WT B6 Research Articles

Loss of IFN-γ Enables the Expansion of Autoreactive CD4+ T Cells to Induce Experimental Autoimmune Encephalomyelitis by a Nonencephalitogenic Myelin Variant Antigen

MHC variant peptides are analogues of immunogenic peptides involving alterations of the MHC-binding residues, thereby altering the affinity of the peptide for the MHC molecule. Recently, our laboratory demonstrated that immunization of WT B6 mice with 45D, a low-affinity MHC variant peptide of MOG(35-55), results in significantly attenuated experimental autoimmune encephalomyelitis (EAE), yet IFN-gamma production is comparable to myelin oligodendrocyte glycoprotein (MOG)(35-55)-immunized mice. In light of these findings, we asked whether IFN-gamma was required for the reduced encephalitogenicity of the weak ligand 45D in EAE. In this study, we report that immunization of mice deficient in IFN-gamma or its receptor with 45D exhibit significant EAE signs compared with 45D-immunized wild-type B6 mice. Moreover, 45D-immunized IFN-gamma(-/-) and IFN-gammaR(-/-) mice demonstrate MOG tetramer-positive CD4(+) T cells within the CNS and display substantial numbers of MOG-specific CD4(+) T cells in the periphery. In contrast, wild-type mice immunized with 45D exhibit reduced numbers of MOG-specific CD4(+) T cells in the periphery and lack MOG tetramer- positive CD4(+) T cells in the CNS. Importantly, the increased encephalitogenicity of 45D in mice lacking IFN-gamma or IFN-gammaR was not due to deviation toward an enhanced IL-17-secreting phenotype. These findings demonstrate that IFN-gamma significantly attenuates the encephalitogenicity of 45D and are the first to highlight the importance of IFN-gamma signaling in setting the threshold level of responsiveness of autoreactive CD4(+) T cells to weak ligands.

A Role for TNF Receptor Type II in Leukocyte Infiltration into the Lung during Experimental Idiopathic Pneumonia Syndrome

Idiopathic pneumonia syndrome (IPS) is a frequently fatal complication following allogeneic stem cell transplantation (allo-SCT). Experimental models have revealed that TNF-α contributes to pulmonary vascular endothelial cell (EC) apoptosis, and modulates the infiltration of donor leukocytes into the lung parenchyma. The inflammatory effects of TNF-α are mediated by signaling through the type I (TNFRI) or type II (TNFRII) TNF receptors. We investigated the relative contribution of TNFRI and TNFRII to leukocyte infiltration into the lung following allo-SCT by using established murine models. Wild-type (wt) B6 mice or B6 animals deficient in either TNFRI or TNFRII were lethally irradiated and received SCT from allogeneic (LP/J) or syngeneic (B6) donors. At week 5 following SCT, the severity of IPS was significantly reduced in TNFRII−/− recipients compared to wt controls, but no effect was observed in TNFRI−/− animals. Bronchoalveolar lavage fluid (BALF) levels of RANTES and pulmonary ICAM-1 expression in TNFRII−/− recipients were also reduced, and correlated with a reduction of CD8 + cells in the lung. Pulmonary inflammation was also decreased in TNFRII−/− mice using an isolated MHC class I disparate model (bm1 → B6), and in bm1 wt mice transplanted with B6 TNF-α−/− donor cells. Collectively, these data demonstrate a role for TNF-α signaling through TNFRII in leukocyte infiltration into the lung following allo-SCT, and suggest that disruption of the TNF-α:TNFRII pathway may be an effective tool to prevent or treat IPS.

TRAIL‐TRAIL Receptor interactions between donor T cells and host B cells are required for induction of autoimmunity in chronic graft‐versus‐host disease

TNF‐related apoptosis‐inducing ligand (TRAIL) mediates the apoptosis of transformed and immune cells and also exerts immunoregulatory functions. Our studies in the parent‐into‐F1 model of chronic graft‐vs‐host disease (cGVHD) demonstrated that TRAIL expression on T cells exacerbates lupus by positively regulating CD4+ T helper cell numbers and sustaining T cell help for B cells. To determine whether direct interactions between TRAIL on donor T cells and TRAIL‐R on autoreactive host B cells are required for induction of autoimmunity we used TRAIL‐R KO as recipients in the bm12‐>B6 model of cGVHD. When cGVHD was induced by injecting Bm12 donor cells into TRAIL R1/DR5‐KO B6 mice, parameters of cGVHD were attenuated compared to cGVHD induced in WT B6 mice. Specifically, the expression of activation markers on host B cells were significantly lower in TRAIL R1/DR5‐KO B6 compared to WT B6 hosts (80±20 vs 362±51 for MHC class II MCF, 19± 6.7 vs 54.1±9.2 for Fas and 38.9±8 vs 65.3±6.4 for B7‐2; p=0.001 for all three comparisons). In addition anti ss‐DNA autoantibody levels were significantly lower in TRAIL R1/DR5‐KO B6 hosts compared to WT B6 hosts. In conclusion, these results suggest that the direct interactions between TRAIL on donor CD4+ T cells and TRAIL‐R on host B cells may be essential for the activation of autoreactive B cells and their differentiation into autoantibody producing cells.

Donor T Cells from B Cell Deficient Mice Inhibit B Cell Development in Normal Recipients after Hematopoietic Cell Transplantation.

