Acute Graft-versus-host Disease Model Research Articles

Inducible Costimulator Gene Transduced Bone Marrow Derived Mesenchymal Stem Cells Attenuate the Severity of Acute Graft-Versus-Host Disease in a Mouse Haploidentical Model.

AbstractAbstract 2996It is reported that both block of costimulator (ICOS) or its ligand signal pathway and transfusion of mesenchymal stem cells (MSCs) can reduce the severity of acute graft-versus-host disease (GvHD). Using a MHC-haploidentical murine model of acute GvHD, we investigated the effects of MSC-ICOS-EGFP, bone marrow derived MSCs transduced with ICOS-gene using an adenoviral vector, on acute GvHD. Lethally irradiated CB6F1 mice were transplanted with bone marrow cells and spleen cells from C57BL/6 mice, and injected with MSC-ICOS-EGFP, MSCs, ICOS-Ig fusion protein or the diluent (control), respectively. Survival in recipients of MSC-ICOS-EGFP was significantly higher than that in GVHD group (74.29±7.39% vs 0, P=0.000, n=35), and also higher than in MSCs, ICOS-Ig, or MSCs+ICOS-Ig groups (74.29±7.39% vs 42.86±8.36% or 48.57±8.45% or 50.43±8.45%, P=0.0039 or 0.0323 or 0.0418, n=35). The lower mortality in recipients of MSC-ICOS-EGFP was related to lower incidence of acute GvHD. MSC-ICOS-EGFP can reduce the number of CD4+T cells in the early stage of GvHD due to the increased apoptosis, and it can produce CD4+CD25+Treg cells meanwhile. The effect on GvHD in recipient of MSC-ICOS-EGFP was also associated with higher serum levels of IL-4, IL-10 and lower levels of IFN-γ, IL-2, IL-12, IL-17A, as well as inducing expression of GATA-3, STAT6 transcription factors while inhibiting expression of T-bet, STAT4, ROR-rt in spleen. In vitro, the CD4+T cells of donor were sharply inhibited by MSC-ICOS-EGFP and the B7h blockade. In MLRs we can also detect lower levels of IFN-γ, IL-2and higher levels of IL-4, IL-10. These data indicate that MSC-ICOS-EGFP is a more prospective strategy for acute GvHD prevention, which had a synergism effect between MSCs and ICOS-Ig. The mechanisms of MSC-ICOS-EGFP on acute GvHD were closely associated with modulation of CD4+T cells, CD4+CD25+Tregs, and regulation of the Th1/Th2/Th17 balance in the early stage of GvHD. Disclosures:No relevant conflicts of interest to declare.

TH17 Cells Are Critical for Skin-Specific Pathological Injury in Acute Graft-Versus-Host Disease

Interleukin-17 (IL-17), which is important for host defens, has been implicated in autoimmune and chronic inflammatory diseases. As knockout mice lack IL-17 expression in δγT, NKT-like cells, studies investigating the association between TH17 cells and cutaneous graft-versus-host disease (GVHD) in animal models have reported conflicting results. To determine the role of TH17 cells in cutaneous GVHD, we developed an acute GVHD model using C57BL/6(H-2b) donors to BABL/c (H-2d) recipients. Blood samples and skin were examined for inflammation and infiltrating cells using histology and fluorescence-activated cell sorter (FACS) on days 6 and 15 after bone marrow transplantation. We found donor T cells to mediate severe cutaneous inflammation, which was ameliorater by administration of halofuginone (HF) to the recipients. Mechanistically, we demonstrate the severe tissue damage during this disorder to be associated with the production of IL-17 and the expansion of IL-17-producing CD4+ cells. Specific inhibition of TH17 differentiation and function by HF reduced disease severity. Thus, TH17 cells are sufficient to induce acute cutaneous GVHD.

Inhibition of Cathepsin S Reduces Allogeneic T Cell Priming but Not Graft-versus-Host Disease Against Minor Histocompatibility Antigens

Cathepsin (Cathepsin) S, L, and B proteases mediate antigen presentation on major histocompatibility complex (MHC) class II by degrading the invariant chain Ii, which blocks peptide loading. The ability of the Cathepsin S inhibitor LHVS (morpholinurea-leucine-homophenylalanine-vinylsulfone phenyl) to impede antigen presentation has led its development as a therapy for autoimmune diseases. There is substantial evidence that donor T cell recognition of host minor histocompatibility antigens (miHA) and subsequent destruction of host tissue mediates graft-versus-host disease (GVHD). We hypothesized that enzymes involved in antigen presentation may play a role in the development of GVHD. Using the C57BL/6 → BALB.B minor mismatch acute GVHD (aGVHD) model, we found that the cathepsin S activity of spleens from allogenetically transplanted mice were significantly increased 1 week after transplantation compared with syngeneic mice. Although LHVS decreased T cell priming responses against both single OVA antigen and miHA invitro, LHVS did not reduce the severity of aGVHD. In fact, LHVS exacerbated a CD4(+)-T cell-dependent model of GVHD similar to chronic GVHD. This suggests that cytokines rather than T cells may mediate much of the damage in the aGVHD model and that therapeutics based on inhibition of antigen presentation for GVHD must be approached with caution.

