Minor Histocompatibility Loci Research Articles

Use of paclitaxel carried in lipid nanoparticles to treat aortic allograft transplantation in rats.

The aim of this study was to test whether lipid core nanoparticles loaded with paclitaxel (LDE-PTX) protect rat aortic allograft from immunological damage. Fisher and Lewis rats were used differing in minor histocompatibility loci. Sixteen Lewis rats were allocated to four-animal groups: SYNG (syngeneic), Lewis rats receiving aorta grafts from Lewis rats; ALLO (allogeneic), Lewis rats receiving aortas from Fisher rats; ALLO+LDE (allogeneic transplant treated with LDE), Lewis rats receiving aortas from Fisher rats, treated with LDE (weekly injection for 3 weeks); ALLO+LDE-PTX (allogeneic transplant treated with LDE-PTX), Lewis rats receiving aortas from Fisher rats treated with LDE-PTX (4 mg/kg weekly for 3 weeks). Treatments began on transplantation day. Thirty days post-transplantation, SYNG showed intact aortas. ALLO and ALLO+LDE presented intense neointimal formation. In ALLO+LDE-PTX, treatment inhibited neointimal formation; narrowing of aortic lumen was prevented in ALLO and ALLO+LDE. LDE-PTX strongly inhibited proliferation and intimal invasion by smooth muscle cells, diminished 4-fold presence of apoptotic/dead cells in the intima, reduced the invasion of aorta by macrophages and T-cells and gene expression of pro-inflammatory tumour necrosis factor-alpha (TNFα), interferon gamma (IFNγ) and interleukin-1 beta (IL-1β). LDE-PTX was effective in preventing the vasculopathy associated with rejection and may offer a potent therapeutic tool for post-transplantation.

Enhancement of Antigen Presentation by Deletion of Viral Immune Evasion Genes Prevents Lethal Cytomegalovirus Disease in Minor Histocompatibility Antigen-Mismatched Hematopoietic Cell Transplantation.

Hematoablative treatment followed by hematopoietic cell transplantation (HCT) for reconstituting the co-ablated immune system is a therapeutic option to cure aggressive forms of hematopoietic malignancies. In cases of family donors or unrelated donors, immunogenetic mismatches in major histocompatibility complex (MHC) and/or minor histocompatibility (minor-H) loci are unavoidable and bear a risk of graft-vs.-host reaction and disease (GvHR/D). Transient immunodeficiency inherent to the HCT protocol favors a productive reactivation of latent cytomegalovirus (CMV) that can result in multiple-organ CMV disease. In addition, there exists evidence from a mouse model of MHC class-I-mismatched GvH-HCT to propose that mismatches interfere with an efficient reconstitution of antiviral immunity. Here we used a mouse model of MHC-matched HCT with C57BL/6 donors and MHC-congenic BALB.B recipients that only differ in polymorphic autosomal background genes, including minor-H loci coding for minor-H antigens (minor-HAg). Minor-HAg mismatch is found to promote lethal CMV disease in absence of a detectable GvH response to an immunodominant minor-HAg, the H60 locus-encoded antigenic peptide LYL8. Lethality of infection correlates with inefficient reconstitution of viral epitope-specific CD8+ T cells. Notably, lethality is prevented and control of cytopathogenic infection is restored when viral antigen presentation is enhanced by deletion of immune evasion genes from the infecting virus. We hypothesize that any kind of mismatch in GvH-HCT can induce “non-cognate transplantation tolerance” that dampens not only a mismatch-specific GvH response, which is beneficial, but adversely affects also responses to mismatch-unrelated antigens, such as CMV antigens in the specific case, with the consequence of lethal CMV disease.

Insufficient Antigen Presentation Due to Viral Immune Evasion Explains Lethal Cytomegalovirus Organ Disease After Allogeneic Hematopoietic Cell Transplantation.

Reactivation of latent cytomegalovirus (CMV) poses a clinical problem in transiently immunocompromised recipients of hematopoietic cell (HC) transplantation (HCT) by viral histopathology that results in multiple organ manifestations. Compared to autologous HCT and to syngeneic HCT performed with identical twins as HC donor and recipient, lethal outcome of CMV infection is more frequent in allogeneic HCT with MHC/HLA or minor histocompatibility loci mismatch between donor and recipient. It is an open question if a graft-vs.-host (GvH) reaction exacerbates CMV disease, or if CMV exacerbates GvH disease (GvHD), or if interference is mutual. Here we have used a mouse model of experimental HCT and murine CMV (mCMV) infection with an MHC class-I mismatch by gene deletion, so that either HCT donor or recipient lack a single MHC class-I molecule, specifically H-2 Ld. This particular immunogenetic disparity has the additional advantage that it allows to experimentally separate GvH reaction of donor-derived T cells against recipient's tissues from host-vs.-graft (HvG) reaction of residual recipient-derived T cells against the transplanted HC and their progeny. While in HvG-HCT with Ld-plus donors and Ld-minus recipients almost all infected recipients were found to control the infection and survived, almost all infected recipients died of uncontrolled virus replication and consequent multiple-organ viral histopathology in case of GvH-HCT with Ld-minus donors and Ld-plus recipients. Unexpectedly, although anti-Ld-reactive CD8+ T cells were detected, mortality was not found to be associated with GvHD histopathology. By comparing HvG-HCT and GvH-HCT, investigation into the mechanism revealed an inefficient reconstitution of antiviral high-avidity CD8+ T cells, associated with lack of formation of protective nodular inflammatory foci (NIF) in host tissue, selectively in GvH-HCT. Most notably, mice infected with an immune evasion gene deletion mutant of mCMV survived under otherwise identical GvH-HCT conditions. Survival was associated with enhanced antigen presentation and formation of protective NIF by antiviral CD8+ T cells that control the infection and prevent viral histopathology. This is an impressive example of lethal viral disease in HCT recipients based on a failure of the immune control of CMV infection due to viral immune evasion in concert with an MHC class-I mismatch.