Allogeneic hematopoietic cell transplant (HCT) recipients often exhibit B cell (BC) lymphopenia due, in part, to graft-versus-host-disease (GVHD). Here, we studied the impact of donor T cells (TC) on BC deficiency post minor antigen-mismatched HCT. Following lethal irradiation, BALB.B mice were given FACS purified hematopoietic stem cells (HSC: cKIT+Thy1.1loLin-Sca-1+) alone, with whole splenocytes (SP), CD4 or CD8 TC from minor antigen-mismatched C57BL/6 (B6) mice. Chimerism analyses were performed on day (d) 30, 60, and 90. When pure HSC were transplanted, BCs reconstituted promptly (median 33% of lymphocytes [d30]; 61% [d60]; 74% [d90]), whereas TC engraftment was retarded and did not achieve full donor chimerism. Addition of SP or CD4 TCs, or to a lesser degree CD8 TCs, delayed BC reconstitution, with extremely low percentages of BCs beyond d60. This BC suppression correlated with the degree of acute GVHD, and BC numbers increased with recovery from GVHD. Additionally, this BC suppression was in stark contrast to TC development, with TC transfer resulting in early conversion to full donor chimerism. To test if previous events in the donor sensitize TCs against BC features (e.g. minor antigens), thereby promoting anti-BC cytotoxicity post-HCT, TCs from B6 muMT mice were co-transplanted with HSC. muMT mice are devoid of mature BCs because they lack the mu chain; consequently, their TCs were not exposed to BCs prior to transfer. Remarkably, BC engraftment was completely prevented through d90. TCs regenerated faster, but the vast majority originated from spleen and not HSC. To differentiate this lack of BC engraftment from GVHD-associated, alloreactive BC lymphopenia, syngenic B6 recipients were used. Again, initially complete blockade of BC engraftment was observed, although this suppression was overcome earlier post-HCT as compared to the minor-mismatched pair (median % BC d60: 'HSC only' recipients 52%; +CD4 17%; +CD8 48%). To clarify if this phenomenon was a purely cytotoxic reaction of muMT TC against BCs, we used WT B6 HSC +/− SP as donors and lethally or sublethally irradiated muMT mice as recipients. All groups, including sublethally irradiated animals, where host muMT TC were still present, engrafted BCs making a direct anti-BC cytotoxicity unlikely as the sole cause of the BC inhibition. FACS analysis of bone marrow was used to assess the developmental stages of BCs (Hardy fractions (Fr.) A-F) and revealed GVHD recipients with peripheral B lymphopenia have a shift of B220+ cells from more mature Fr. D-F to immature Fr. A-C stages and a lower proportion of IgM expressing BC. Recipients of the muMT TCs showed, in addition to a shift to more immature stages, a clear block in BC development with an absent switch to the expression of IgM (stage D to E)(Fig. 1). In conclusion, muMT TCs are capable of blocking BC maturation when transferred into WT mice, suggesting defective TC activity in muMT animals necessary for the co-development of both BCs and TCs. Furthermore, this study provides evidence that mature TCs are capable of interfering with BC regeneration post-HCT. Hence, our HCT combinations using WT and muMT B6 mice provide a powerful tool to study the role of TC function in the process of donor BC development post-HCT.

Hyperexpression of Foxp3 and IDO During Acute Rejection of Islet Allografts

We investigated the hypothesis that Foxp3+ cells are an integral component of antiallograft immunity but are dominated by pathogenic effectors. Wild-type H-2b C57BL/6 (B6) mice or B6 mice with a targeted disruption of c-Rel gene (c-Rel-/-) were used as recipients of islet grafts from allogeneic DBA/2 (H-2d) mice or syngeneic B6 mice. We developed kinetic quantitative polymerase chain reaction assays and measured intragraft expression of mRNA for Foxp3, IDO, cytolytic molecules, proinflammatory cytokines, and chemokines/receptors. Intraislet levels of mRNA for Foxp3, IDO, CD3, CD25, tumor necrosis factor-alpha, RANTES, IP-10, and CXCR3 were highest in DBA/2 islet allografts from WT B6 recipients compared to DBA/2 islet allografts from c-Rel-/- B6 recipients or syngeneic B6 islet grafts from WT B6 mice. The ratio of granzyme B or IFN-gamma to Foxp3 was higher with the DBA/2 islet allografts from the WT B6 recipients compared to DBA/2 islet allografts from c-Rel-/- B6 recipients or B6 islet grafts from WT B6 recipients. Foxp3+ cells are an integral component of acute rejection of allografts but may be dominated by pathogenic effectors.

Modulation of malaria-induced immunopathology by concurrent nematode infection (46.8)

Abstract Gastrointestinal nematode infections, highly prevalent in malaria endemic areas, are known to modulate host immune responses to unrelated pathogens. However, the complex relationship between worms and malaria and the impact on malarial incidence vs. severity are unclear. Previously, we observed that C57BL/6 (B6) mice infected with the gastrointestinal nematode Heligosomoides polygyrus (Hp) for 2 wks before blood-stage Plasmodium chabaudi AS (Pc) infection developed significantly higher parasitemia compared to mice infected with Pc alone. Despite this, malaria-induced body weight loss, anemia and hepatosplenic damage were not exacerbated in co-infected mice. Indeed, there was less severe hypothermia and hypoglycemia and more robust reticulocytosis in co-infected mice. Co-infection with Hp suppressed type 1 immune responses induced by P. chabaudi infection with significantly reduced plasma levels of IFN-γ, TNF-α and nitric oxide, but increased IL-6 and IL-10 levels. Co-infected STAT-6−/− mice, which have disrupted Th2 responses, experienced only slightly more severe hypothermia, hypoglycemia, and anemia than co-infected WT B6 mice while in vivo administration of anti-IL-10R (1B1.2) mAb resulted in higher mortality in co-infected WT mice. Although concurrent nematode infection resulted in increased parasitemia due to impaired anti-malarial type 1 immunity, our data suggest that malaria-associated pathology is alleviated by a complex immunoregulatory network, possibly involving IL-10, and, to a lesser extent, Th2 cytokines.