Immunomodulatory Effects of Palifermin (Recombinant Human Keratinocyte Growth Factor) in an SLE‐Like Model of Chronic Graft‐Versus‐Host Disease

Keratinocyte growth factor (KGF) promotes epithelial cell proliferation and survival. Recombinant human KGF, also known as palifermin, protects epithelial cells from injury induced by chemicals, irradiation and acute murine graft-versus-host disease (GVHD). Findings from our studies and others have shown that palifermin also has immunomodulatory properties. In a model of acute GVHD, we showed that it shifts the immune response from one in which Th1 cytokines dominate to mixed Th1 and Th2 cytokine profile. Using the DBA/2→(C57BL/6 × DBA/2)F(1)-hybrid model of chronic, systemic lupus erythematosus-like GVHD, we showed that palifermin treatment is associated with higher levels of Th2 cytokines, the production of anti-nuclear antibodies, cryoglobulinemia and the development of more severe pathological changes in the kidney. The aim of our current study was to gain a better understanding of the immunobiology of KGF by further characterizing the palifermin-mediated effects in this model of chronic GVHD. Because the pathological changes we observed resemble those seen in thymic stromal lymphopoietin (TSLP) transgenic mice, we had originally hypothesized that palifermin might augment TSLP levels. Surprisingly, we did not observe an increase in thymic TSLP mRNA expression in palifermin-treated recipients. We did, however, observe some differences in the percentages of CD4(+) CD25(+) Foxp3(+) regulatory T cells in the spleen at some time points in palifermin-treated recipients. Most importantly, we found that TGFβ levels were higher in palifermin-treated recipients early in the GVH reaction, raising the possibility that KGF might indirectly induce the development of fibrosis and glomerulonephritis through a pathway involving TGFβ.

Both Tissue-Derived and Bone Marrow-Derived Host IL-17 Producing Cells Are Required for Preventing Acute Graft-Versus-Host Disease

Abstract 2970Th17 is a newly identified T cell lineage, which secretes the proinflammatory cytokine IL-17. Th17 have been shown to play a crucial role in some immune-mediated diseases. We have reported that host-derived IL-17 has a protective effect against acute graft-versus-host disease (GVHD). In contrast, donor-derived IL-17 exacerbates chronic GVHD in rodent models. Briefly, in acute GVHD model, lethally irradiated IL-17 knockout (KO) recipient mice receiving allogeneic BM with low dose splenocytes developed more severe acute gut GVHD compared to wild type (WT) host mice and finally half of them died (p<0.05). To demonstrate the mechanisms of protective role of host derived IL-17 against acute GVHD in further detail, first we determined in vivo gut epithelial permeability by using FITC-dextran. No significant difference was observed between WT mice and IL-17KO mice, even in GVHD induced mice. Next, to exclude the possibility that alloreactivity of host IL-17KO derived mature dendritic cells (DCs) could be much more than that of WT DCs, mixed leukocyte reaction (MLR) was performed using stimulators from WT or IL-17KO mature DCs and responders from WT allogeneic splenocytes. There was no significant difference between WT mature DCs and IL-17KO mature DCs in thymidine uptake and percentage of responder cells producing IFN-γ or TNF-α. However, when DCs were cultured from BM cells with GM-CSF, co-stimulatory molecules such as CD80 and CD86 were expressed much stronger and from earlier time point on IL-17KO DCs compared with WT DCs. This suggested that DCs from IL-17KO tend to be activated much easier, resulting in much severe GVHD. In addition, when recombinant IL-17 was added into MLR mixture (Stimulator: Balb/c IL-17KO mature DCs, Responder: B6 IL-17KO splenocytes), proliferation was significantly decreased, demonstrating that IL-17 has inhibitory effect on alloreaciton. Next, much higher number of infiltrated monocyte/macrophage cluster was observed in spleen and gut in IL-17KO host mice. Residual host-typed peritoneal macrophages in IL-17KO host mice were highly activated with the expression of TNF-α, while activation of donor-typed macrophages was much less with the production of anti-inflammatory cytokine IL-10. Activated monocytes/macrophages in IL-17KO recipients might migrate into inflamed tissue much easier, partially because lack of IL-17 dose not block some macrophage chemotactic factors such as IP-10, RANTES. Finally to explore which host IL-17 producing cells are necessary for protection from acute GVHD, that is, BM-derived IL-17 producing cells or tissue derived IL-17 producing cells such as Paneth cells in the gut, [IL-17KO→WT], [WT→IL-17KO], [WT→WT], and [IL-17KO→IL-17KO] chimeric mice (Balb/c background) were created by reconstituting lethally irradiated WT or IL-17 KO Balb/c (H-2d) with BM cells from WT or IL-17 KO Balb/c mice. Four months later, chimeras were re-irradiated with 8Gy TBI and injected with WT C57BL/6(H-2b) BM plus low dose splenocytes to induce acute GVHD. Although [WT→WT] chimeric recipients were all alive, all [IL-17KO→IL-17KO] chimeric mice died of severe GVHD. In both [WT→IL-17KO] and [IL-17KO→WT] chimeras, half of them died with less GVHD compared to [IL-17KO→IL-17KO] recipients as shown below. This result indicated that as well as tissue derived IL-17, host BM derived IL-17 is necessary for protection from acute GVHD. In conclusion, host-derived IL-17 has a protective effect against acute GVHD due to in part by regulating APCs (monocytes/macrophages/DCs) activation and migration into GVHD target tissues. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Donor but Not the Host Derived Interleukin-10 (IL-10) Contributes to Regulation of Graft-Versus-Host Disease (GVHD)

