-independent allogeneic immune responses can be, at least in part, dependent on the TRANCE pathway of costimulation.     

Role of CXCL9/CXCR3 chemokine biology during pathogenesis of acute lung allograft rejection

Acute allograft rejection is a major complication postlung transplantation and is the main risk factor for the development of bronchiolitis obliterans syndrome. Acute rejection is characterized by intragraft infiltration of activated mononuclear cells. The ELR-negative CXC chemokines CXCL9, CXCL10, and CXCL11) are potent chemoattractants for mononuclear cells and act through their shared receptor, CXCR3. Elevated levels of these chemokines in bronchoalveolar lavage fluid have been associated with human acute lung allograft rejection. This led to the hypothesis that the expression of these chemokines during an allogeneic response promotes the recruitment of mononuclear cells, leading to acute lung allograft rejection. We performed studies in a rat orthotopic lung transplantation model of acute rejection, and demonstrated increased expression of CXCL9 and CXCL10 paralleling the recruitment of mononuclear cells and cells expressing CXCR3 to the allograft. However, CXCL9 levels were 15-fold greater than CXCL10 during maximal rejection. Inhibition of CXCL9 decreased intragraft recruitment of mononuclear cells and cellular expression of CXCR3, resulting in lower acute lung allograft rejection scores. Furthermore, the combination of low dose cyclosporin A with anti-CXCL9 therapy had more profound effects on intragraft leukocyte infiltration and in reducing acute allograft rejection scores. This supports the notion that CXCL9 interaction with cells expressing CXCR3 has an important role in the recruitment of mononuclear cells, a pivotal event in the pathogenesis of acute lung allograft rejection.     

Alteration of V beta usage and cytokine production of CD4+ TCR beta beta homodimer T cells by elimination of Bacteroides vulgatus prevents colitis in TCR alpha-chain-deficient mice

A major pathogenic factor for the development of inflammatory bowel disease (IBD) is the breakdown of the intestinal homeostasis between the host immune system and the luminal microenvironment. To assess the potential influence of luminal Ags on the development of IBD, we fed TCR alpha(-/-) mice an elemental diet (ED). ED-fed TCR alpha(-/-) mice showed no pathologic features of IBD, and their aberrant mucosal B cell responses were suppressed. Similar numbers of CD4(+), TCR betabeta homodimer T cells (betabeta T cells) were developed in the colonic mucosa of ED-fed mice; however, Th2-type cytokine productions were lower than those seen in diseased regular diet (RD)-fed mice. The higher cytokine production in diseased RD-fed mice could be attributed to the high incidence of Bacteroides vulgatus (recovered in 80% of these mice), which can induce Th2-type responses of colonic CD4(+), betabeta T cells. In contrast, ED-fed TCR alpha(-/-) mice exhibited a diversification of Vbeta usage of betabetaT cell populations from the dominant Vbeta8 one associated with B. vulgatus in cecal flora to Vbeta6, Vbeta11, and Vbeta14. Rectal administration of disease-free ED-fed mice with B. vulgatus resulted in the development of Th2-type CD4(+), betabeta T cell-induced colitis. These findings suggest that the ED-induced alteration of intestinal microenvironments such as the enteric flora prevented the development of IBD in TCR alpha(-/-) mice via the immunologic quiescence of CD4(+), betabeta T cells.     

Adjuvant costimulation during secondary antigen challenge directs qualitative aspects of oral tolerance induction, particularly during the neonatal period

In this report we demonstrate that although passive feeding of specific Ag to mice as neonates or adults can induce oral tolerance in both the cellular and humoral arms of the immune response, quantitative and, in particular, qualitative aspects of the tolerance process are determined by the nature of the inflammatory costimuli provided at the time of secondary Ag challenge. Moreover, this dependency upon nonspecific costimulation is more profound in Ag-fed neonates than in their adult counterparts. Thus, administration of Ag in the Th1-selective adjuvant CFA to prefed animals resulted in significant inhibition of IgG2a, IL-2, and IFN-gamma responses, whereas IL-5 responses were increased. In contrast, rechallenge with Ag in the Th2-selective adjuvant aluminum hydroxide resulted in significant inhibition of IgG1, IgE, IL-2, and IL-5 responses, whereas IFN-gamma responses were increased. Additionally, although soluble Ag challenge of prefed adults revealed marginal tolerogenic effects, the same challenge protocol in animals prefed as neonates elicited enhanced Th2-dependent IgG1 production. These results suggest that inflammatory stimulation at the time of Ag challenge is obligatory to trigger oral tolerance mechanisms, particularly in animals fed as neonates and also that the type of adjuvant used at the time of challenge selects for the type of Th cell population to be inhibited.     

De novo expression of MHC class II molecules by microglial cells during acute rat renal allograft rejection

The expression of MHC class I and class II molecules in the cerebral cortex of rats was investigated at daily intervals from day 3 to day 6 after fully allogeneic (DA→LEW) and isogeneic (LEW→LEW) kidney transplantation. MHC class II molecules were temporarily induced on the previously negative microglial cells and on the endothelia of arterioles and venules during acute rejection. On the endothelia of all brain vessels MHC class I expression was enhanced. MHC class I⁺ cells with microglial cell morphology were discernible within the diffusely MHC class I⁺ brain parenchyma. In contrast, the brain parenchyma of isograft recipients and untreated control animals did not express detectable levels of MHC molecules. In conclusion, we demonstrate that a strong immune reaction in the periphery is able to activate microglial cells in the central nervous system.     

