Alloreactive T cell responses and acute rejection of single class II MHC-disparate heart allografts are under strict regulation by CD4+ CD25+ T cells

Skin but not vascularized cardiac allografts from B6.H-2bm12 mice are acutely rejected by C57BL/6 recipients in response to the single class II MHC disparity. The underlying mechanisms preventing acute rejection of B6.H-2bm12 heart allografts by C57BL/6 recipients were investigated. B6.H-2bm12 heart allografts induced low levels of alloreactive effector T cell priming in C57BL/6 recipients, and this priming was accompanied by low-level cellular infiltration into the allograft that quickly resolved. Recipients with long-term-surviving heart allografts were unable to reject B6.H-2bm12 skin allografts, suggesting potential down-regulatory mechanisms induced by the cardiac allografts. Depletion of CD25+ cells from C57BL/6 recipients resulted in 15-fold increases in alloreactive T cell priming and in acute rejection of B6.H-2bm12 heart grafts. Similarly, reconstitution of B6.Rag(-/-) recipients with wild-type C57BL/6 splenocytes resulted in acute rejection of B6.H-2bm12 heart grafts only if CD25+ cells were depleted. These results indicate that acute rejection of single class II MHC-disparate B6.H-2bm12 heart allografts by C57BL/6 recipients is inhibited by the emergence of CD25+ regulatory cells that restrict the clonal expansion of alloreactive T cells.     

Monokine induced by IFN-gamma is a dominant factor directing T cells into murine cardiac allografts during acute rejection.

The use of chemokine antagonism as a strategy to inhibit leukocyte trafficking into inflammatory sites requires identification of the dominant chemokines mediating recruitment. The chemokine(s) directing T cells into cardiac allografts during acute rejection remain(s) unidentified. The role of the CXC chemokines IFN-gamma inducible protein 10 (IP-10) and monokine induced by IFN-gamma (Mig) in acute rejection of A/J (H-2(a)) cardiac grafts by C57BL/6 (H-2(b)) recipients was tested. Intra-allograft expression of Mig was observed at day 2 posttransplant and increased to the time of rejection at day 7 posttransplant. IP-10 mRNA and protein production were 2.5- to 8-fold lower than Mig. Whereas allografts were rejected at day 7-9 in control recipients, treatment with rabbit antiserum to Mig, but not to IP-10, prolonged allograft survival up to day 19 posttransplant. At day 7 posttransplant, allografts from Mig antiserum-treated recipients had marked reduction in T cell infiltration. At the time of rejection in Mig antiserum-treated recipients (i.e., days 17-19), intra-allograft expression of macrophage-inflammatory protein-1alpha, -1beta, and their ligand CCR5 was high, whereas expression of CXCR3, the Mig receptor, was virtually absent. Mig was produced by the allograft endothelium as well as by recipient allograft-infiltrating macrophages and neutrophils, indicating the synergistic interactions between innate and adaptive immune compartments during acute rejection. Collectively, these results indicate that Mig is a dominant recruiting factor for alloantigen-primed T cells into cardiac allografts during acute rejection. Although Mig antagonism delays acute heart allograft rejection, the results also suggest that the alloimmune response circumvents Mig antagonism through alternative mechanisms.     

Intraallograft chemokine RNA and protein during rejection of MHC-matched/multiple minor histocompatibility-disparate skin grafts

Chemokines direct leukocyte recruitment into sites of tissue inflammation and may facilitate recruitment of leukocytes into allografts following transplantation. Although the expression of chemokines during rejection of MHC-disparate allografts has been examined, chemokine expression in MHC-matched/multiple minor histocompatibility Ag-disparate allografts has not been tested. The intraallograft RNA expression of several C-X-C and C-C chemokines was tested during rejection of full thickness skin grafts from B10. D2 donors on control Ig-, anti-CD4 mAb-, and anti-CD8 mAb-treated BALB/c recipients. In all recipients, two patterns of intragraft chemokine expression were observed during rejection of these grafts: 1) macrophage-inflammatory protein-1alpha, macrophage-inflammatory protein-1beta, GRO-alpha (KC), JE, and IFN-gamma-inducible protein (IP-10) were expressed at equivalent levels in allo- and isografts for 2-4 days posttransplant and then returned to low or undetectable levels; and 2) IP-10 and monokine induced by IFN-gamma (Mig) were expressed in the allografts 3 days before rejection was completed, suggesting a possible role in recruiting primed T cells into the allograft. Three days before completion of rejection, intraallograft IP-10 protein was restricted to the epidermis, whereas Mig was located in the lower dermis and associated with the intense infiltration of mononuclear cells. Treatment of B10.D2 recipients with rabbit antiserum to Mig, but not to IP-10, delayed rejection of the allografts 3-4 days. The results suggest that Mig mediates optimal recruitment of T cells into MHC-matched/multiple minor histocompatibility Ag-disparate allografts during rejection.     