Abstract 2975IL-10 is a key immune-regulatory cytokine and its gene polymorphisms correlate with severity of clinical graft-versus-host (GVHD). IL-10 is made by a variety of donor and host cells, but the functional relevance of its source in acute GVHD is not well understood. We utilized well characterized MHC matched, minor mismatched C3H.SW →B6 model of acute GVHD to first examine the relevance host hematopoietic derived IL-10 on acute GVHD induced by alloreactive donor T effector cells in the absence of donor regulatory T (Tregs) cells. We generated [WTB6→B6-Ly5.2] and [IL-10−/−B6→B6-Ly5.2] chimeras such that only the host hematopoietic derived cells were incapable of producing IL-10 and utilized them as recipients. The chimeras were lethally irradiated and transplanted with 5×106 T cell-depleted (TCD) BM and 2×105 CD8+ T cells from either the allogeneic C3H.SWor the syngeneic B6 donors. All of the allogeneic animals demonstrated similar clinical severity and mortality from GVHD (65% vs 72%, P=NS). Because IL-10 can also be derived from non-hematopoietic cells, we next determined whether the absence of IL-10 secretion by both hematopoietic and non-hematopoietic cells would modulate GVHD severity. To this end, the WT B6 and the IL-10−/− B6 animals were irradiated with 10Gy and transplanted 5×106 T cell-depleted bone marrow (TCDBM) and 2×105 CD8+ T cells from either allogeneic C3H.SW or the syngeneic B6 donors. All of the allogeneic animals demonstrated similar severity and mortality from GVHD regardless of whether all of the host cells can make IL-10 (P=0.1, NS).We next examined relevance of IL-10 deficiency in donor cells. 1×106 CD25+depleted donor T effectors from either WT or IL-10−/− B6 mice were infused along with 5×106 TCDBM from WT B6 into lethally irradiated allogeneic C3H.SW or syngeneic B6 animals. The allogeneic animals that received either WT or IL-10−/− B6 effector cells demonstrated similar GVHD severity and mortality (P=0.6). Further, when T effectors from WT B6 mice were infused along with TCDBM from either WT or IL-10−/− B6 donors, the allogeneic C3H.SW animals once again demonstrated similar GVHD mortality (70% vs 80%, P=NS). Thus deficiency of IL-10 in either host tissues or in the donor grafts did not alter CD25+ depleted donor T effector cell mediated severity of GVHD.We next examined the relevance of the source of IL-10 in donor natural (Treg) mediated regulation of GVHD. Donor CD4+CD25+ Tregs from C3H.SW donors induced equivalent protection from GVHD mortality in both WT and IL-10−/− hosts (90% vs. 100%, P=NS). To examine the relevance of IL-10 derived from Tregs, we transplanted 0.25×106 CD4+CD25+ Tregs from either B6 or IL-10−/− donors along with 1×106 CD25 depleted T effectors (1 :4 ratio) into C3H.SW animals. 100% of the allogeneic animals that received WT donor Tregs survived while only 60% of those that receive IL-10−/− donor Tregs survived (P=0.001). However, when the Il-10−/− donor Treg numbers were increased to 1×106 (1 :1 ratio), all of the allogeneic animals survived (P=0.008) demonstrating that donor Treg derived IL-10 is not obligatory but is necessary for optimal reduction of GVHD.To determine the role of donor BM derived IL-10 in Treg mediated protection from GVHD, Tregs and T effector cells from WT B6 donors (1 :4 ratio) were transplanted along with 5×106 TCD BM from either WT or IL-10−/− B6 donors into lethally irradiated allogeneic C3H.SW animals. The allogeneic recipients of WT BM were protected from GVHD mortality, donor Tregs failed to protect the allogeneic animals that received IL10−/− donor BM (85% vs. 20%, P<0.04) in whom the rate of mortality was similar to the animals that did not receive any donor Tregs (P=0.96). Mechanistic studies demonstrated that donor Tregs continued to express similar levels of foxp3 and expansion early (day 7) after BMT. However the their expansion was significantly reduced at later time-points (day 14 and 21) when the hosts received BM from IL-10−/− donors. Together these data suggest that IL-10 from either host or donors does not contribute to the induction of GVHD severity by the T effector cells. However, only donor BM and donor Treg derived IL-10 is important for donor Treg mediated suppression of GVHD. Disclosures:No relevant conflicts of interest to declare.

Cell Autonomous Role of TGFβ and IL-2 Receptor in the In Vivo Generation of CD4 and CD8 Inducible Regulatory T Cells During Graft-Versus-Host Disease,