CD8+ but Not CD4+ T Cells Require Cognate Interactions with Target Tissues To Mediate GVHD across Only Minor H Antigens but CD4+ and CD8+ T Cells Both Require Direct Leukemic Contact for GVL.

There has been debate as to whether CD4+ or CD8+ T cells require direct cognate interactions with their tissue targets to mediate GVHD. In one report, GVHD mediated by CD4 or CD8 cells did not require target tissue MHCI or MHCII expression in MHC-disparate models (Teshima, et al 2002). However, GVHD lethality may have been due to cytokines produced by high-frequency alloreactive T cells primed by MHC-disparate host APCs. In contrast, MHC-matched, multiple minor H antigen (miHA) disparate GVHD mediated by CD4 or CD8 cells has been reported to be reduced or absent in host → donor chimeric recipients of a second transplant with donor BM and T cells. In this case, host antigen was restricted to hematopoietic cells and the authors concluded that tissue miHA expression must be required (Korngold and Sprent, 1982; Jones et al, 2003). However these experiments did not address whether T cells directly interacted with MHC on target tissues. Rather they demonstrated that a continuing source of host antigen was essential. To resolve this we created bone marrow chimeras in which hematopoiesis was wild type (wt) while the parenchyma was either MHC I- (B6→B6 beta-2-microglobulin−/− (β2M−/−)) or MHC II− (B6→B6 IAb−/−) and used these chimeras as recipients in GVHD-inducing second transplants. We found that B6→B6β2M−/− chimeras were completely resistant clinically and pathologically to CD8-mediated GVHD induced by a second transplant with C3H.SW (H-2b) BM and CD8+ T cells whereas control B6→B6 chimeras developed severe disease. Thus, CD8 cells require direct cognate interactions with target host tissues to induce MHC-matched, miHA disparate GVHD. In contrast, B6→B6 IAb−/− chimeras developed similar clinical and histologic GVHD (liver, ear, skin and bowel) as did control B6→B6 chimeras when retransplanted with 129/J (H-2b) bone marrow and purified CD4+ T cells. Notably we observed lymphocytic infiltrates in involved organs. Therefore, CD4 cells can mediate tissue damage without directly recognizing alloantigen presented by MHCII on target epithelial cells. This suggests an indirect mechanism, perhaps mediated by T cell release of factors after stimulation in tissues by donor-derived APCs presenting host antigens. Alternatively, donor CD4 cells may activate miHA-bearing macrophages to release inflammatory mediators. To investigate the requirement for cognate recognition in GVL, we created murine CML via retroviral-mediated bcr-abl (p210) transduction of bone marrow from wt B6, B6 IAb−/− and B6 β2M−/− mice. Using the C3H.SW→B6 and 129→B6 GVHD models we found that both CD8-and CD4-mediated GVL requires leukemic expression of MHCI and MHCII, respectively. Thus both CD8-mediated GVHD and GVL required cognate T cell:target interactions. However, for CD4 cells only GVL, but not GVHD required target cell MHCII expression. This indicates that CD4-mediated GVL and GVHD have distinct mechanisms of action. Further understanding of these may provide insight in how to deliver GVL with less GVHD.

Nuclear Translocation of RelB within Host and Donor Antigen Presenting Cells Is Critical for the Initiation and Maintenance of Acute Graft-Versus-Host Disease.

Either donor or host antigen presenting cells (APC) are sufficient for the initiation of CD4 dependent graft versus host disease (GVHD). However the molecular transcription pathways within APC required for this effect are unknown. The NF-kB/Rel family member RelB is associated with dendritic cell (DC) maturation and is critical for the induction of potent APC function. DC from RelB−/− mice had markedly reduced levels of CD40 and to a lesser extent CD80/CD86 following in vitro activation. Following total body irradiation, the number of residual splenic DC with nuclear RelB was increased 5-fold relative to untreated mice. We therefore examined the role of RelB within donor and host APC in GVHD utilizing two well established bone marrow transplant models of CD4-dependant GVHD. To study the requirement of RelB within host APC we generated chimeric mice by transplanting wild-type (wt) or RelB−/− B6 bone marrow into irradiated wt B6 mice. Following immune reconstitution 4–6 months later, the number and frequency of DC (CD11chi and CD11cdimB220+) was equivalent in RelB−/− and RelB+/+ chimeras, although RelB−/− chimeras were specifically deficient in CD11chiCD4+ DC. Chimeras were subsequently transplanted with allogeneic Balb/c bone marrow and purified T cells. The absence of RelB within host APC significantly improved survival (survival day 60: 83% v 19%, P< .0001) and GVHD clinical scores were significantly reduced in RelB−/− chimeras for the first 4 weeks after transplant but subsequently rose to levels equivalent to those in surviving RelB+/+ chimeras. All RelB−/− and RelB+/+ chimeras that received syngeneic grafts survived without clinical evidence of GVHD. Sera from RelB−/− chimera recipients of allogeneic grafts contained reduced IFNg (117 ± 23 vs 253 ± 45 pg/ml; P< 0.02) and increased IL-5 (358 ± 105 vs 112 ± 20 pg/ml; P<0.05) compared to RelB+/+ chimera recipients (mean ± SE). Furthermore, CD4 T cells purified from the spleens of RelB−/− chimera recipients produced 2.6 fold more IL-4 (451 ± 31 vs 168 ± 17 pg/ml; P=0.01) than those from RelB+/+ chimera recipients. Taken together these data suggest the absence of nuclear RelB translocation within host APC abrogates GVHD and this is associated with the induction of donor Th2 differentiation. To study the role of RelB within donor APC we transplanted wt or RelB−/− B6 bone marrow and wt purified T cells into irradiated B6D2F1 recipients. In this model, GVHD severity was identical for the first 4 weeks after transplant but subsequently GVHD clinical scores in the recipients of RelB−/− donor APC returned toward levels seen in syngeneic recipients (clinical scores at day 49: 1.0 ± 0.6; n=6 vs 3.75 ± 0.4; n=6; RelB−/− vs RelB+/+P=0.01). This attenuation of acute GVHD in recipients of RelB−/− donor-derived APC was associated with the reconstitution of donor DC on day 21. These data suggest the inhibition of the nuclear RelB translocation within APC represents a potential new therapeutic target for the prevention of allograft rejection and GVHD.