Early chemokine cascades in murine cardiac grafts regulate T cell recruitment and progression of acute allograft rejection

The identification of early inflammatory events after transplant in solid tissue organ grafts that may direct T cell recruitment and promote acute allograft rejection remain largely unknown. To better understand temporal aspects of early inflammatory events in vascularized organ grafts, we tested the intragraft expression of four different chemokines in heterotopically transplanted A/J (H-2(a)) and syngeneic heart grafts in C57BL/6 (H-2(b)) recipient mice from 1.5 to 48 h after transplant. Similar temporal expression patterns and equivalent levels of chemokine expression were observed in both syngeneic and allogeneic cardiac allografts during this time period. Expression of the neutrophil chemoattractant growth-related oncogene alpha (KC) was observed first and reached peak levels by 6 h after transplant and was followed by the monocyte/macrophage chemoattractant protein-1 (JE) and then macrophage inflammatory proteins 1beta and 1alpha. Administration of rabbit KC antiserum to allograft recipients within 30 min of cardiac transplantation attenuated downstream events including intra-allograft expression of the T cell chemoattractants IFN-gamma-inducible protein-10 and monokine induced by IFN-gamma, cellular infiltration into the allograft, and graft rejection. Similarly, depletion of recipient neutrophils at the time of transplantation significantly extended allograft survival from day 8 to 10 in control-treated recipients up to day 21 after transplant. These results indicate the induction of highly organized cascades of neutrophil and macrophage chemoattractants in cardiac grafts and support the proposal that early inflammatory events are required for optimal recruitment of T cells into allografts during the progression of acute rejection of cardiac allografts.     

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.     

Tolerance induction of allo-class II H-2 antigen-reactive L3T4+ helper T cells and prolonged survival of the corresponding class II H-2-disparate skin graft

The present study investigates the effects of i.v. presensitization with class II H-2-disparate allogeneic cells on various L3T4+ T cell functions including the capability of rejecting the corresponding allogeneic skin graft. C57BL/6 (B6) mice were i.v. presensitized with class II H-2 disparate B6-C-H-2bm12 (bm12) spleen cells. Such presensitization did not affect the bm12-specific L3T4+ T cell-mediated proliferative and interleukin 2 (IL-2)-producing capacities. A single cell suspension of (B6 x bm12)F1 spleen cells was depleted of APC by two round-passages over Sephadex G-10 columns. This APC-depleted fraction of (B6 x bm12)F1 cells failed to stimulate B6 responding cells in mixed lymphocyte reactions (MLR). The addition of recombinant IL-1 to the MLR restored anti-bm12 MLR responses, indicating that APC-depleted (B6 x bm12)F1 cells bear bm12 alloantigens but are unable to stimulate B6 anti-bm12 L3T4+ T cells. A single i.v. administration of APC-depleted (B6 x bm12)F1 cells into B6 mice resulted in almost complete abrogation of the capacity of recipient B6 lymphoid cells to give anti-bm12 MLR and IL2 production. This suppression was bm12 alloantigen-specific and attributed to the elimination or functional impairment of anti-bm12 T cell clones rather than the induction of suppressor cells. The tolerance was also observed in graft-rejection responses. The strikingly prolonged survival of bm12 skin grafts was produced when grafts were implanted into B6 mice which had been presensitized with APC-depleted, but not with untreated (B6 x bm12)F1 spleen cells. These results indicate that allo-class II H-2 antigen-reactive L3T4+ T cells are rendered tolerant by i.v. presensitization with APC-depleted fraction of the corresponding allogeneic cells.     