Abstract 4015FoxP3+ regulatory T cells (Tregs) suppress graft-versus-host disease (GVHD) while preserving graft-versus-tumor effects, making them an attractive target for GVHD therapy. The donor-derived Treg pool can potentially be derived from expansion of pre-existing natural Tregs (nTregs) or from de novo generation of inducible Tregs (iTregs) from donor conventional T cells (Tconvs) in the transplant recipient. Although the co-adoptive transfer of nTregs or in vitro -derived iTregs has been shown to prevent the development of GVHD, the relative contribution of these two Treg subsets in protection against GVHD has been unclear. To investigate the contribution of the different FoxP3+ Treg subsets, we used a MHC-mismatched mouse model of acute GVHD. Lethally irradiated (500cGy × 2) B6D2F1.SJL (H-2bxd) host mice were injected with T cell-depleted bone marrow cells and FACS-sorted Tconvs (WT or Foxp3-deficient) with or without FACS-sorted Tregs of C57BL/6 (H-2b) mouse origin. Weight loss in mice receiving Foxp3-deficient Tconvs alone was significantly more pronounced compared to other groups. The presence of either donor-derived nTregs or iTregs alone protected against GVHD-induced weight loss but was suboptimal compared to the presence of both donor-derived nTregs and iTregs.Next, we sought to determine how the donor-derived Treg pool was established during acute GVHD and tracked the appearance of Tregs in the secondary lymphoid organs at different time points post transplant. On Day 8 post GVHD induction, ∼5% of the donor-derived CD4+ T cells in the spleen were FoxP3+. We found that the Treg pool was comprised equally of donor-derived nTregs and iTregs. Unexpectedly, we found a significant fraction of CD8+FoxP3+ T cells (1–3% of all CD8+ T cells) in the spleen and in the lymph nodes. These CD8+FoxP3+ T cells representing ∼70% of the iTreg pool on Day 8 post GVHD induction. These CD8+FoxP3+ T cells shared phenotypic markers with their CD4+ counterparts and displayed suppressive activity, suggesting that they were bona fide iTregs. Both CD4+ and CD8+ Tregs expanded in vivo with IL-2 treatment and required IL-2 and TGFβ receptor expression for their generation.In summary, we found that donor derived-iTregs are generated during GVHD and contribute to suppression of acute GVHD induced-weight loss. Surprisingly, CD8+Foxp3+T cells were a major contributor to the donor derived-iTreg pool after transplantation. The generation of CD8+ and CD4+ iTregs occurred at least in part by a cell autonomous IL-2 and TGFβ receptor-dependent mechanism. Thus, our data suggest that in addition to increasing nTregs, concomitant strategies aimed at enhancing the conversion of donor-derived Tconvs to iTregs for example by engaging the IL-2 and TGFβ signaling pathways might be beneficial for the treatment of GVHD. Disclosures:No relevant conflicts of interest to declare.

A Plasmin Inhibitor Prevents Lethal Acute Graft-Versus-Host Disease in Mice

Abstract 1897Proinflammatory cytokines released upon cell damage can cause excessive tissue destruction in acute graft-versus-host disease (GVHD). Certain key cytokines for GVHD like TNF-α and Fas-ligand, are secreted by ectodomain shedding from matrix metalloproteinases (MMPs). Many MMP inhibitors have been developed and used over the last three or four decades, but its widespread use is hampered by severe side effects. We reported that the fibrinolytic system, above all plasmin formation promotes the activation of several MMPs. Therefore we hypothesized that plasmin inhibition might be a novel means to control acute GVHD in part by suppressing MMP and inflammatory cytokine secretion. We found that the fibrinolytic system is activated during the development of acute GVHD in mice and in humans. Here, we examined a novel plasmin inhibitor (YO-2) that inhibits MMP activation and inflammatory cytokines release for lethal acute GVHD model in mice. Administration of YO-2 into (BALB/c × C57BL/6) F1 that received C57BL/6 spleen cells showed markedly reduced mortality and weight loss in association with minimal signs of GVHD pathology in the liver, intestine, and hematopoietic tissues. Furthermore, our results suggest that administaration of YO-2 blocked the processing of inflammatory cytokines through MMP inhibition in vitro and in vivo. These data have major implications for transplantation medicine, as pharmacological inhibition of plasmin does not require the need for intensive immunosuppression. Our results suggest that YO-2 could be a novel molecular therapeutic agent for GVHD. Disclosures:No relevant conflicts of interest to declare.

Protective role of T-bet and Th1 cytokines in pulmonary graft-versus-host disease and peribronchiolar fibrosis.

T-box expressed in T cells (T-bet) is a critical transcription factor for T helper (Th) 1 responses. Although Th1 cells are thought to contribute to certain alloimmune responses, their role in pulmonary graft-versus-host disease (GVHD) is uncertain. We have established a murine model of acute pulmonary GVHD after hematopoietic cell transplant (HCT) and inhaled LPS exposure. We tested the hypothesis that pulmonary GVHD can occur independent of Th1 cells using T-bet-deficient donors. B10.BR(H2(k)) mice underwent allogeneic (Allo) or syngeneic (Syn) HCT with cells from either C57Bl/6J(H2(b)) mice (Allo wild-type [WT] or SynWT) or C57Bl/6J mice lacking T-bet (AlloTbet(-/-) or SynTbet(-/-)). After HCT, mice were exposed daily to aerosolized LPS and subsequently bronchoalveolar lavage and lung tissue were analyzed for cytokines, lymphocytic inflammation, pathology, and fibrosis. Independent of LPS exposure, AlloTbet(-/-) mice developed pulmonary GVHD manifested by lymphocytic inflammation. Furthermore, AlloTbet(-/-) mice developed features of chronic pulmonary GVHD, including increased peribronchiolar fibrosis and collagen content. LPS exposure increased neutrophil recruitment and decreased static compliance in AlloTbet(-/-) mice as compared with LPS-exposed AlloWT mice or LPS-exposed SynTbet(-/-) mice. In addition, LPS-exposed AlloTbet(-/-) mice had increased pulmonary IL-17, IL-13, and Th17 cells, and diminished regulatory T cells compared with the other groups. Our results demonstrate that Th1 cytokines are dispensable in pulmonary GVHD. In the absence of T-bet, there is increased production of Th17 and Th2 cytokines that is associated with peribronchiolar fibrosis and is further enhanced by LPS. These results suggest that the interplay between local innate immunity and non-Th1 T cell subsets contribute to chronic pulmonary GVHD.