Myeloablative hematopoietic stem cell transplantation improves survival but is not curative in a pre-clinical model of myelodysplastic syndrome.

Allogeneic hematopoietic stem cell transplantation (A-HSCT) remains the only curative option for patients with myelodysplastic syndrome (MDS). We used the NUP98-HOXD13 (NHD13) murine model for MDS to study HSCT in a pre-clinical setting. NHD13 recipients transplanted with syngeneic bone marrow (S-HSCT) following myeloablative irradiation showed disease remission, with normalization of peripheral blood parameters and marked decrease in circulating leukocytes derived from the MDS clone. Despite the disease remission and improved survival compared to non-transplanted NHD13 controls, all mice eventually relapsed, indicating persistence of a long-lived radio-resistant MDS clone. In an effort to induce a graft versus leukemia (GVL) effect, A-HSCT with donor bone marrow that was mismatched at minor histocompatibility loci was compared to S-HSCT. Although recipients in the A-HSCT showed a lower early relapse rate than in S-HSCT, all mice in both groups eventually relapsed and died by 54 weeks post-transplant. To obtain a more significant GVL effect, donor splenocytes containing reactive T-cells were transplanted with allogeneic bone marrow. Although the relapse rate was only 20% at post-transplantation week 38, suggesting a GVL effect, this was accompanied by a severe graft versus host disease (GVHD) Taken together, these findings indicate that a myeloablative dose of ionizing radiation is insufficient to eradicate the MDS initiating cell, and that transplantation of donor splenocytes leads to decreased relapse rates, at the cost of severe GVHD. We suggest that NHD13 mice represent a feasible pre-clinical model for the study of HSCT for MDS.

Blockade of p38 Mitogen-Activated Protein Kinase Inhibits Murine Sclerodermatous Chronic Graft-versus-Host Disease

Bone marrow transplantation (BMT) of B10.D2 mice into sublethally irradiated BALB/c mice across minor histocompatibility loci is a well-established animal model for human sclerodermatous chronic graft-versus-host disease (Scl-cGVHD) and systemic sclerosis (SSc). The p38 mitogen-activated protein kinase (MAPK) pathway is a key regulator of inflammation and cytokine production. Furthermore, the activation of p38 MAPK plays an important role in collagen production in SSc. We investigated the effects of p38 MAPK inhibitor, VX-702, on Scl-cGVHD mice. VX-702 was orally administered to Scl-cGVHD mice from day 7 to 35 after BMT. We compared skin fibrosis of Scl-cGVHD mice between the VX-702-treated group and control group. Allogeneic BMT increased the phosphorylation of p38 MAPK in the skin. The administration of VX-702 attenuated the skin fibrosis of Scl-cGVHD compared to the control mice. Immunohistochemical staining showed that VX-702 suppressed the infiltration of CD4+ T cells, CD8+ T cells, and CD11b+ cells into the dermis of Scl-cGVHD mice compared to the control mice. VX-702 attenuated the mRNA expression of extracellular matrix and fibrogenic cytokines, such as IL-6 and IL-13, in the skin of Scl-cGVHD mice. In addition, VX-702 directly inhibited collagen production from fibroblasts invitro. VX-702 was shown to be a promising candidate for use in treating patients with Scl-cGVHD and SSc.

The effects of mesenchymal stem cells in a murine sclerodermatous model of graft-versus-host disease

Abstract Human chronic graft-versus-host disease (CGVHD) shares clinical characteristics with a murine sclerodermatous GVHD (scl GVHD) model that is characterized by skin and lung fibrosis. We used B10.D2 → BALB/c model of scl GVHD, which differ at minor histocompatibility loci, to address the therapeutic effect of mesenchymal stem cells (MSCs) on the development of CGVHD. MSCs were intravenously treated on days 3, 5 and 7 post-transplantation. The clinical severity of cutaneous scl GVHD was significantly attenuated after the treatment of MSCs. Pathologically, animals treated with MSCs showed less fibrosis and inflammatory cell infiltrations in skin and ear but not in visceral organs including lung and liver. Accordingly, the mRNA expression of collagen type 1 α1, 1 α2 and 3 α1 as well as collagen production were significantly reduced after the MSC treatment only in skin compared to the allogeneic scl GVHD controls. MSCs down-regulated mRNA of proinflammatory cytokines mainly in skin tissues and among them TGF-β expression was consistently reduced 28 days after HSCT. The effects of MSCs on molecular markers implicated in persistent TGF-β signaling and fibrosis, such as phosphatase and tensin homolog (PTEN), p-Smad2/3 and matrix metaloprotease-1 (MMP-1) were observed at the skin tissues. MSCs inhibited infiltration of immune cells into skin through down-regulating CCR4, CCR8 and CCR10 on donor CD4 T cells and CCR1 on CD11b monocytes. Moreover, MSCs diminished the expression of mRNA for chemokines such as CCL1, CCL8, CCL17, CCL22 and CCL3, in skin. In conclusion, treatment of MSCs early after transplant attenuated cutaneous scl GVHD by selectively blocking of immune cell migration and down-regulating the chemokines and chemokine receptors.

Genome-Wide Analysis in Swine Associates Corneal Graft Rejection with Donor-Recipient Mismatches in Three Novel Histocompatibility Regions and One Locus Homologous to the Mouse H-3 Locus.

In rodents, immune responses to minor histocompatibility antigens are the most important drivers of corneal graft rejection. However, this has not been confirmed in humans or in a large animal model and the genetic loci are poorly characterised, even in mice. The gene sequence data now available for a range of relevant species permits the use of genome-wide association (GWA) techniques to identify minor antigens associated with transplant rejection. We have used this technique in a pre-clinical model of corneal transplantation in semi-inbred NIH minipigs and Babraham swine to search for novel minor histocompatibility loci and to determine whether rodent findings have wider applicability. DNA from a cohort of MHC-matched and MHC-mismatched donors and recipients was analysed for single nucleotide polymorphisms (SNPs). The level of SNP homozygosity for each line was assessed. Genome-wide analysis of the association of SNP disparities with rejection was performed using log-likelihood ratios. Four genomic blocks containing four or more SNPs significantly linked to rejection were identified (on chromosomes 1, 4, 6 and 9), none at the location of the MHC. One block of 36 SNPs spanned a region that exhibits conservation of synteny with the mouse H-3 histocompatibility locus and contains the pig homologue of the mouse Zfp106 gene, which encodes peptide epitopes known to mediate corneal graft rejection. The other three regions are novel minor histocompatibility loci. The results suggest that rejection can be predicted from SNP analysis prior to transplant in this model and that a similar GWA analysis is merited in humans.