Absence of recipient CCR5 promotes early and increased allospecific antibody responses to cardiac allografts

Acute rejection is mediated by T cell infiltration of allografts, but mechanisms mediating the delayed rejection of allografts in chemokine receptor-deficient recipients remain unclear. The rejection of vascularized, MHC-mismatched cardiac allografts by CCR5(-/-) recipients was investigated. Heart grafts from A/J (H-2(a)) donors were rejected by wild-type C57BL/6 (H-2(b)) recipients on day 8-10 posttransplant vs day 8-11 by CCR5(-/-) recipients. When compared with grafts from wild-type recipients, however, significant decreases in CD4(+) and CD8(+) T cells and macrophages were observed in rejecting allografts from CCR5-deficient recipients. These decreases were accompanied by significantly lower numbers of alloreactive T cells developing to IFN-gamma-, but not IL-4-producing cells in the CCR5(-/-) recipients, suggesting suboptimal priming of T cells in the knockout recipients. CCR5 was more prominently expressed on activated CD4(+) than CD8(+) T cells in the spleens of allograft wild-type recipients and on CD4(+) T cells infiltrating the cardiac allografts. Rejecting cardiac allografts from wild-type recipients had low level deposition of C3d that was restricted to the graft vessels. Rejecting allografts from CCR5(-/-) recipients had intense C3d deposition in the vessels as well as on capillaries throughout the graft parenchyma similar to that observed during rejection in donor-sensitized recipients. Titers of donor-reactive Abs in the serum of CCR5(-/-) recipients were almost 20-fold higher than those induced in wild-type recipients, and the high titers appeared as early as day 6 posttransplant. These results suggest dysregulation of alloreactive Ab responses and Ab-mediated cardiac allograft rejection in the absence of recipient CCR5.     

Antibody-mediated rejection of cardiac allografts in CCR5-deficient recipients

Rejected MHC-mismatched cardiac allografts in CCR5(-/-) recipients have low T cell infiltration, but intense deposition of C3d in the large vessels and capillaries of the graft, characteristics of Ab-mediated rejection. The roles of donor-specific Ab and CD4 and CD8 T cell responses in the rejection of complete MHC-mismatched heart grafts by CCR5(-/-) recipients were directly investigated. Wild-type C57BL/6 and B6.CCR5(-/-) (H-2(b)) recipients of A/J (H-2(a)) cardiac allografts had equivalent numbers of donor-reactive CD4 T cells producing IFN-gamma, whereas CD4 T cells producing IL-4 were increased in CCR5(-/-) recipients. Numbers of donor-reactive CD8 T cells producing IFN-gamma were reduced 60% in CCR5(-/-) recipients. Day 8 posttransplant serum titers of donor-specific Ab were 15- to 25-fold higher in CCR5(-/-) allograft recipients, and transfer of this serum provoked cardiac allograft rejection in RAG-1(-/-) recipients within 14 days, whereas transfer of either serum from wild-type recipients or immune serum from CCR5-deficient recipients diluted to titers observed in wild-type recipients did not mediate this rejection. Wild-type C57BL/6 and B6.CCR5(-/-) recipients rejected A/J cardiac grafts by day 11, whereas rejection was delayed (day 12-60, mean 21 days) in muMT(-/-)/CCR5(-/-) recipients. These results indicate that the donor-specific Ab produced in CCR5(-/-) heart allograft recipients is sufficient to directly mediate graft rejection, and the absence of recipient CCR5 expression has differential effects on the priming of alloreactive CD4 and CD8 T cells.     

Requirement of the thymus for the recovery of anti-alloantigen helper T cells from tolerance induced by intravenous presensitization with allogeneic cells

Intravenous presensitization of C57BL/6 (B6) mice with class I H-2-disparate B6-C-H-2bm1 (bm1) whole spleen cells and class II H-2-disparate B6-C-H-2bm12 (bm12) spleen cells depleted of APC resulted in almost complete elimination of the respective anti-bm1 and anti-bm12 reactivities. However, the reduced alloreactivities as assessed by Th cell capacities (proliferative responses and IL-2 production) were recovered around 8 wk after the i.v. presensitization in euthymic B6 mice. In contrast, background levels of bm1- or bm12-specific reactivities were revealed to last more than 12 wk after the presensitization in B6 mice which had been thymectomized prior to the i.v. presensitization. Such a reduced alloreactivity was also observed in the capacity to reject bm1 or bm12 skin grafts, and prolongation of graft survival was strikingly enhanced in the thymectomized group compared to that induced in the nonthymectomized group. However, there was an important difference in such prolongation in the thymectomized hosts between bm1 and bm12 grafts; a considerable percentage (greater than 80%) of bm12 skin grafts continued to take more than 5 mo, whereas about 90% of bm1 grafts were rejected by around 5 mo along with the emergence of weak, but detectable anti-bm1 Th and cytotoxic T cell activities. These results indicate that 1) i.v. presensitization regimen is capable of eliminating in vitro and in vivo alloreactive capabilities but these alloreactivities can be recovered in euthymic hosts with T cell repopulating potential and 2) there are differential requirements of the thymus for repopulating anti-bm1 (Lyt-2+) and anti-bm12 (L3T4+) T cell activities.     