Host CD25 +CD4 +Foxp3 + Regulatory T Cells Primed by anti-CD137 mAbs Inhibit Graft-versus-Host Disease

CD25(+)CD4(+)Foxp3(+) regulatory T cells (Tregs) play a pivotal role in the maintenance of self-tolerance and regulation of immune responses. Previous studies have demonstrated that CD137 signals can promote proliferation and survival of Tregs in vitro. Here, we show that in vivo CD137-induced expansion of Tregs in naive mice was dependent upon IL-2 secreted by memory T cells. Tregs primed by anti-CD137 mAbs had a higher immunosuppressive capacity. Preconditioning with anti-CD137 mAbs significantly inhibited graft-versus-host disease (GVHD) in the C57BL/6 → (C57BL/6 × DBA/2) F1 acute GVHD model. In this disease model, a high proportion of host Tregs remained long-term in the recipient spleen, whereas donor hematopoietic cells replaced other host bone marrow-derived cells. Transient depletion of Tregs before transfer of donor cells completely abrogated the inhibitory effect of anti-CD137 mAbs on GVHD. In addition, adoptive transfer of anti-CD137-primed Tregs ameliorated GVHD. Our results demonstrate that it is possible to enhance the survival and/or the immunosuppressive activity of host Tregs in nonmyeloablative GVHD, and that 1 way of accomplishing this is through the prophylactic use of anti-CD137 mAbs in nonmyeloablative GVHD.

An absence of donor TH17 differentiation ameliorates dermal tissue damage

To explore the functions of TH17 cell in cutaneous graft-versus-host disease (GVHD). A model of acute GVHD (aGVHD) was established with a major histocompatibility complex class I/II-disparate allogeneic bone marrow transplantation (BMT). Bone marrow monocytes and splenic T cells from donor C57/BL6 were enriched. The recipient BABL mice were irradiated ((60)Co source) with 7.5 Gy total body irradiation (TBI) and injected with 5 × 10(6) marrow monocytes and 5 × 10(5) T cells. The experimental mice were divided into 3 groups: lethal total body irradiation (TBI); allogeneic bone marrow transplantation (BMT) and recipients of halofuginone (HF). The symptoms of aGVHD were observed daily and detailed histopathologic analyses of recipient skin were performed at Day 6 post-transplantation. And Tri-color flow cytometry (FCM) was performed at Day 6 post-transplantation to measure the levels of interleukin (IL)-17, interferon (IFN)-γ and TH1/TH17. Clinical GVHD symptoms were observed in recipient mice. The administration of HF to lethally irradiated recipients led to very modest GVHD-induced cutaneous changes manifested predominantly by fur loss. However, the experimental animals receiving only allogeneic BMT showed significant fur loss and pathologic skin conditions. Consistent with the clinical evaluations, the histopathologic results demonstrated significantly increased pathologic cutaneous lesions in recipients undergoing only BMT. The median ratios of TH1/TH17 cells were 17.57 and 5.31 in the HF and BMT groups respectively. The difference had statistical significance (P < 0.05). The serum levels of IL-17 were(1.47 ± 0.18) and (2.81 ± 0.19) pg/ml in the TBI and BMT groups respectively (P < 0.05). But IL-17 could not be detected in the HF group. The serum levels of IFN-γ were (3.86 ± 0.32), (42.97 ± 0.42) and (9.89 ± 0.51) pg/ml in the TBI, BMT and HF groups respectively. The inter-group differences had statistical significance (P < 0.05). An absence of TH17 cell may alleviate the cutaneous GVHD but exacerbate the systemic GVHD.

The Protective Role of Host Toll-Like Receptor-4 in Acute Graft-Versus-Host Disease

Mutations in Toll-like receptor (TLR)-4 have been associated with the hyporesponsiveness of macrophages to lipopolysaccharide, possibly reducing the risk of acute graft-versus-host disease (GVHD). However, TLR-4 mutations may also increase the risk of intestinal damage and microbial infection, thereby accelerating acute GVHD. In this study, we investigated the role of TLR-4 in triggering acute GVHD using C3H/HeJ mice with disrupted TLR-4 and C3H/HeN mice with intact TLR-4 as recipients in an acute GVHD model. TLR-4 expression was significantly increased in the intestines and livers from acute GVHD mice. TLR-4-mutant C3H/HeJ hosts that received C57BL/6 (B6) donor cells developed significantly more severe GVHD than TLR-4-intact C3H/HeN hosts receiving B6 donor cells. Antibiotic treatment prolonged the survival of C3H/HeN-host GVHD mice but reduced the survival of C3H/HeJ-host GVHD mice. C3H/HeJ-host GVHD mice showed increased lipopolysaccharide levels in the blood, donor cell and CD68+ cell infiltration, tumor necrosis factor-α mRNA expression, and more apoptotic cells in the intestine compared with C3H/HeN host GVHD mice. In contrast, intestinal cyclooxygenase-2, prostaglandin E2, and hepatocyte growth factor expression in C3H/HeJ-host GVHD mice were significantly decreased compared with C3H/HeN-host GVHD mice. Our results indicated that host TLR-4 is crucial for the induction of tissue protective factors and for protection against intestinal cell apoptosis during acute GVHD.

SiglecH Positive Myeloid Cells Mobilized by Pegylated Murine GM-CSF Have Potent Allo-Suppressive Properties In Vitro.