Critical role of host γδ T cells in experimental acute graft-versus-host disease

Abstractγδ T cells localize to target tissues of graft-versus-host disease (GVHD) and therefore we investigated the role of host γδ T cells in the pathogenesis of acute GVHD in several well-characterized allogeneic bone marrow transplantation (BMT) models. Depletion of host γδ T cells in wild-type (wt) B6 recipients by administration of anti-T-cell receptor (TCR) γδ monoclonal antibody reduced GVHD, and γδ T-cell-deficient (γδ-/-) BM transplant recipients experienced markedly improved survival compared with normal controls (63% vs 10%, P < .001). γδ T cells were responsible for this difference because reconstitution of γδ-/- recipients with γδ T cells restored GVHD mortality. γδ-/- recipients showed decreased serum levels of tumor necrosis factor α (TNF-α), less GVHD histopathologic damage, and reduced donor T-cell expansion. Mechanistic analysis of this phenomenon demonstrated that dendritic cells (DCs) from γδ-/- recipients exhibited less allostimulatory capacity compared to wt DCs after irradiation. Normal DCs derived from BM caused greater allogeneic T-cell proliferation when cocultured with γδ T cells than DCs cocultured with medium alone. This enhancement did not depend on interferon γ (IFN-γ), TNF-α, or CD40 ligand but did depend on cell-to-cell contact. These data demonstrated that the host γδ T cells exacerbate GVHD by enhancing the allostimulatory capacity of host antigen-presenting cells. (Blood. 2005;106:749-755)

Donor-derived IL-15 is critical for acute allogeneic graft-versus-host disease

AbstractInterleukin-15 (IL-15) is a pleiotropic proinflammatory cytokine with inefficient posttranscriptional processing. We hypothesized that endogenous IL-15 could affect disease progression in the well-described C57Bl/6 (B6) → (C57Bl/6 × DBA/2) F1 hybrid (B6D2F1) murine model of acute allogeneic graft-versus-host disease (GVHD). B6D2F1 allogeneic recipients received transplants of IL-15-/- B6 bone marrow cells or B6 bone marrow cells expressing a murine IL-15 transgene (IL-15 tg) modified for efficient translation and secretion. Mice that received transplants of IL-15-/- B6 bone marrow cells displayed a significantly longer median survival time (MST) compared with mice that received transplants of wild-type (wt) B6 bone marrow; in contrast, mice that received transplants of IL-15 tg B6 bone marrow cells had a dramatically decreased MST. This decrease in survival was associated with a substantial activation and expansion of effector-memory (CD44highCD62Llow) CD8+ T lymphocytes. Finally, in vivo depletion of either CD4+ or CD8+ T lymphocyte subsets significantly prolonged survival in mice receiving IL-15 tg B6 marrow, while depletion of both CD4+ and CD8+ T cells provided complete protection from acute GVHD. We thus show that acute GVHD is attenuated in the absence of donor bone marrow–derived IL-15 and conclude that donor-derived IL-15 is a critical mediator of T-cell function in acute GVHD.

Host γd T cells Exacerbate Experimental Acute Graft-Versus-Host Disease through Activation of Host Antigen Presenting Cells.