Tumor allograft rejection is mainly mediated by CD8+ cytotoxic T lymphocytes stimulated with class I alloantigens in cooperation with CD4+ helper T cells recognizing class II alloantigens

Presence of the three major pathways (self-Ia restricted, allo-K/D restricted, and allo-Ia restricted pathways) in generating class I-restricted CTL has been reported. The present study was conducted in order to clarify which of the three is the main pathway in mediating tumor allograft rejection. One million EL-4 tumor cells derived from C57BL/6 (B6;H-2b) were inoculated into the various strains of mice that were genetically different from B6. Class I (K/D) Ag-disparate but IA Ag-matched B6.C-H-2bm1 (bm1;Kbm1, IAb, IE-, Db) mice or B10.A (5R) (5R; b, b, k, d) mice could not reject 1 x 10(6) EL-4 tumor cells in spite of the strong generation of CTL against the B6 Ag, suggesting the inability of the self-Ia restricted pathway and the allo-K/D restricted pathway in rejecting tumor allografts. The strains of mice being capable of rejecting EL-4 tumor were disparate from B6 mice in both class I and class II (IA) Ag, suggesting the importance of the allo-Ia restricted pathway in rejecting tumor allografts. To generate CTL against Kb Ag via the allo-Ia restricted pathway in the bm1 mice, 2 x 10(7) B6.H-2bm12 (bm12; b, bm12, -, b) spleen cells were injected into the bm1 mice as a supplementary source of allogeneic APC that possibly raise CTL through CD4+ Th cells of bm1 origin. These bm1 mice became capable of rejecting 1 x 10(6) EL-4 tumor cells. The same was observed in the combination of bm12----B10.A (5R) (b, b, k, d) mice. To further elucidate the role of the class II restricted CD4+ Th cells, anti-CD4 antibody was repeatedly i.v. administered into the C3H/He (C3H; H-2k) or the DBA/2 (DBA; H-2d) mice on days 0, 1, and 4. Injection of anti-CD4 antibody led 1 x 10(6) EL-4 tumor cells to grow and kill the C3H and DBA mice. These results suggest that the main effector CTL pathway involved in tumor allograft rejection is allo-Ia restricted pathway where CD8+ precursor CTL were stimulated by the class II-restricted CD4+ Th cells.     

Property of class I H-2 alloantigen-reactive Lyt-2+ helper T cell subset. Abrogation of its proliferative and IL-2-producing capacities by intravenous injection of class I H-2-disparate allogeneic cells

The present study investigates the distinctiveness of Class I H-2 alloantigen-reactive Lyt-2+ helper/proliferative T cell subset in the aspect of tolerance induction. Primary mixed lymphocyte reactions (MLR) revealed that Lyt-2+ and L3T4+ T cell subsets from C57BL/6 (B6) mice were exclusively capable of responding to class I H-2 [B6-C-H-2bm1 (bm1)]- and class II H-2 [B6-C-H-2bm12 (bm12)]-alloantigens, respectively. Anti-bm12 MLR was not affected by i.v. injection of bm12 spleen cells into recipient B6 mice. In contrast, a single i.v. administration of bm1 spleen cells into B6 mice resulted in the abrogation of the capacity of recipient B6 spleen and lymph node cells to give anti-bm1 MLR. This suppression was bm1 alloantigen-specific, since lymphoid cells from B6 mice i.v. presensitized with bm1 cells exhibited comparable anti-bm12 primary MLR to that obtained by normal B6 lymphoid cells. Such tolerance was rapidly (24 h after the i.v. injection of bm1 cells) inducible and lasting for at shortest 3 wk. Addition of lymphoid cells from anti-bm1-tolerant B6 mice to cultures of normal B6 lymphoid cells did not suppress the proliferative responses of the latter cells, indicating that the tolerance is not due to the induction of suppressor cells but attributed to the elimination or functional impairment of anti-bm1 proliferative clones. The tolerance was also demonstrated by the failure of tolerant lymphoid cells to produce IL-2. It was, however, found that anti-bm1 CTL responses were generated by tolerant lymphoid cells which were unable to induce the anti-bm1 MLR nor to produce detectable level of IL-2. These results demonstrate that class I H-2 alloantigen-reactive Lyt-2+ Th cell subset exhibits a distinct property which is expressed by neither Lyt-2+ CTL directed to class I H-2 nor L3T4+ Th cells to class II H-2 alloantigens.     