AbstractAbstract 1459Granulocyte-macrophage colony stimulating factor (GM-CSF) is a growth factor regulating proliferation and differentiation of hematopoietic stem and progenitor cells. Pegylation of murine GM-CSF (peg-mGM) has been shown to prolong its half-life. We have observed that patients transplanted with stem cell products mobilized by GM-CSF have a lower incidence of acute GvHD (Devine et al. BMT 2005), we hypothesized that peg-mGM mobilized stem cell products may be protective from acute GvHD in MHC mismatched murine models.C57/Bl6 (B6) mice treated with peg-mGM (5mcg IP qd × 4) resulted in increased CFU-C by 26.5 fold compared to PBS treated mice (n=6, 95%CI 10.25–42.73). In addition, peg-mGM increased CD4+FoxP3+ Tregs in both blood and spleen by 2 fold compared to PBS (p<0.05). Importantly, the function of these peg-mGM Tregs, as measured in a mixed lymphocyte reaction (MLR) was found to be equivalent to Tregs from PBS treated control mice (t-test, n=3, p=0.27). Furthermore, we have observed an increased expression of Ki67 in murine Tregs in vivo in response to peg-mGM treatment consistent with peripheral expansion of Tregs. We hypothesized that a peg-mGM mobilized product would cause less graft versus host disease (GvHD). We have assessed the in vivo impact of peg-mGM on GvHD, by using the B6 to B6D2F1 acute GvHD model. B6 donors were treated with peg-mGM or PBS for 4 days; on the 5th day splenocytes were isolated. An equivalent dose of CD3+ T-cells along with congenic T cell depleted bone marrow was administered to recipients (n=5/group) that had undergone myeloablative conditioning (11 Gy TBI). We observed less weight loss and improved survival in the peg-mGM treated group (Log-rank chi-square 53.22, df=9, p<0.001). This effect may be explained by an increase in Tregs delivered in the peg-mGM group but also potentially to additional accessory cells that could be present in the graft and mediating an allo-suppressive response. We have phenotyped cells mobilized by peg-mGM and have observed a novel cellular phenotype consistent with a monocyte/macrophage mobilized in the peripheral blood that is SiglecH+, B220-, Ly6c+, CD11b+, GR-1 intermediate, and F4/80+/−. This subset appears within 2 days of the start of peg-mGM treatment and was not observed in mice mobilized with PBS, G-CSF or plerixafor. To evaluate the function of this novel population and its potential suppressor properties we performed an MLR. B6 mice were mobilized with peg-mGM for 4 days, on the 5th day whole splenocytes were isolated and single cell sorted for SiglecH+B220- population or B220+SiglecH- (control cells). These cells were incubated with irradiated Balb/C splenocytes (stimulators) and CFSE labeled congenic B6 CD4+CD25- T-conventional (Tcon) cells (responders) at varying ratios of stimulators:responders:suppresors. Freshly isolated naive Tregs were used as the positive control. At the end of 6 days of incubation wells containing the SiglecH+B220- cells at 1:1:1 or 1:1:2 ratios suppressed allo-reactive Tcon cells as potently as Tregs at a 1:1:1 ratio (t-test, n=3, p=0.49 and p=0.98 respectively) compared to no suppressor controls (t-test, n=3, p=0.02 and p=0.005 respectively)(Figure). Finally, we performed an ELISA for IFN-alpha on these sorted cells, which was not significantly increased (12 pg/mL) after stimulation with the TLR9 agonist, CpG.These data suggest that reduced GvHD observed in mouse and man after infusion of GM-CSF mobilized stem cell products may be due to both in vivo expansion of Tregs and robust (<1% to 15–20%) mobilization of a novel population of myeloid suppressor cells. SiglecH, a member of the CD33 like immunoglobulin superfamily of lectin proteins, may identify a unique myeloid suppressor cell mobilized by GM-CSF in mice. [Display omitted] Disclosures:Schroeder:Genzyme: Research Funding; Celgene: Research Funding.

Roles of Host Nonhematopoietic Cells in Autoimmunity and Donor Cell Engraftment in Graft-versus-host Disease

BackgroundGraft-versus-host disease (GVHD) is initiated when alloreactive donor T cells are primed by host APCs to undergo clonal expansion and maturation. Since there is a controversy regarding the role of nonhematopoietic cells in GVHD, we wanted to investigate the influence of MHC disparity on nonhematopoietic cells on the pathogenesis of GVHD in the MHC-haplomismatched C57BL/6 (H-2b) or DBA/2 (H-2d)→unirradiated (C57BL/6×DBA/2) F1(BDF1; H-2b/d) murine model of acute GVHD (aGVHD) or chronic GVHD (cGVHD).MethodsWe generated (BDF1→C57BL/6), (BDF1→DBA/2), and (BDF1→BDF1) chimeras and examined GVHD-related parameters and donor cell engraftment in those chimeras.ResultsUsing this experimental system, we found that 1) severe aGVHD across MHC Ag barrier depends on the expression of nonhematopoietically rather than hematopoietically derived alloAgs for maximal GVHD manifestations; 2) host APCs were sufficient to break B cell tolerance to self molecules in cGVHD, whereas host APCs were insufficient to induce autoimmunity in aGVHD; 3) donor cell engraftment was greatly enhanced in the host with MHC-matched nonhematopoietic cells.ConclusionTaken together, our results provide an insight into how MHC disparity on GVHD target organs contribute to the pathogenesis of GVHD.