Large numbers of T cells bearing γd T cell receptors are present in graft-versus-host disease (GVHD) target tissues. We investigated the potential role of host γd T cells during acute GVHD in a well-characterized GVHD model following full intensity conditioning (11 Gy TBI). BM and spleen T cells from BALB/c (H2d) donors were transplanted into wild type (wt) B6, aß T cell deficient B6 (aß −/−) or γd T cell deficient B6 (γd −/−) hosts. γd −/− hosts demonstrated significantly better day 35 survival (85%) than wt (40%) or aß−/− hosts (18%) (P<0.05). Reconstitution of γd −/− B6 hosts with B6 type γd T cells 24 hr prior to BMT restored lethal GVHD (50 % day 35 survival). In vivo, γd −/− B6 hosts demonstrated at least a five fold reduction in donor T cell expansion and cytokine production. In vitro, T cells proliferated less when co-cultured with allogeneic γd −/− dendritic cells (DCs) than with wt DCs (40,127 ± 1634 vs. 72,503 ± 1296, P<0.05). BM-derived DCs cultured with γd T cells caused greater proliferation of allogeneic T cells than DCs cultured with aß T cells (15.1 ± 21 x 104 vs. 5.1 ± 1.2 x 104, P<0.05). We next tested the effect of γd T cells on host DCs in vivo using a model system in which only the DCs injected prior to BMT expressed the alloantigen that stimulated the GVHD reaction. MHC Class II −/− B6 mice that had been depleted of γd T cells were given 11 Gy TBI and injected one day prior to BMT with B6 DCs that had been co-cultured either with γd T cells or with medium. On day 0 both groups of recipient mice were injected with BM plus splenic T cells from allogeneic bm12 donors. On day +5, CD4+ donor T cells expanded four times more in recipients of DCs co-cultured with γd T cells than in recipients of control DCs and serum levels of TNF-a were significantly higher (36.7 + 6.8 vs. 21.3 + 3.7 pg/ml, P<0.05). Together these data demonstrate that γd T cells amplify the stimulatory function of host DCs and increase the severity of GVHD, suggesting that a new therapeutic target for the prevention of the major BMT toxicity.

Effects of B7.2−/− Mature Dendritic Cells on Tolerance Induction to Alloantigens in Fetal Mice Following In Utero Transplantation with Lineage Depleted Bone Marrow.

In utero hematopoietic stem cell transplantation (IUT) has the potential to cure a variety of marrow stem cell defects without using marrow ablative therapy. However IUT for diseases other than SCID has been unsuccessful. To better understand the barriers to successful IUT we wanted to define the role of the B7.1/B7.2 co-stimulatory molecules in inducing tolerance to allogeneic donor bone marrow cells in the fetal murine recipient. We studied the relative role of B7.1 and B7.2 expression on dendritic cells (DC) on engraftment and in generating donor specific tolerance in fetal mice. Mature DC (mDC) from B7.1−/− or B7.2−/− donors and wild type (wt) lineage depleted (lin−) C57Bl/6 (B6) bone marrow (BM) were injected into gestational day (GD) 14 Balb/c fetuses. Recipients of lin− wt BM and B7.1−/− mDC had a significantly lower survival (47.4%, p<0.01) associated with mild-moderate GvHD compared to the recipients of B7.2−/− mDC and lin− BM (82.3%) where none developed GvHD. Engraftment results in blood at 6 weeks post IUT showed, B7.1−/− recipients had multilineage engraftment (4.7±0.8% T cells and 5.7± 1.1% granulocytes) in their blood, but by 12 weeks, only donor CD3+ (predominantly CD8+) cells (2.1±1.3%) were present. The percent H2Kb+ (donor) T cells (predominantly CD4+) in the blood of recipients of lin− wt BM and B7.2−/− was 11.8±8.5% at 6 weeks p<0.001 and 6.5±2.5% at 12 weeks, p=0.006. The circulating donor CD4+ cells were Th2 (CD4+CD25−IL4+IL10+) and Treg (CD4+CD25+IL4−IL10−). Both fractions inhibited the T cell proliferative response in the MLR. Long term engraftment in thymic tissues was found in the tolerant recipients of lin− wt BM and B7.2−/− mDC (13.4±8.3% donor CD3+ T cells). We also found prolonged (rejection by day 36) acceptance of donor skin grafts in 7 of 12 recipients of B7.2−/− mDC and 2 of 5 recipients of B7.2−/− mDC and lin−BM. All third party C3H grafts were rejected by day 14 and 80% of the Balb/c (self) skin grafts were permanently accepted. We hypothesized that tolerized animals would behave similarly to recipients of megadoses of syngeneic BM with an increase in multilineage engraftment. We injected a total of 200x106 male wt B6 lin− BM cells over 5 days into adult IUT recipients of B7.1−/− or B7.2−/− mDC ± lin− wt BM and wt age-matched allogeneic and syngeneic (female) controls. Mice that had received B7.2−/− mDC + lin− BM in utero showed multi-lineage engraftment in the blood. In contrast, the in utero recipients of B7.1−/− mDC + lin− BM showed no significant engraftment (p<0.05). In conclusion, donor DC costimulatory molecules significantly affect survival, engraftment and GvHD; and these responses to B7.2−/− mDC and lin− BM appear to be mediated by both Th2 and Treg donor cells.

Unique gene expression profiles of heart allograft rejection in the interferon regulatory factor-1-deficient mouse