MyD88 in Donor Bone Marrow Cells Is Critical for Protection from Acute Intestinal Graft-Versus-Host Disease

Allogeneic hematopoietic stem cell transplantation is an important therapeutic modality used to treat malignancies of hematopoietic origin, such as leukemia and lymphoma. However, development of graft-versus-host disease (GVHD) causes non-relapse mortality and substantial morbidity of recipients. Myeloid differentiation factor 88 (MyD88), a major adaptor mediating TLR signaling, is also known to deliver pro-inflammatory signals. Activation of inflammatory signaling through MyD88 plays a key role in the expansion of myeloid-derived suppressor cells (MDSC) which are a heterogeneous population of immature myeloid ells with anti-inflammatory activity. To explore the contribution of MyD88 expressed by donor bone marrow (BM) cells to development of GVHD, we induced GVHD using T-cell-depleted BM (TCD-BM) isolated from MyD88-deficient (MyD88KO) mice and T cells isolated from wild-type (WT) mice. We employed C57BL/6 (H-2b) → B6D2F1 (H-2b/d) mouse model of GVHD, which differ at major and minor histocompatibility loci. Lethally irradiated B6D2F1 recipient mice were transplanted with either T cell-depleted bone marrow (TCD BM, 5 x 106) from either WT or MyD88KO mice together with WT spleen T cells (1 x 106). Transplantation with MyD88KO TCD BM aggravated GVHD; serious gut damage was evident, with infiltration of T cells specifically into the intestines of recipients. GVHD hosts transplanted with MyD88KO TCD BM exhibited markedly reduced expansion of MDSC. GVHD aggravation after transplantation with MyD88KO TCD-BM, associated with high-level T cell infiltration into the intestine and insignificant expansion of MDSC, was reproduced in another minor histocompatibility mismatch model (C57BL/6 → BALB.B). We next examined allogeneic T cells in the spleens of GVHD hosts in terms of the expression levels of CCR9, which are known to be associated with T cell migration to the intestinal mucosa and the proportion of CCR9 positive cells in CFSE low CD8+ T cells was higher in recipients of MyD88KO TCD BM than WT controls. In parallel, the levels of CCL25 were more highly expressed in the gut of MyD88KO recipients than WT controls. Mixed leukocyte cultures of CFSE- labeled C57BL/6 T cells and irradiated B6DF1 feeder cells were prepared in the presence of MDSC isolated from MyD88KO or WT mice. T cells, co-incubated with MDSC isolated from MyD88KO BM, exhibited a greater extent of CFSE dilution and less Annexin V staining, compared to T cells co-incubated with cells from WT BM. Moreover, MDSC from recipients of MyD88KO TCD BM exhibited a reduced suppressive function, compared to their WT counterparts. Next, we determined whether insufficient expansion of and ineffective suppression by MDSC caused severe GVHD in recipients of MyD88KO TCD BM. Supplementation of transplanted mice with MDSC from WT mice, not from MyD88KO mice, attenuated the severity of GVHD and reduced intestinal T cell infiltration in recipients of MyD88KO TCD BM. To verify the importance of MyD88-mediated signaling by MDSC in protection against severe GVHD, we determined if transplantation with TCD-BM cells containing high levels of MDSC attenuated the severity of GVHD. Pre-treatment of BM donors with lipopolysaccharide increased the frequencies of MDSC and the amounts of MyD88 transcripts in TCD-BM transplant, and alleviated the severity of GVHD and intestinal T-cell infiltration. To explore whether MDSC expansion levels could be used to predict the severity of intestinal GVHD, the T/MDSC ratios were calculated in blood of patients at the time of engraftment and were significantly higher in patients with intestinal GVHD ≥ grade 2. In conclusion, we have shown that MyD88-dependent MDSC expansion from donor BM is critical for protection against fatal acute intestinal GVHD. DisclosuresNo relevant conflicts of interest to declare.

Close To Clinics: Acute Gvhd In a Novel Chemotherapy Based Minor Mismatch Transplantation Model