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.     

CD4 T cell-mediated rejection of cardiac allografts in B cell-deficient mice

CD4 T cell-dependent mechanisms promoting allograft rejection include expression of inflammatory functions within the graft and the provision of help for donor-reactive CD8 T cell and Ab responses. These studies tested CD4 T cell-mediated rejection of MHC-mismatched cardiac allografts in the absence of both CD8 T and B lymphocytes. Whereas wild-type C57BL/6 recipients depleted of CD8 T cells rejected A/J cardiac grafts within 10 days, allografts were not rejected in B cell-deficient B6.muMT(-/-) recipients depleted of CD8 T cells. Isolated wild-type C57BL/6 and B6.muMT(-/-) CD4 T cells had nearly equivalent in vivo alloreactive proliferative responses. CD4 T cell numbers in B6.muMT(-/-) spleens were 10% of that in wild-type mice but were only slightly decreased in peripheral lymph nodes. CD8 T cell depletion did not abrogate B6.muMT(-/-) mice rejection of A/J skin allografts and this rejection rendered these recipients able to reject A/J cardiac allografts. Redirection of the alloimmune response to the lymph nodes by splenectomy conferred the ability of B6.muMT(-/-) CD4 T cells to reject cardiac allografts. These results indicate that the low number of splenic CD4 T cells in B6.muMT(-/-) mice underlies the inability to reject cardiac allografts and this inability is overcome by diverting the CD4 T cell response to the peripheral lymph nodes.     

The Immunosuppressant Protosappanin A Diminished Recipient T Cell Migration into Allograft via Inhibition of IP-10 in Rat Heart Transplant

The immunosuppressant Protosappanin A (PrA), isolated from the medicinal herb, promotes cardiac allograft survival, diminishes inflammatory cell infiltration, and inhibits interferon γ-induced protein 10 kDa (IP-10) mRNA expression in rats cardiac grafts. Binding of the chemokine IP-10 to its cognate receptor, CXCR3, plays crucial roles in allograft immunity, especially by mediating the recruitment of effector T cells to allografted tissues. In this study, we attempted to determine whether PrA-mediated inhibition of IP-10 contributes to the effect of reduced T cell infiltration into cardiac allograft within a rat model. Administration of PrA (25 mg/kg daily) via oral gavage following heart transplantation significantly reduced the increase of IP-10 mRNA level in allograft and prevented IP-10 secretion by peripheral blood mononuclear cells (PBMC) isolated from recipient rats seven days posttransplantation. Furthermore, in vitro experiments demonstrated that PrA addition to control PBMC prevented IP-10 secretion. Chemotactic migration assays were utilized to evaluate recipient T cell migration towards PBMC supernatant. PrA administration impaired PBMC supernatant-induced T cell migration. Additional in vitro experiments revealed that PrA slightly reduced naïve T cell migration towards chemokines. The presence of IP-10 in PBMC supernatant prevented PrA from reducing T cell migration in PrA-treated recipients. Neither CXCR3 chemokine ligand Mig nor non-CXCR3 chemokine ligand SDF-1 had any effect on T cell migration in PrA-treated recipients. The addition of anti-CXCR3 antibody restored PrA-mediated inhibition of T cell migration. Immunofluorescence microscopy showed that IP-10 was expressed mainly in CD68 positive infiltrating monocytes. Furthermore, PrA consistently reduced CXCR3⁺T cell infiltration into cardiac allografts. The reduced intensity of CXCR3 staining in PrA-treated allografts contributed to the previously depressed naïve T cell migrating activity induced by PrA. Collectively, these data indicate that PrA inhibition of IP-10 activity reduced recipient T cell migration and infiltration of cardiac allografts, thus partially explaining the immunosuppressive effect of PrA.     