Interferon regulatory factor-1 (IRF1) is a transcription factor for many genes involved in innate and adaptive immune responses. By using DNA array technology, we have previously demonstrated that IRF1 is significantly upregulated during acute rejection in rat heart allografts and is restored to isograft levels when recipients are treated with the immunosuppressants tacrolimus or cyclosporin A (CsA). To understand the precise role of IRF1 in transplant rejection, we investigated the rejection responses of mice completely deficient of IRF1 protein. Heterotopic heart transplantations were performed using C57BL/6J wild-type (WT B6) and IRF1-deficient (IRF1 −/−) mice as recipients, and C3H mice as donors. Graft survival was determined by abdominal palpation and rejection was confirmed by histology. On day 6 after transplantation, isografts and allografts were harvested and subjected to gene expression analysis by a commercial nylon array and by real-time RT-PCR. Median survival time of heart allografts was 8 days in the WT B6 mice and 10 days in the IRF1 −/− mice. The gene expression profiles of allografts from the WT B6 and IRF1 −/− recipients were nearly identical to each other and very different from the profile of the isograft control. Both WT B6 and IRF1 −/− profiles showed 13 genes upregulated ( IFN-γ, MCP-2, MIP-1α, MIP-1β, CCR5, MIG, IP-10 and others) and one gene downregulated (SDF2) among the 76 genes detectable on the array. In more detailed analyses, distinct cytokine and chemokine gene expression profiles were identified in the allografts from the WT B6 and IRF1 −/− recipients. Whereas IL-4, IL-6, IL-13, MCP-1, MCP-3, and MPIF-2 were upregulated, RANTES, IL-2Rγ and gp130 were downregulated in allografts from the IRF1 −/− recipients when compared to the WT B6 control. Although the inactivation of the IRF1 gene did not sufficiently prevent acute allograft rejection in this model, a unique cytokine and chemokine gene expression profile was found in the absence of IRF1.

Heart, but Not Skin, Allografts from Donors Lacking Flt3 Ligand Exhibit Markedly Prolonged Survival Time

Fms-like tyrosine kinase 3 ligand (Flt3L) administration leads to dramatic increases in dendritic cells (DC) in lymphoid and nonlymphoid tissues. Conversely, mice lacking Flt3L (Flt3L(-)/(-)) show severe reductions in both myeloid (CD11c(+)CD8alpha(-)) and lymphoid-related DC (CD11c(+)CD8alpha(+)) in the thymus and secondary lymphoid organs. In this study marked reductions in CD11c(+) interstitial cardiac DC and in dermal, but not epidermal, DC (Langerhans cells) were also observed. CD11c(+) cells that migrated from Flt3L(-/-) skin explants expressed lower surface MHC class II and costimulatory molecules and naive T cell allostimulatory activity than migratory wild-type (wt) C57BL/6 (B6) CD11c(+) cells. We examined the survival of Flt3L(-)/(-) heart or tail skin grafts (H2(b)) in allogeneic wt (BALB/c; H2(d)) recipients. The outcome of transplantation of BALB/c organs into Flt3L(-)/(-) recipients was also determined. Flt3L(-)/(-) mice rejected BALB/c heart or skin grafts with similar kinetics as B6 wt recipients. Trafficking of donor DC into host spleens or draining lymph nodes was markedly reduced after transplantation of Flt3L(-)/(-) heart, but not skin grafts, respectively. Compared with wt hearts, survival of Flt3L(-)/(-) hearts was markedly prolonged in BALB/c recipients (median survival time, 37 and 15 days, respectively; p < 0.001). Skin graft survival was unaffected. Rejection of Flt3L(-/-) hearts was precipitated by infusion of wt donor DC at the time of transplant. Thus, severe depletion of interstitial heart DC resulting from targeted gene disruption prolongs, but does not indefinitely extend, heart survival. Acute rejection of wt grafts in Flt3L(-/-) recipients reflects presumably an intact role of the direct pathway of allorecognition.

The contribution of cytotoxic and noncytotoxic function by donor T-cells that support engraftment after allogeneic bone marrow transplantation

The present studies were designed for investigation of the requirements for cytotoxic function in donor T-cells transplanted to support engraftment after infusion of allogeneic bone marrow. The experiments examined the capacity of donor CD8 T-cells lacking Fas ligand and/or perforin function to facilitate donor B6 congenic (B6-Ly5.1) BM engraftment across major histocompatibility complex class I/II barriers after transplantation. T-cell-depleted BM cells from B6-Ly5.1 donors were transplanted into sublethally irradiated (5.5 Gy) BALB/c recipients together with different lymphocyte populations from wild-type B6 (B6-wt) donors or donors lacking functional cytotoxic pathways. Early presence of lineage-committed donor progenitor cells was assessed by the presence of day 5 splenic colony-forming units-granulocyte-macrophage (CFU-GM). Recipients of BMT without donor T-cells did not demonstrate significant CFU-GM activity 5 days post-BMT. Lineage-committed progenitor cells in recipient spleens could be supported by addition to the BM of wild-type (B6-wt) and cytotoxically single- (perforin, B6-pko or FasL, B6-gld) or double-deficient (B6-cdd) CD8 T-cells. However, B220+-enriched B-cells could not support the presence of day 5 donor CFU-GM. For further assessment of the capacity of cytotoxically impaired T-cells to participate in the engraftment process, the ability of these and normal CD8 cells to support the homing of donor cells to the BM was examined after infusion of carboxyfluorescein diacetete succinimidyl ester-labeled progenitors. In a syngeneic model lacking resistance, cytotoxically impaired donor T-cells supported increased numbers of progenitor cells in the marrow equivalent to the support provided by wild-type donor T-cells. Examination of peripheral chimerism indicated that during the first month after B6-->BALB/c BMT, donor chimerism was detected in BMT recipients receiving unfractionated T-cells or CD8+ T-cells from B6-wt donors, and chimerism was maintained at least 80 days after BMT. In contrast, B6-cdd unfractionated or CD8+ T-cells failed to maintain long-term B6 donor chimerism in the host. Experiments with highly enriched populations of positively selected CD8+ T-cells from B6-pko, B6-gld, or B6-cdd donors demonstrated that although each of these T-cell populations could promote the initial presence of donor CFU-GM early post-BMT, B6-pko and B6-cdd CD8+ T-cell populations were not able to support long-term peripheral chimerism. These results demonstrate that donor T-cells lacking major cytotoxic effector pathways have functions that support initial donor progenitor cell presence in the host hematopoietic compartment after BMT. They also demonstrate that support of long-term donor BM engraftment requires CD8+ T-cells with intact cytotoxic, that is, perforin, function. Finally, syngeneic B6-->B6 BMT suggests activation of CD8+ T-cells posttransplantation apparently is required to support enhanced progenitor cell activity. This study provides new findings concerning the role of cytotoxic function in the process of facilitating allogeneic donor BM engraftment.