GVHD (graft-vs-host disease) is one of the major complications after allogeneic hematopoietic stem cell transplantation leading to substantial morbidity and mortality. Several murine models are used to investigate this systemic inflammatory disease in vivo and to identify possible targets for clinical treatment. However, current well-established murine GVHD models have their clinical limitations as they are based on major histocompatibility mismatches and only lethal irradiation as conditioning. Development of new treatment options would be forwarded by the availability of a more clinically relevant murine model.To reflect the clinical situation of transplantation and GVHD, we established a new murine GVHD model which is based on Chemotherapy conditioning and MHC-matched bone marrow transplantation (BMT). In LP/J -> C57BL/6 mouse model, our chemotherapy transplantation protocol resulted in stable full donor chimerism and development of acute GVHD.C57BL/6 recipients were conditioned with a 7-day busulfan-cyclophosphamide Chemotherapy protocol. Mice received for 5 days 20 mg/kg/day busulfan intraperitoneally (i.p.) and the last three days additionally 100 mg/kg/day cyclophosphamide. After a two-day resting phase, recipients were transplanted with 1.5 x 107 bone marrow and 2 x 106 splenic T cells from LP/J donors. Recipients showed good engraftment with already 80 % engraftment in peripheral blood at day 15 after BMT. 90 % donor chimerism in peripheral blood and bone marrow could be shown already at day 30 after BMT.LP/J and C75BL/6 mice share the same haplotype (H2b) with only mismatches at different minor histocompatibility loci. As in clinical BMT, GVHD still arises after MHC-matched transplantation because of multiple minor mismatches. We could show in our LP -> C57BL/6 model typical features of acute GVHD. Allogeneic transplanted animals showed significant decreased survival compared to syngeneic transplanted animals (Figure 1A). GVHD animals exhibited GVHD-typical weight loss (Figure 1B) and a significant increased clinical GVHD index consisting of five clinical parameters: weight loss, posture, activity, fur and skin (Figure 1C). Our model showed a strong skin phenotype, especially between day 15 and 25 after BMT extensive scurf spread over the whole body combined with fur loss. Pronounced weight loss and highest clinical GVHD index were monitored between day 15 and 25, implicating this period as acute phase of GVHD. Histopathologic analysis of GVHD organs (large bowel, liver and skin) confirmed acute GVHD symptoms in our model as histopathological scores after Lerner (1974,Transplant Proc 6:367-371) were significantly increased in GVHD animals compared to BMT recipients without GVHD (Figure 1D). [Display omitted] We next characterized systemic inflammation in our model by determining the number of circulating inflammatory cells in peripheral blood at different time points by flow cytometry. CD8+ T cells were significantly increased in GVHD animals in acute phase (day 15 after BMT), whereas CD4+ T cells were elevated in late phase (day 50-60 after BMT). These data suggests, that in our model two inflammatory phases occur, an early acute CD8-driven and a late CD4-driven phase.Finally, we could demonstrate GVHD-typical T cell infiltration in GHVD target organs in acute phase (Figure 2). In large and small bowel, liver and skin CD4 and CD8 infiltration was significantly elevated in GVHD animals, however, CD8 infiltration was more prominent. We conclude that CD8 T cells play a prominent role in acute phase of GVHD in our model. [Display omitted] In summary, we could establish a novel chemotherapy based minor mismatch GVHD model which shows good engraftment, typical features of acute GVHD and systemic and target organ specific inflammation. Our murine GVHD model closely resembles the clinical situation of MHC-matched stem cell transplantation and may help to better understand pathogenic mechanisms in GVHD and develop new treatment options. Disclosures:No relevant conflicts of interest to declare.

A Critical Role of MyD88 Signaling in Donor T Cells for Graft-Versus-Host Disease (GVHD) and Graft-Versus-Leukemia Effect (GVL) After Experimental Allogeneic Hematopoietic Stem Cell Transplantation (allo-HSCT)

AbstractAbstract 1893The therapeutic potential of allogeneic hematopoietic stem cell transplantation (allo-HSCT) relies on the graft-versus-leukemia effect (GVL) to eradicate residual tumor cells by immunologic mechanisms. However, graft-versus-host disease (GVHD) remains the major toxicity of allo-HSCT. Alloreactive donor T cells are important effector cells in the development of GVHD, and proinflammatory cytokines enhance the generation of donor antihost cytotoxic function. Myeloid differentiation factor (MyD88) is a cytoplasmic adaptor molecule essential for integrating and transducing the signals generated by the Toll-like receptor (TLR) family. TLR engagement on professional antigen-presenting cells induces their maturation, resulting in optimal T-cell activation. However, recent advances indicate that the adjuvant effects of certain TLR agonists may also be attributed to the activation of TLRs and MyD88 directly in T cells. Both CD4 and CD8 T cells express functional TLRs. It remains to be defined whether direct TLR signaling on donor T cells is critical for GVHD or GVL activity.We used C57BL/6 (H-2b) → B6D2F1 (H-2b/d) experimental allo-HSCT model, which differs at major and minor histocompatibility loci, to address the role of donor T cell MyD88 signaling on GVHD and GVL. Lethally irradiated recipient mice were transplanted TCD-BM (5 × 106) together with either wild-type (WT) or MyD88 knock out (KO) mice spleen T cells (1 × 106) on day 0 and then host-type P815 mastocytoma or L1210 leukemia (H-2d) cells were injected either intravenously (3 × 103) or subcutaneously (1 × 106) on day 1 to generate a GVHD/GVL model. First of all, clinical GVHD scores were comparable between recipients of WT T cells and MyD88 KO T cells. At 70 days post-allo-HSCT, 50 % of allogeneic recipients of WT T cells died due to severe GVHD, but necropsy showed no evidence of tumor. In contrast, 83.5% of those of MyD88 KO T cells died with gross evidence of tumors (P<.05). Moreover, subcutaneous tumors in the allogeneic recipients receiving MyD88 KO T cells exhibited markedly increased growth in vivo compared to those receiving WT T cells (tumor volume on day 41, 15205.6 vs. 373.9 mm3, P<.01). GVHD mortality is critically dependent on donor CD4 T cells in this donor/recipient strain combination (B6 → B6D2F1) and CD8 T cells that mediate cytotoxicity are more potent effectors of GVL. The percentages of donor T cells to undergo proliferation or apoptosis in response to alloantigens in vivo between the two T cell types was examined; apoptosis of CD8 T cells in recipients of MyD88 KO T cells was significantly enhanced compared to those of WT T cell recipients (P<.01) whereas apoptosis of CD4 T cells was comparable between two groups. Resultingly, the percentages of CD8 T cells in recipients of MyD88 KO T cells were significantly lower (P<.01). We next examined the effects of MyD88 signaling in donor T cells on cytolytic activity to host antigens. Splenocytes harvested from WT mice showed stronger cytolytic activity against P815 targets compared to those from MyD88 KO mice (P<.01). After allogeneic mixed leukocyte reaction, responder T cells from MyD88 KO mice showed markedly reduced IFN-γ, MCP-1 and IL-17A production with a significant augmentation in IL-10 secretion. We further evaluated the effect of T-cell MyD88 deficiency on GVL mediated by the intensity of total body irradiation (TBI) conditioning (1300 vs. 900 cGy, Exp Hematol 2011; 39: 1018–29). Enhanced GVL in the allogeneic recipients receiving 1300 cGy TBI was not shown in the recipients of MyD88 KO T cells.In summary, these results highlight a critical role for MyD88 signaling in T-cell activation and cytotoxicity, offering the opportunity for improving GVL activity by targeting TLR-MyD88 signaling within donor T cells. Furthermore, these data demonstrated that MyD88 deficiency in T cells can impair cytolytic function or subsequent GVL activity of CD8 T cells without significant change in the severity of CD4-dependent GVHD. This difference is attributed to the fact that MyD88 deficiency in T cells causes an enhanced apoptosis of donor CD8 T cells but not donor CD4 T cells in vivo after HSCT. Disclosures:No relevant conflicts of interest to declare.