Absence of donor Th17 leads to augmented Th1 differentiation and exacerbated acute graft-versus-host disease

AbstractTh17 is a newly identified T-cell lineage that secretes proinflammatory cytokine IL-17. Th17 cells have been shown to play a critical role in mediating autoimmune diseases such as EAE, colitis, and arthritis, but their role in the pathogenesis of graft-versus-host disease (GVHD) is still unknown. Here we showed that, in an acute GVHD model of C57BL/6 (H-2b) donor to BALB/c (H-2d) recipient, IL-17−/− donor T cells manifested an augmented Th1 differentiation and IFN-γ production and induced exacerbated acute GVHD. Severe tissue damage mediated by IL-17−/− donor T cells was associated with increased Th1 infiltration, up-regulation of chemokine receptors by donor T cells, and enhanced tissue expression of inflammatory chemokines. Administration of recombinant IL-17 and neutralizing IFN-γ in the recipients given IL-17−/− donor cells ameliorated the acute GVHD. Furthermore, the regulation of Th1 differentiation by IL-17 or Th17 may be through its influence on host DCs. Our results indicate that donor Th17 cells can down-regulate Th1 differentiation and ameliorate acute GVHD in allogeneic recipients, and that treatments neutralizing proinflammatory cytokine IL-17 may augment acute GVHD as well as other inflammatory autoimmune diseases.

Murine bone marrow derived stromal progenitor cells fail to prevent or treat acute graft‐versus‐host disease

Human and murine stromal progenitor cells (SPCs) can suppress alloresponse in vitro, suggesting that SPCs may have clinical application toward prevention or treatment of graft-versus-host disease (GVHD). However, th eresults of in vivo studies have been conflicting. This study utilized an established murine model of acute GVHD to assess the ability of bone marrow derived murine SPCs (mSPCs) to prevent or treat GVHD. GVHD was established by transplantation of B6 bone marrow and spleen cells into lethally irradiated (900 cGy) B6 x BALB/c F(1) recipients. mSPCs were administered using various dose and timing protocols designed to either prevent or treat GVHD. After transplantation, mice were monitored daily for weight and survival. Differences in symptom severity were compared using a clinical GVHD scoring system. All GVHD control mice died of lethal GVHD. All groups treated with mSPCs for the prevention of GVHD went on to develop clinical GVHD with no alteration of the disease course or severity compared to controls. Administration of mSPC after the development of GVHD failed to improve the disease course. We conclude that in this model, the ability of SPCs to suppress alloresponse in vitro does not correlate with in vivo prevention or treatment of acute GVHD.

Predominant donor CD103+CD8+ T cell infiltration into the gut epithelium during acute GvHD: a role of gut lymph nodes

The existence of donor effector cell subsets responsible for either gut or skin graft-versus-host disease (GvHD) is still undetermined. We examined the trafficking and role of donor CD8(+) intra-epithelial lymphocytes (IELs) in the gut and skin epithelia concerning alpha E beta 7 integrin (CD103) expression, using a rat acute lethal GvHD model. Most CD103(+) donor cells were CD8(+) and showed a proliferative activity in the target epithelia. On the other hand, activated donor T cells in the host lymphoid tissues did not express CD103, indicating the presence of CD8(+) IEL precursors in the lymphoid tissues that may up-regulate CD103 only after migrating to the target organs. At the late stage of GvHD, while >80% of the donor CD8(+) IELs were CD103(+) in the gut epithelium, both CD4(+) and CD103(+)CD8(+) T cells evenly accumulated in the skin epidermis. The CD103 expression by donor CD8(+) IELs especially in the gut was also correlated with the clinical GvHD manifestations. Furthermore, the selective removal of gut lymph nodes (LNs) but not skin LNs suppressed the infiltration of CD103(+) donor IELs in the gut and alleviated intestinal GvHD. In conclusion, CD103(+)CD8(+) donor T cells predominantly infiltrate into the gut epithelium and are responsible for the manifestations of intestinal GvHD. This pathology is at least partly dependent on the gut LNs.

Anti-Murine Thymocyte Globulin Prevents Acute Graft-Versus-Host Disease and Induces Foxp3+ T Cells in Target Organs.

Rabbit anti-human thymocyte globulins (ATG) (Thymoglobulin®) are widely used for treatment and prevention of solid organ transplant rejection. However, more recently these therapies have been shown to also reduce the severity of graft-versus-host disease (GVHD) following allogeneic stem cell transplantation. However, dose and schedule of ATG administration in the clinic has not been well defined, and thus, the studies reported here were undertaken to explore optimal dosing and timing regimens as well as possible mechanisms of action of anti-thymocyte globulin in an in vivo murine model of GVHD. We demonstrate that a murine version of ATG, rabbit anti-murine thymocyte globulin (mATG), completely inhibits the development of acute GVHD in a model of allosplenocyte transfer into immunodeficient recipient mice (C57BL/6→BALB/c RAG-2−/ −). This protection is observed even when mATG administration is delayed for up to three days following allosplenocyte transfer. Administration of mATG six or more days following induction of GVHD still ameliorates disease in up to 50% of the animals, depending on the timing of mATG treatment. Murine ATG also remains completely protective down to doses of 1mg/kg if administered at the time of allosplenocyte transfer. Although T cell depletion is still observed at this low, but efficacious dose of mATG, we also find significant increases in Foxp3+ CD4+ regulatory T cells in the spleen (30 fold over control) as well as increased Foxp3+ expression in liver and intestines (3 fold over control). These results demonstrate a potent protective effect of murine ATG in this model of acute GVHD and suggest that the induction of regulatory T cells may participate in the protective effects observed.