T-cell subsets mediate graft-versus-myeloid leukemia responses via different cytotoxic mechanisms

Analysis of the cytotoxic effector mechanisms by which T-cell subsets mediate graft-versus-leukemia (GVL) activity is complicated by systems that use unfractionated T cells and leukemias that express alloantigens in addition to tumor-specific antigens. In this study, we used MMB1.10, a myeloid leukemia of C57Bl/6 (B6) origin, to examine the cytolytic pathways employed by syngeneic GVL-mediating, and therefore tumor antigen-specific, T-cell subsets. Wright-Giemsa staining and flow cytometric analysis indicated that MMB1.10 cells exhibited the morphology and markers most consistent with a monocytic-myeloid origin. Although reverse transcription-polymerase chain reaction analysis revealed that MMB1.10 cells expressed tumor necrosis factor (TNF) receptor types I and H, in vitro assays suggested that these cells were resistant to TNF-alpha-mediated cytotoxicity. For study of in vivo GVL responses, mice were challenged with MMBl.10 cells, lethally irradiated, and administered anti-Thy-1-treated (T-cell-depleted) bone marrow (ATBM) either alone or in combination with T-cell subsets from MMB1.10-presensitized mice. In regard to CD4+ donor T cells, 4 x 10(6) MMB1.10-presensitized wild-type (wt) cells exhibited increased GVL responses and survival values relative to tumor-challenged recipients of ATBM only. CD4 T cells from either perforin-deficient (pfp0) or Fas ligand (FasL)-deficient (gld) mice exhibited a lower level of GVL activity but did not produce any long-term survivors. Recipients of 5 x 10(6) wt B6 CD8+ T cells had significantly improved survival relative to tumor-challenged mice that received ATBM only. The same dose of gld CD8+ T cells exhibited a reduced but significant level of GVL activity, whereas cells from mice that were perforin-deficient or cytotoxicity doubly deficient (cdd) (ie, lacking perforin and FasL) exhibited no discernable GVL activity. Doubling the gld CD8+ T-cell dose to 10(7) cells resulted in further improved survival of recipients. We conclude that GVL effects mediated by CD4+ T cells can depend on either perforin- or FasL-mediated mechanisms, whereas the CD8+ T-cell subset is heavily dependent on perforin-mediated cytotoxicity. Biol Blood Marrow Transplant 2000;6(3):231-40.

PROLONGATION OF MURINE CARDIAC AND SKIN ALLOGRAFT SURVIVAL BY ANTI-CD40 MONOCLONAL ANTIBODY

512 CD40/CD40L interactions are crucial for the development of CD4+ T cell dependent immune responses such as allograft rejection. For example, simultaneous administration of a CD40L monoclonal antibody (mAb) and the CTLA4-Ig fusion protein synergistically prolongs the survival of skin and cardiac allografts. In this report we establish the efficacy of an anti-CD40 mAb alone and in combination with CTLA4-Ig as an alternative to anti-CD40 ligand as a strategy to inhibit rejection. Immunohistochemical analysis demonstrates that CD40 is strongly induced on both donor and recipient cells in the rejecting allograft. Furthermore, transplants using CD40-/- mice indicate that CD40 on the recipient is functionally more important than on donor tissues. Hearts from CD40-/- donors are rejected promptly, whereas CD40-/- recipients reject wild type (wt) cardiac allografts more slowly than wt B6,129 F1 control recipients. The ability of an anti-CD40 mAb, 7E1, to inhibit allograft rejection was tested in the BALB/c to C3H/HeJ strain combination. Recipient mice were treated with 7E1 (250µg/dose) intraperitoneally (IP) on days 0, 2, 4& 6. 7E1 significantly prolonged cardiac allograft survival relative to untreated controls. However, histological assessment of the 7E1 treated animals demonstrated significant interstitial fibrosis and coronary vasculopathy by postoperative day (POD) 140. As previously reported with a CD40L mAb (MR1), 7E1 synergized with CTLA4-Ig in inhibiting these pathologic changes. C3H/HeJ recipients of BALB/c skin grafts were treated with anti-CD40 mAb, alone and in combination with CTLA4-Ig (250µg/dose IP on days 0, 2, 4& 6). Anti-CD40 mAbs alone prolonged skin graft survival (MST=30d) compared to MR1 treatment (MST-14d). Untreated controls rejected the skin grafts promptly by POD11. As in the cardiac allograft model, 7E1 and CTLA4-Ig synergize to improve skin graft survival (MST=93d). 7E1 was found to prevent the formation of donor specific alloantibodies in the cardiac allograft recipients. Alloantibodies were undetectable on POD 29, 35 & 57, whereas alloantibodies were detected in the control BALB/c cardiac allograft recipients at a dilution of 1:512 at POD 29, 34 & 57. Flow cytometric analysis of peripheral blood of cardiac allograft recipients treated with 7E1 demonstrated partial B cell depletion on POD 8 and 21 which returned to normal by POD55. 7E1 treatment had no effect on T cell and macrophage compartments. These studies suggest that the use of anti-CD40 mAbs is an effective alternative to MR1 in the blockade of the gp39-CD40 pathway alone or in combination with CTLA4Ig.