Donor–Recipient Sex Mismatch in Kidney Transplantation

The lack of reliable human proxies for minor (ie, non-HLA) histocompatibility loci hampers the ability to leverage these factors toward improving transplant outcomes. Despite conflicting reports of the effect of donor-recipient sex mismatch on renal allografts, the association between acute rejection of renal allografts and the development of human alloantibodies to the male H-Y antigen suggested to us that donor-recipient sex mismatch deserved re-evaluation. To evaluate whether the relationships between donor sex and allograft failure differed by recipient sex. We studied recipients of deceased-donor (n = 125,369) and living-donor (n = 63,139) transplants in the United States Renal Data System. Using Cox proportional hazards models stratified by donor type, we estimated the association between donor-recipient sex mismatch and death-censored allograft failure with adjustment for known risk factors, with and without the use of multiple imputation methods to account for potential bias and/or loss of efficiency due to missing data. The advantage afforded by male donor kidneys was more pronounced among male than among female recipients (8% vs 2% relative risk reduction; interaction P < 0.01). This difference is of the order of magnitude of several other risk factors affecting donor selection decisions. Donor-recipient sex mismatch affects renal allograft survival in a direction consistent with immune responses to sexually determined minor histocompatibility antigens. Our study provides a paradigm for clinical detection of markers for minor histocompatibility loci.

MyD88-Dependent Expansion of An Immature GR-1+CD11b+ Population From Donor Bone Marrow Reduces Early Mortality After Allogeneic Hematopoietic Stem Cell Transplantation,

Abstract 4011Graft-versus-host disease (GVHD) is a common complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells with anti-inflammatory activity. MyD88 is a cytoplasmic adaptor molecule essential for integrating and transducing the signals generated by the toll-like receptor (TLR) family. Activation of inflammatory signaling through MyD88, presumably through ligation of multiple TLRs, plays a key role in the expansion of MDSCs.We therefore investigated how the MyD88-dependent expansion of MDSCs from donor bone marrow (BM) contributes to protection of acute GVHD. To test this, we employed an intestinal GVHD murine model, C57BL/6 (H-2b) → B6D2F1 (H-2b/d), which differs at major and minor histocompatibility loci. Lethally irradiated recipient mice were transplanted with wild-type (WT) or MyD88 knock out (KO) mice T cell-depleted (TCD)-BM together with WT spleen T cells. Morbidity and mortality of GVHD was significantly worse in recipients of MyD88 KO TCD-BM with higher intestinal pathologic grading. Animals that underwent syngeneic HSCT did not show early mortality regardless of presence of MyD88 in BM, which ruled out myelosuppression-associated toxicity. The expression of Gr-1+CD11b+ in blood, mesenteric lymph nodes and liver on day 13 was significantly reduced in the recipients of MyD88 KO TCD-BM compared with those of WT TCD-BM while the percentage of donor T cells infiltrating colon and liver was significantly higher. In parallel, the percentages of donor T cells to undergo apoptosis in response to alloantigens in vivo were significantly decreased in recipients of MyD88 KO TCD-BM. Injection of MDSCs from BM of non-tumor bearing donor markedly inhibited GVHD lethality in recipients of MyD88 KO TCD-BM. Moreover, in vivo administration of lipopolysaccharide (LPS), a TLR ligand, to donor mice expanded GR-1+CD11b+ in BM with enhanced expression of MyD88 mRNA. Recipients of TCD-BM from WT mice injected LPS showed attenuated GVHD severity as measured by weight loss and survival compared to those of TCD-BM from WT mice injected diluent.In summary, MyD88-dependent expansion of GR-1+CD11b+ population from donor TCD-BM appears to be critical for survival after allo-HSCT. Incomplete expansion of GR-1+CD11b+ population in target organs correlates with decreased apoptosis and increased infiltration of donor T cells into the target organs. Disclosures:No relevant conflicts of interest to declare.

Effects of Pravastatin on Murine Chronic Graft-Versus-Host Disease

Chronic graft-versus-host disease (CGVHD) is a serious and increasingly common complication after allogeneic (allo) hematopoietic stem-cell transplantation, but currently available therapies have demonstrated limited efficacy. Furthermore, the statins have been reported to be effective in various immune-mediated disease models, but their therapeutic potentials versus CGVHD have not been determined. We used a B10.D2→BALB/c model of CGVHD, which differs at minor histocompatibility loci, to address the therapeutic effect of statins on the development of CGVHD. Pravastatin (PST, 30 mg/kg/day) was intraperitoneally injected for 5 days per week from the day of transplantation until 4 weeks after allo hematopoietic stem-cell transplantation. The onset of clinical cutaneous GVHD was significantly slower in PST-treated recipients than in allo-controls (36 days vs. 25 days, respectively, P<0.05), and pathologic changes in skin disease confirmed this clinical result. Animals injected with PST showed less submucosal fibrosis in lungs than allo-controls. In addition, collagen deposition in skin and lungs was markedly attenuated by PST treatment. PST also significantly reduced protein concentrations and numbers of inflammatory and epithelial cells in bronchoalveolar lavage fluid. Significantly lower numbers of donor CD11b and CD4, but not CD8 cells, were observed in skin and bronchoalveolar lavage fluid after PST treatment. The protein concentrations of monocyte chemoattractant protein-1 (MCP-1) and regulated on activation normal T cell expressed and secreted (RANTES) in skin and lungs were substantially reduced in PST-treated animals when compared with allo-controls. This study suggests that the CGVHD-protecting effect of PST involves the down-regulation of chemokines and the reduction of collagen synthesis.