Role of nitric oxide and superoxide in acute cardiac allograft rejection in rats

The role of NO and superoxide (O(2)(-)) in tissue injury during cardiac allograft rejection was investigated by using a rat ex vivo organ perfusion system. Excessive NO production and inducible NO synthase (iNOS) expression were observed in cardiac allografts at 5 days after cardiac transplantation, but not in cardiac isografts, as identified by electron spin resonance spectroscopy and Northern blotting. Cardiac isografts or allografts obtained on Day 5 after transplantation were perfused with Krebs bicarbonate buffer with or without various antidotes for NO or O(2)-, including N(omega)-monomethyl-L-arginine (L-NMMA; 1 mM), 2-phenyl-4,4,5, 5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO; 100 microM), 4-amino-6-hydroxypyrazolo[3,4-d]pyrimidine (AHPP; a xanthine oxidase inhibitor; 100 microM), and superoxide dismutase (SOD; 100 units/ml). Treatment of the cardiac allografts with PTIO showed most remarkable improvement of the cardiac injury as revealed by significant reduction in aspartate transaminase, lactate dehydrogenase, and creatine phosphokinase concentrations in the perfusate. Similar but less potent protective effect on the allograft injury was observed by treatment with L-NMMA, AHPP, and SOD. Immunohistochemical analyses for iNOS and nitrotyrosine indicated that iNOS is mainly expressed by macrophages infiltrating the allograft tissues, and nitrotyrosine formation was demonstrated not only in macrophages but also in cardiac myocytes of the allografts, providing indirect evidence for the generation of peroxynitrite during allograft rejection. Our results suggest that tissue injury in rat cardiac allografts during acute rejection is mediated by both NO and O(2)(-), possibly through peroxynitrite formation.     

Role of STAT4 and STAT6 signaling in allograft rejection and CTLA4-Ig-mediated tolerance

STAT4(-/-) mice have impaired type 1 T cell differentiation, whereas STAT6(-/-) mice fail to generate type 2 responses. The role of type 1 and type 2 T cell differentiation in acute cardiac allograft rejection and in the induction of tolerance was examined in wild-type, STAT4(-/-), and STAT6(-/-) recipients. All recipients rejected the grafts promptly. Analysis of in situ cytokine gene expression in the allografts confirmed decreased levels of IFN-gamma in STAT4(-/-) recipients and undetectable levels of IL-4 and IL-5 in STAT6(-/-) mice. Blockade of the CD28/B7 costimulatory pathway prolonged cardiac graft survival for >100 days in 100% of wild-type and STAT4(-/-) mice. However, 14% of CTLA4-Ig-treated STAT6(-/-) mice rejected their grafts between 20 and 100 days. Moreover, of those animals followed past 100 days, 60% of the STAT6(-/-) mice rejected their grafts. Splenocytes harvested on day 145 posttransplant from CTLA4-Ig-treated rejecting STAT6(-/-) recipients were transfused into syngeneic SCID mice transplanted with donor or third party cardiac allografts. Both donor and third party grafts were rejected, indicating that the initial graft loss may be due to an immunological rejection. In contrast, when splenocytes from CTLA4-Ig-treated wild-type or nonrejecting STAT6(-/-) mice were transferred into SCID recipients, donor allografts were accepted, but third party hearts were rejected. Thus, long-term prolongation of cardiac allograft survival by CTLA4-Ig is STAT4-independent but, at least in part, STAT6-dependent. These data suggest that the balance of type 1 and type 2 T lymphocyte differentiation is not critical for acute rejection but influences the robust tolerance induced by CD28/B7 blockade in this model.     

Analysis of early events occurring during neonatal induction of tolerance to histoincompatible cells in mice

Mice have been analyzed at different times post neonatal inoculation of F₁ hybrid lymphoid cells for their ability to respond to foreign MHC antigens expressed on the tolerizing cell population, and to impart that response phenotype to normal adult spleen cells. At early times following neonatal challenge with F₁ spleen cells all mice produced serum immunoglobulins of F₁ donor origin which inhibited the response of normal adult cells. The antigenic specificity of the inhibitory factors suggests a role for an antireceptor antibody in the inhibition seen, and the presence of such serum-mediated inhibition is indicative of (and may be causally related to) the late appearance of a cell-mediated suppressor mechanism in these mice.     

Competitive displacement of pT alpha by TCR-alpha during TCR assembly prevents surface coexpression of pre-TCR and alpha beta TCR

During alphabeta T cell development, CD4(-)CD8(-) thymocytes first express pre-TCR (pTalpha/TCR-beta) before their differentiation to the CD4(+)CD8(+) stage. Positive selection of self-tolerant T cells is then determined by the alphabeta TCR expressed on CD4(+)CD8(+) thymocytes. Conceivably, an overlap in surface expression of these two receptors would interfere with the delicate balance of thymic selection. Therefore, a mechanism ensuring the sequential expression of pre-TCR and TCR must function during thymocyte development. In support of this notion, we demonstrate that expression of TCR-alpha by immature thymocytes terminates the surface expression of pre-TCR. Our results reveal that expression of TCR-alpha precludes the formation of pTalpha/TCR-beta dimers within the endoplasmic reticulum, leading to the displacement of pre-TCR from the cell surface. These findings illustrate a novel posttranslational mechanism for the regulation of pre-TCR expression, which may ensure that alphabeta TCR expression on thymocytes undergoing selection is not compromised by the expression of pre-TCR.