PROLONGATION OF MURINE CARDIAC AND SKIN ALLOGRAFT SURVIVAL BY ANTI-CD40 MONOCLONAL ANTIBODY

512 CD40/CD40L interactions are crucial for the development of CD4+ T cell dependent immune responses such as allograft rejection. For example, simultaneous administration of a CD40L monoclonal antibody (mAb) and the CTLA4-Ig fusion protein synergistically prolongs the survival of skin and cardiac allografts. In this report we establish the efficacy of an anti-CD40 mAb alone and in combination with CTLA4-Ig as an alternative to anti-CD40 ligand as a strategy to inhibit rejection. Immunohistochemical analysis demonstrates that CD40 is strongly induced on both donor and recipient cells in the rejecting allograft. Furthermore, transplants using CD40-/- mice indicate that CD40 on the recipient is functionally more important than on donor tissues. Hearts from CD40-/- donors are rejected promptly, whereas CD40-/- recipients reject wild type (wt) cardiac allografts more slowly than wt B6,129 F1 control recipients. The ability of an anti-CD40 mAb, 7E1, to inhibit allograft rejection was tested in the BALB/c to C3H/HeJ strain combination. Recipient mice were treated with 7E1 (250μg/dose) intraperitoneally (IP) on days 0, 2, 4 & 6. 7E1 significantly prolonged cardiac allograft survival relative to untreated controls. However, histological assessment of the 7E1 treated animals demonstrated significant interstitial fibrosis and coronary vasculopathy by postoperative day (POD) 140. As previously reported with a CD40L mAb (MR1), 7E1 synergized with CTLA4-Ig in inhibiting these pathologic changes. C3H/HeJ recipients of BALB/c skin grafts were treated with anti-CD40 mAb, alone and in combination with CTLA4-Ig (250μg/dose IP on days 0, 2, 4 & 6). Anti-CD40 mAbs alone prolonged skin graft survival (MST=30d) compared to MR1 treatment (MST=14d). Untreated controls rejected the skin grafts promptly by POD11. As in the cardiac allograft model, 7E1 and CTLA4-Ig synergize to improve skin graft survival (MST=93d). 7E1 was found to prevent the formation of donor specific alloantibodies in the cardiac allograft recipients. Alloantibodies were undetectable on POD 29, 35 & 57, whereas alloantibodies were detected in the control BALB/c cardiac allograft recipients at a dilution of 1:512 at POD 29, 34 & 57. Flow cytometric analysis of peripheral blood of cardiac allograft recipients treated with 7E1 demonstrated partial B cell depletion on POD 8 and 21 which returned to normal by POD55. 7E1 treatment had no effect on T cell and macrophage compartments. These studies suggest that the use of anti-CD40 mAbs is an effective alternative to MR1 in the blockade of the gp39-CD40 pathway alone or in combination with CTLA4Ig.

Selenium poisoning during pregnancy: Report on an at-risk pregnancy and review of the literature

Adult immunocompetent male C57Bl/6 mucopolysaccharidosis, type I (MPSI) mice develop aortic insufficiency (AI), dilated ascending aortas and decreased cardiac function, findings not observed in immune incompetent adult male NSG MPSI mice. We sought to determine why.Cardiac ultrasound measurements of ascending aorta and left ventricular dimensions and Doppler interrogation for AI were performed in 6-month-old male B6 MPSI (N = 12), WT (N = 6), NSG MPSI (N = 8), NSG (N = 6) mice. Urinary glycosaminoglycans, RNA sequencing with quantitative PCR were performed and aortic pathology assessed by routine and immunohistochemical staining on subsets of murine aortas.Ascending aortic diameters were significantly greater, left ventricular function significantly decreased, and AI significantly more frequent in B6 MPSI mice compared to NSG MPSI mice (p < 0.0001, p = 0.008 and p = 0.02, respectively); NSG and B6 WT mice showed no changes. Urinary glycosaminoglycans were significantly greater in B6 and NSG MPSI mice and both were significantly elevated compared to WT controls (p = 0.003 and p < 0.0001, respectively). By RNA sequencing, all 11 components of the inflammasome pathway were upregulated in B6 MUT, but only Aim2 and Ctsb in NSG MUT mice and none in WT controls. Both B6 and NSG MUT mice demonstrated variably-severe intramural inflammation, vacuolated cells, elastin fragmentation and disarray, and intense glycosaminoglycans on histological staining. B6 MPSI mice demonstrated numerous medial MAC2+ macrophages and adventitial CD3+ T-cells while MAC2+ macrophages were sparse and CD3+ T-cells absent in NSG MPSI mice.Aortic dilation, AI and decreased cardiac function occur in immunocompetent B6 MPSI male mice but not in immune incompetent NSG MPSI mice, unrelated to GAG excretion, upregulation of Ctsb, or routine histologic appearance. Upregulation of all components of the inflammasome pathway in B6 MUT, but not NSG MUT mice, and abundant medial MAC2 and adventitial CD3 infiltrates in B6, but not NSG, MPSI aortas differentiated the two strains. These results suggest that the innate and adaptive immune systems play a role in these cardiac findings which may be relevant to human MPSI.