The Role of Pretransplant Conditioning On Graft-Versus-Leukemia Effects: Migration Matters.

Abstract 3563Poster Board III-500The therapeutic potential of allogeneic hematopoietic stem cell transplantation (HSCT) relies on the graft-versus-leukemia (GVL) effect to eradicate residual tumor cells by immunologic mechanisms. However, the relationship of conditioning intensity to GVL effect has not been clearly established independent of immunosuppression or the tolerance induced by mixed donor-host chimerism. Using a murine allogeneic HSCT model, we have compared two total body irradiation (TBI) doses (1,300 vs. 900 cGy), both of which provided complete donor engraftment and elimination of host lympho-hematopoetic cells.We used C57BL/6 (H-2b) → B6D2F1 (H-2b/d) model of GVHD, which differ at major and minor histocompatibility loci, to address the role of conditioning intensity on the GVL effect. Lethally irradiated (either 900 or 1300 cGy) recipient mice were transplanted with either C57BL/6 (allogeneic) or B6D2F1 (syngeneic) bone marrow (5 × 106) and spleen T cells (1 × 106) on day 0 and then P815 (H-2d) mastocytoma cells (1 × 106) injected subcutaneously on day 1 to generate a GVL model.As expected, GVHD morbidity after the higher TBI dose was aggravated compared to the lower TBI dose (P<.05). Among the syngeneic recipients, the injection of P815 cells into the recipient skin led to progressive tumor growth and death of about 100% 21 days after transplant regardless of the TBI dose. In contrast, tumor growth was remarkably suppressed and tumor death was not observed in the allogeneic recipients. Surprisingly, tumors in the allogeneic recipients receiving 1300 cGy TBI exhibited markedly delayed growth in vivo compared to those with 900 cGy (tumor volume on day 42, 428 vs. 8735mm3, P<.01), which was associated with an increase in the in vivo cytotoxicity using comparing the clearance of infused allogeneic B cells labeled with CFSE reflecting the enhanced alloimmune reactivity. To ask whether the diminished GVL effect after the lower TBI dose was due to reduced production of inflammatory cytokines, we measured the levels of TNF-α or IFN-γ in recipient sera on days 6, 28 and 42 after transplantation and did not find any significant difference according to the intensity of radiation dose (P>.05). In parallel, the in vitro P815-specific TNF-α or IFN-γ responses of splenocytes were comparable between the two doses. The percentages of donor T cells to undergo proliferation or apoptosis in response to alloantigens in vivo between the two TBI doses also were comparable (P>.05). Collectively, these data indicate that the impaired ability of alloreacive T cells to inhibit tumor growth after the lower TBI dose was not attributed to an intrinsic defect in T-cell expansion and activation. We next analyzed the spleen for the number of donor CD4+ and CD8+ T cells and observed no difference between the two TBI doses. In contrast to spleen, the number of CD8+ but not CD4+ T cells from the recipients that had received 1300 cGy was significantly increased in the skin (P<05). The effector function of donor CD8+ and CD4+ cells in both spleen and tumor tissue was examined by intracellular staining for IFN-γ. In the spleen, the percentages of CD8+ and CD4+ T cells expressing IFN-γ were not different between the two TBI doses. (5.9% vs 4.8%, P>.05, and 7.6% vs. 6.5%, P>.05 respectively) By contrast, 45.5% and 50.3% of CD8+ and CD4+ T cells, respectively, isolated from the tumor tissue of recipients receiving the higher TBI dose were IFN-γ; secreting cells, whereas only 25.5% and 16.3% of those cells from the tumor tissue of recipients treated with the lower dose showed this phenotype (P<.01 and <.05, respectively). After the higher TBI dose, secondary lymphoid organ homing receptors including CD62L and CCR7 were down-regulated on donor CD8+ T cells while CD44 expression was up-regulated compared to the lower TBI dose, which may facilitate migration to the tumor sites.In summary, the higher TBI dose (1300 vs. 900 cGy) resulted in significantly enhanced GVL effect, and the alterations in effector T cell trafficking into tumor tissue are the most likely mechanism. Moreover, T-cell activation and function were largely comparable between these conditioning regimens. This provides the rationale for targeting T cell trafficking by inflammation, possibly in combination with integrin or chemokine receptor agonists as a new therapeutic approach in leukemia relapse after allogeneic HSCT. Disclosures:No relevant conflicts of interest to declare.

Role of perforin-mediated cell apoptosis in murine models of infusion-induced bone marrow failure

To investigate the role of perforin-mediated cell apoptosis in murine models of immune-mediated bone marrow (BM) failure. We compared C57BL/6J (B6) mice carrying a perforin gene deletion (Prf(-/-)) with wild-type (WT) controls for cellular composition in lymphohematopoietic tissues. Lymph node (LN) cells from Prf(-/-) mice were coincubated with BM cells from B10-H2(b)/LilMcdJ (C.B10) mice in an apoptosis assay in vitro. We then infused Prf(-/-) and WT B6 LN cells into sublethally irradiated C.B10 and CByB6F1 recipients with mismatches at the minor and major histocompatibility loci, respectively, in order to induce BM failure. Cellular composition was analyzed by flow cytometry. Prf(-/-) mice showed normal lymphoid cell composition, but Prf(-/-) LN cells had reduced ability to induce C.B10 BM cell apoptosis in vitro. Infusion of 5 to 10 x 10(6) Prf(-/-) LN cells produced obvious BM failure in C.B10 and CByB6F1 recipients; pancytopenia and BM hypocellularity were only slightly less severe than those caused by infusion of 5 x 10(6) WT B6 LN cells. Infused Prf(-/-) LN cells showed less T-cell expansion, normal T-cell activation, and higher proportions of T cells expressing gamma-interferon, tissue necrosis factor-alpha, and Fas ligand CD178, in comparison to infused WT B6 LN cells. Fas expression was equally high in residual BM cells in recipient of both Prf(-/-) and B6 LN cells. Perforin deficiency alters T-cell expansion but upregulates T-cell Fas ligand expression. Perforin-mediated cell death appears to play a minor role in mouse models of immune-mediated BM failure.