Donor T cell activation initiates small bowel allograft rejection through an IFN-gamma-inducible protein-10-dependent mechanism

The poor success in controlling small bowel (SB) allograft rejection is partially attributed to the unique immune environment in the donor intestine. We hypothesized that Ag-induced activation of donor-derived T cells contributes to the initiation of SB allograft rejection. To address the role of donor T cell activation in SB transplantation, SB grafts from DO11.10 TCR transgenic mice (BALB/c, H-2L(d+)) were transplanted into BALB/c (isografts), or single class I MHC-mismatched (L(d)-deficient) BALB/c H-2(dm2) (dm2, H-2L(d-)) mutant mice (allografts). Graft survival was followed after injection of control or antigenic OVA(323-339) peptide. Eighty percent of SB allografts developed severe rejection in mice treated with antigenic peptide, whereas <20% of allografts were rejected in mice treated with control peptide (p < 0.05). Isografts survived >30 days regardless of OVA(323-339) administration. Activation of donor T cells increased intragraft expression of proinflammatory cytokine (IFN-gamma) and CXC chemokine IFN-gamma-inducible protein-10 mRNA and enhanced activation and accumulation of host NK and T cells in SB allografts. Treatment of mice with neutralizing anti-IFN-gamma-inducible protein-10 mAb increased SB allograft survival in Ag-treated mice (67%; p < 0.05) and reduced accumulation of host T cells and NK cells in the lamina propria but not mesenteric lymph nodes. These results suggest that activation of donor T cells after SB allotransplantation induces production of a Th1-like profile of cytokines and CXC chemokines that enhance infiltration of host T cells and NK cells in SB allografts. Blocking this pathway may be of therapeutic value in controlling SB allograft rejection.     

Inhibition of NF-kappaB-dependent T cell activation abrogates acute allograft rejection.

Using a heterotopic model of transplantation, we investigated the role of T cell activation in vivo during allograft rejection in I-kappaB(DeltaN)-transgenic mice that express a transdominant inhibitor of NF-kappaB in T cells. Our results show indefinite prolongation of graft survival in the I-kappaB(DeltaN)-transgenic recipients. Interestingly, at the time of rejection of grafts in wild-type recipients, histology of grafts in the I-kappaB(DeltaN)-transgenic recipients showed moderate rejection; nevertheless, grafts in the I-kappaB(DeltaN) recipients survived >100 days. Analysis of acute phase cytokines, chemokine, chemokine receptors, and immune responses shows that the blockade of NF-kappaB activation in T cells inhibits up-regulation of many of these parameters. Interestingly, our data also suggest that the T cell component of the immune response exerted positive feedback regulation on the expression of multiple chemokines that are produced predominantly by non-T cells. In conclusion, our studies indicate NF-kappaB activation in T cells is necessary for acute allograft rejection.     

Studies of two H-2Db mutants: B6. C-H-2bm13 and B6.C-H-2bm14

Two new C57BL/6 H-2 mutants, B6.C-H-2bm13 and B6.C-H-2bm14 are described. They arose independently in C57BL/6 as spontaneous mutations of the gain and loss type. Complementation studies map the mutations in both bm13 and bm14 to the H-2Db gene. However, these two mutant strains are not identical, but occurred as independent mutations at the same locus, as shown by reciprocal graft rejection and by the inability of the (bm13 X bm14)F1 hybrid to accept C57BL/6 grafts. Serological studies by direct testing (cytotoxicity and hemagglutination) and by quantitative absorption demonstrated a decrease in the H-2Db private specificity H-2.2 in both bm13 and bm14 when compared to C57BL/6. This was confirmed by SDS-PAGE analysis using antisera detecting the H-2.2 specificity. Attempts to produce antibodies to either the gained or lost specificities of the two mutant strains failed.     

The novel costimulatory programmed death ligand 1/B7.1 pathway is functional in inhibiting alloimmune responses in vivo