Preemptive HMG-CoA Reductase Inhibition Significantly Attenuates a Murine Chronic Graft-Versus-Host Disease

Chronic graft-versus-host disease (cGVHD) is a serious and increasingly common complication of allogeneic stem cell transplantation. Treatment of cGVHD remains a large challenge. Even though cGVHD differs among patients, the clinical complications of cGVHD often include fibrosis and scleroderma-like changes. A murine sclerodermatous graft-versus-host disease (Scl GVHD) model sharing critical characteristics with human cGVHD is characterized by skin thickening and lung fibrosis. Statins, 3-hydroxy-3-methyl-CoA (HMG-CoA) reductase inhibitors which are a class of cholesterol lowering drugs decreasing mortality from cardiovascular disease and stroke, have strong immunomodulatory effects on antigen-presenting cells and T cells interfering with synthesis of L-mevalonate and its downstream isoprenoid metabolites. It has been demonstrated that some statins inhibit the production of proinflammatory cytokines and improved chronic inflammatory disorders. We used B10.D2 → BALB/c model of cGVHD, which differ at minor histocompatibility loci, to address the therapeutic effect of statins on the development of cGVHD. Lethally irradiated (700 cGy) BALB/c mice were transplanted with either B10.D2 (allogeneic) or BALB/c (syngeneic) bone marrow (1.5 × 106) and spleen cells (3 × 106) to generate Scl GVHD. We have noted skin thickening as early as day 14, however 28 days was usually required to observe significant skin thickening in allogeneic recipients with Scl GVHD. The skin thickenings were still lingering on but weaken as late as 56 days post-alloSCT, whereas the loss of normal lacy alveolar pattern of lungs remained constant up to that time. Syngeneic recipients did not show any significant changes in each organ. In vivo treatment of pravastatin (30 mg/kg/day, intraperitoneally) for 10 days early after transplant led to greater suppression of the incidence and severity of clinical skin cGVHD compared with allogeneic controls injected with diluent. The occurrence of clinical cutaneous GVHD was significantly slower in onset in the recipients of pravastatin treatment than it in the control animals (36 days vs. 25 days, respectively, P<0.05) Blinded pathologic scoring of skin disease on day 28 confirmed the clinical result. Skin from pravastatin recipients had an average pathology score of 2.5 ± 0.3 compared with 6.9 ± 0.7 for the allogeneic controls (P<0.01). To qualify the cells infiltrating skin or lungs in early Scl GVHD, we examined the number of CD11b+ or CD3+ cells isolated from each organ of the animals with Scl GVHD with or without pravastatin treatment on day 14, 28 and 56 post-alloSCT. Compared with the allogeneic controls with diluent treatment, the injection of pravastatin significantly reduced the number of cells infiltrating each organ on day 14; for bronchoalveolar lavage fluid 3.2 ± 0.3 ×104 vs. 2.2 ± 0.3 ×104 (P<0.05, CD11b+) and 1.3 ± 0.1 ×104 vs. 0.33 ± 0.1 ×104 (P<0.05, CD3+), for skin 56 ± 4.3 ×104 vs. 10 ± 2.3 ×104 (P<0.01, CD11b+) and 12 ± 3.1 ×104 vs. 3 ± 1.8 ×104 (P<0.01, CD3+). On days 28 and 56, the infiltrating cells were also significantly reduced in the animals treated with pravastatin (data not shown). To test whether statins might produce an effect on attracting these monocytes to skin or lungs by influencing the chemokine expression, we performed semiquantitative RT-PCR analyses and ELISA on day 14 to examine the expression of MCP-1 and RANTES in skin and lungs from Scl GVHD animals treated by either pravastatin or the diluent. MCP-1 and RANTES mRNA levels as well as protein concentrations from each organ were significantly reduced in recipients treated with pravastatins compared to the allogeneic controls. In conclusion, preemptive HMG CoA reductase inhibition prevented murine Scl GVHD by effectively blocking the influx of monocytes into target organs and by down-regulating the expression of MCP-1 and RANTES, thereby reducing new collagen synthesis. The Scl GVHD model is valuable for testing the effect of statins on early fibrosing diseases, and chemokines may be the potential targets in cGVHD protection effect of statins.

Fas- and Fas Ligand Rather Than Perforin-Mediated Apoptosis Has a Dominant Role in Target Cell Death in Murine Models of Immune- Mediated Bone Marrow Failure

Fas-Fas ligand and perforin-granzyme are two important cell death pathways associated with cytotoxic T cell induced target cell apoptosis. In patients with immune-mediated aplastic anemia, the development of bone marrow (BM) failure is associated with up-regulation in Fas ligand expression on effector cytotoxic T cells and elevated Fas expression on target BM cells. In some aplastic anemia patients, peripheral blood T lymphocytes also carry polymorphisms in the perforin gene which have been associated with familial hemophagocytosis. These findings suggested that Fas ligand/Fas might be the key signaling molecules mediating cell destruction while perforin might also play a role in the development of BM failure in patients with aplastic anemia. We have modeled immune-mediated BM failure in the mouse by infusing allogeneic lymph node (LN) cells from C57BL/6 (B6) donors into sublethally-irradiated CByB6F1 and C.B10 recipients that are mismatched at either major histocompatibility (MHC) or minor histocompatibility (minor-H) loci. Expansion and activation of allogeneic T cells results in increased production of the inflammatory cytokines gamma interferon and tissue necrosis factor alpha in recipient BM, massive BM cell destruction, severe marrow hypoplasia, and fatal pancytopenia. In the current study, we directly tested the roles of Fas, Fas ligand and perforin in the development of BM failure by using murine models with spontaneous mutations at the lymphoproliferation (lpr) and generalized lymphoproliferative disease (gld) loci, or with germline deletion of the gene perforin (prf−/−). Fas and Fas ligand-deficient lpr and gld mutant mice had no evidence of hematopoietic deficiency despite their autoimmune environment and marked lymphoproliferation. LN cells from lpr and gld mice caused significantly less apoptosis to minor-H mismatched C.B10 BM cells when co-cultured in a cytotoxicity assay in vitro, in comparison to LN cells from wild-type B6 mice. Infusion of lpr, gld, and B6 donor LN cells into sub-lethally irradiated CB10 recipients all caused massive T cell expansion in recipient BM with high level expression of CD11a, indicative of T cell activation, but only B6 LN cells caused severe BM destruction. In contrast, recipients of lpr and gld LN cells had only mild to moderate pancytopenia and marrow hypocellularity. We inferred from these results that disruption of the Fas ligand/Fas signaling pathway effectively abrogated immune mediated marrow destruction. To test the role of perforin in BM failure, we first analyzed prf−/−- mice and found no obvious change in cellular composition in lymphohematopoietic tissues in comparison to wild-type B6 controls. LN cells from prf−/− mice showed reduced ability to induce C.B10 BM cell apoptosis in an in vitro cytotoxicity assay when compared to wild-type B6 LN cells. Infusion of 5–10 million prf−/− LN cells into CByB6F1 and C.B10 recipients produced obvious BM failure in both recipient types with pancytopenia and marrow hypoplasia about 80–90% as severe as in control recipients of 5 million B6 LN cells. In both CByB6F1 and C.B10 recipients, infused prf−/− LN cells resulted in less T cell expansion, a similar level of T cell activation, higher proportions of T cells containing gamma-interferon and tissue necrosis factor-alpha, and a higher proportion of T cells expressing the Fas ligand CD178, in comparison to the infused B6 LN cells. We conclude that Fas-Fas ligand-mediated transmembrane signaling provides the major cell death pathway, while perforin-granzyme-mediated exocytosis plays a minor role, in BM cell destruction in animal models of immune-mediated BM failure.

Cutaneous Gene Expression by DNA Microarray in Murine Sclerodermatous Graft-Versus-Host Disease, a Model for Human Scleroderma

The molecular mechanisms governing skin fibrosis in murine sclerodermatous graft-versus-host disease (Scl GVHD) are not known. We used Affymetrix DNA microarrays representing >14,000 mouse genes to characterize global gene expression in skin during development of Scl GVHD in lethally irradiated BALB/c (H-2d) mice transplanted with B10.D2 (H-2d) bone marrow and spleen cells. These mice develop skin thickening, whereas control mice transplanted with syngeneic BALB/c (H-2d) bone marrow and spleen cells do not develop disease. We found consistent differences between mice with Scl GVHD and controls in cytokine messenger RNAs (mRNAs) for both Th1-like (IFN-gamma) and Th2-like (IL-6, Il-10, and IL-13) inflammatory patterns. mRNAs for chemokines CCL2, CCL5, CCL17, IFN-gamma inducible chemokines (CXCL9/Mig, CXCL10/IP-10, and CXCL11/I-TAC), and for growth factors such as platelet-derived growth factor-c, connective tissue growth factor, fibroblast growth factor 1, epidermal growth factor, nerve growth factor-beta, vascular endothelial growth factor (VEGF)-alpha, and VEGF-beta were elevated, similar to human scleroderma. mRNAs for cell adhesion molecules, such as L-selectin (selectin lymphocyte), P-selectin (selectin platelet), E-selectin (selectin endothelium), and vascular cell adhesion molecule 1, were also upregulated. In separate experiments, we confirmed the increased synthesis of IFN-gamma and IL-2, unchanged IL-10, and absence of tumor necrosis factor-alpha, and IL-4 proteins by flow cytometry of isolated skin T cells. These constellations of immunologic changes provide a "fingerprint" for fibrosing autoimmune disease. They are useful to understand the pathogenesis of Scl GVHD, to identify markers for early diagnosis of disease, and to devise more effective strategies for intervention in early scleroderma and Scl GVHD.

SiPep: a system for the prediction of tissue‐specific minor histocompatibility antigens

Approximately 50 years ago it was found that inbred strains of mice were able to reject tumours and skin grafts from major histocompatibility complex (MHC) identical donors. They proposed that additional transplantation antigens must exist outside the MHC. These were described as minor histocompatibility antigens (mHAgs). Since then, related studies in humans have identified 16 human mHAgs. The aim of this work is to increase the number of known mHAgs by prediction of candidate minor histocompatibility loci by identifying coding single nucleotide polymorphisms (SNPs) where the amino acid variation lies within an MHC-binding peptide and alters the ability of that peptide to bind. We have developed an algorithm called SiPep which uses peptide sequences derived from the flanking regions of known non-synonymous SNPs, various MHC-binding and proteolytic cleavage evaluation methods and protein expression data to predict mHAgs. We have processed 45094 SNPs using the SiPep algorithm and have stored the results in a database called SNPBinder. The facilities to process submitted proteins through the SiPep algorithm as well as the SNPBinder database are available to the public. A set of peptides that are predicted as possible mHAgs by the SiPep algorithm have been tested using refolding assays and gel filtration and the results are presented in this paper. The SiPep tools and SNPBinder database are available free of charge via the internet. An HTML interface providing search facilities can be found at the following address: http://www.sipep.org/.

Experimental model for graft -versus-host disease in rat limb allotransplantation

Graft versus-host disease (GVHD) is a potentially fatal immune-regulated injury occurring unpredictably in solid organ and bone marrow transplant recipients. This model attempts the incidence of potentially lethal GVHD in limb transplants. Two inbred rat strains ACI and Lewis with a strong major and minor histocompatibility locus mismatch were studied. The perspectives of this model are study the incidence of GVHD in limb transplant, determine the best model for the study of the GVHD in limb transplant and prevention and treatment concerning the GVHD.