The programmed death ligand 1 (PDL1)/programmed death 1 (PD1) costimulatory pathway plays an important role in the inhibition of alloimmune responses as well as in the induction and maintenance of peripheral tolerance. It has been demonstrated recently that PDL1 also can bind B7.1 to inhibit T cell responses in vitro. Using the bm12 into B6 heart transplant model, we investigated the functional significance of this interaction in alloimmune responses in vivo. PD1 blockade unlike PDL1 blockade failed to accelerate bm12 allograft rejection, suggesting a role for an additional binding partner for PDL1 other than PD1 in transplant rejection. PDL1 blockade was able to accelerate allograft rejection in B7.2-deficient recipients but not B7.1-deficient recipients, indicating that PDL1 interaction with B7.1 was important in inhibiting rejection. Administration of the novel 2H11 anti-PDL1 mAb, which only blocks the PDL1-B7.1 interaction, aggravated chronic injury of bm12 allografts in B6 recipients. Aggravated chronic injury was associated with an increased frequency of alloreactive IFN-γ-, IL-4-, and IL-6-producing splenocytes and a decreased percentage of regulatory T cells in the recipients. Using an in vitro cell culture assay, blockade of the interaction of PDL1 on dendritic cells with B7.1 on T cells increased IFN-γ production from alloreactive CD4(+) T cells, whereas blockade of dendritic cell B7.1 interaction with T cell PDL1 did not. These data indicate that PDL1 interaction with B7.1 plays an important role in the inhibition of alloimmune responses in vivo and suggests a dominant direction for PDL1 and B7.1 interaction.     

Absence of allograft ICAM-1 attenuates alloantigen-specific T cell priming,but not primed T cell trafficking into the graft,to mediate acute rejection

The expression and function of ICAM-1 are critical components in the initiation and elicitation of many T cell-mediated responses. Whether ICAM-1 expression is required on the T cells or on the APC during T cell priming remains unclear. To address this issue in alloantigen-specific T cell activation, the priming and function of T cells in response to heart allografts from MHC-mismatched wild-type vs ICAM-1(-/-) donors were tested. Wild-type C57BL/6 (H-2(b)) heart allografts were rejected by A/J (H-2(a)) recipients on days 7-9, whereas B6.ICAM-1(-/-) allografts survived until days 18-23 post-transplant. On day 7 post-transplant, infiltrating macrophages and CD4(+) and CD8(+) T cells in the ICAM-1(-/-) allografts were 20-30% those observed in the wild-type allografts. ELISPOT analyses indicated that the number of alloantigen-specific T cells producing IFN-gamma from recipients of ICAM-1-deficient grafts was 60% lower than that from recipients of wild-type allografts. On day 16 post-transplant, these numbers did not markedly increase in ICAM-1-deficient allograft recipients. Consistent with the reduced priming of alloreactive T cells, isolated dendritic cells from ICAM-1(-/-) mice stimulated allogeneic T cell proliferation poorly compared with wild-type dendritic cells. When A/J mice were primed with wild-type dendritic cells and then received wild-type or ICAM-1-deficient heart allografts 3 days later, the primed recipients rejected the wild-type and ICAM-1(-/-) allografts on days 5-6 post-transplant. These results indicate that optimal priming of alloreactive T cells requires allograft expression of ICAM-1, but, once primed, recipient T cell infiltration into the allograft is independent of graft ICAM-1 expression.     

Blockade of CXCR3 receptor:ligand interactions reduces leukocyte recruitment to the lung and the severity of experimental idiopathic pneumonia syndrome

Idiopathic pneumonia syndrome (IPS) is a frequently fatal complication after allogeneic stem cell transplantation (allo-SCT) that responds poorly to standard immunosuppressive therapy. The pathophysiology of IPS involves the secretion of inflammatory cytokines including IFN-gamma and TNF-alpha along with the recruitment of donor T cells to the lung. CXCR3 is a chemokine receptor that is expressed on activated Th1/Tc1 T cell subsets and the expression of its ligands CXCL9 (monokine induced by IFN-gamma (Mig)) and CXCL10 (IFN-gamma-inducible protein 10 (IP-10)) can be induced in a variety of cell types by IFN-gamma alone or in combination with TNF-alpha. We used a lethally irradiated murine SCT model (B6 --> bm1) to evaluate the role of CXCR3 receptor:ligand interactions in the development of IPS. We found that Mig and IP-10 protein levels were significantly elevated in the bronchoalveolar lavage fluid of allo-SCT recipients compared with syngeneic controls and correlated with the infiltration of IFN-gamma-secreting CXCR3(+) donor T cells into the lung. The in vivo neutralization of either Mig or IP-10 significantly reduced the severity of IPS compared with control-treated animals, and an additive effect was observed when both ligands were blocked simultaneously. Complementary experiments using CXCR3(-/-) mice as SCT donors also resulted in a significant decrease in IPS. These data demonstrate that interactions involving CXCR3 and its primary ligands Mig and IP-10 significantly contribute to donor T cell recruitment to the lung after allo-SCT. Therefore, approaches focusing on the abrogation of these interactions may prove successful in preventing or treating lung injury that occurs in this setting.