A Critical Role for STAT-3 Dependent Induction of Indoleamine 2, 3 Dioxygenase in Histone Deacetylase Inhibitors Mediated the Regulation of GVHD.

Histone deacetylase (HDAC) inhibitors reduce experimental acute graft-versus-host disease (GVHD) and recent work by us and others suggest that HDAC inhibitors regulate dendritic cell (DC) function. However, the critical cellular and molecular mechanisms underpinning these observations are not known. We investigated the mechanisms by utilizing two HDAC inhibitors, suberoylanilide hydroxamic acid (SAHA) and ITF 2357. Pretreatment of murine bone marrow (BM) and human peripheral blood mononuclear cell derived DCs with either HDAC inhibtors and stimulated with TLR ligands such as LPS caused a significant reduction in the secretion of TNF-α compared to the untreated controls (P< 0.01). Pre-treatment also significantly reduced the DC mediated in vitro and in vivo stimulation of allogeneic T cell proliferation (P<0.05). SAHA and ITF 2357 increased expression of indoleamine 2, 3-dioxygenase (IDO) at both mRNA and protein levels. Blockade of IDO induction with specific small interfering RNA (siRNA) in the wild type (WT) DCs and those derived from IDO deficient (IDO−/−) animals confirmed a functional role for IDO in the HDAC inhibitor mediated regulation TNFα secretion and allo-T cell proliferation. DNA-protein interaction analysis with ChIP assay demonstrated that both acetylated histone(H) 4 and STAT3 bound to murine IDO promoter. Using TESS DNA soft-wear analysis we found two potential STAT3 binding Gamma Activated Sites (GAS sites) in the IDO promoter and it was recently reported that acetylation of STAT3 is sufficient for its activation (Yuan, et al. Science 2005). We therefore sought to determine whether direct acetylation of STAT3 by the HDAC inhibitors is critical for the induction of IDO in DCs. SAHA or ITF2357 treatment induced acetylation, activation and dimerization of STAT-3 as determined by protein-protein interaction studies. Co-culture studies with JSI-124, an inhibitor of STAT3 signaling, demonstrated that STAT3 is critical for induction of IDO by the HDAC inhibitors. Functional relevance was confirmed by the lack of HDAC inhibitor induced suppression of DC function when co-treated with JSI-124. We next cloned 1500bp DNA fragment upstream of mouse IDO gene start codon and attached it to luciferase gene and peformed mutagenesis studies to evaluate for luciferase activity. Deletion of the GAS regions or treatment with JSI-124 impaired luciferase activity of the IDO promoter constructs demonstrating that STAT3 is necessary and sufficient for transcriptional induction of IDO by the HDAC inhibitors. To specifically address the in vivo relevance of IDO induction by HDAC inhibitors in only the host type DCs, we generated [B6 → B6] and [IDO−/−B6 → B6] BM chimeras and utilized them in a well characterized [BALB/c B6] mouse model of acute GVHD. [B6 → B6] and [IDO−/− B6 → B6] animals received 800 Gy on day −1 and were treated orally with of 50mg/kg of ITF 2357 or diluent on days −1 to +2. Mice were transplanted on day 0 with 3 x 106 T cells and 5 x 106 BM from either syngeneic B6 or allogeneic BALB/c donors. Treatment with ITF 2357 resulted in significantly better survival in the allogeneic [B6 → B6] animals (80% vs. 40%, P < 0.02) but did not confer any survival benefit to the [IDO−/− B6 → B6] animals when compared with diluent treated recipients [20% vs. 30%, P = NS]. Our data thus demonstrate a novel molecular pathway in modulation of GVHD through a STAT3 dependent induction of IDO in the host DCs.

A New Mice Model of Graft Versus Host Disease Based on Chemotherapy Conditioning.

Background: Transplantation related complications such as graft versus host disease (GVHD) are limiting factors for allogeneic haematopoietic stem cell transplantation (HSCT). The majority of mice models of GVHD are based on the use of radiotherapy as a conditioning regimen. However, these models may not explain mechanisms of HSCT related complications following chemotherapy-based conditioning regimen.Aims: The aim of this study was establishment of a mice model of GVHD using the chemotherapy as conditioning regimen.Methods: Recipient female BALB/c (H-2Kd) were conditioned using busulfan (Bu) in total dose of 80 or 100 mg/kg followed by cyclophosphamide (Cy) in total dose of 200 or 300 mg/kg. Twenty millions of bone marrow (BM) cells and 30×106 spleen cells of male C57BL/6 (H-2Kb) were injected into the recipient mice in allogeneic setting. As a control, syngeneic HSCT using female BALB/c as recipients and donors and the same conditioning regimen and cell doses as in allogeneic setting was used. Acute GVHD manifestations and histo-pathological changes in skin, liver and intestine were evaluated at different time points after BMT. The chimerism and tissue mapping were studied by FACS, immunohistochemistry and FISH analysis. Affected skin was examined by FACS analysis to find donor T cells. Cytokines level were measured by using luminex assay.Results: Infusion of 20×106 BM cells and 30×106 spleen cells from C57BL/6 mice to BALB/c mice conditioned with 80 or 100mg/kg Bu plus 200mg/kg Cy induced lethal acute GVHD between days 5–7 post transplantation. Histological and clinical signs of GVHD were found in 85% and 100% of the allogeneic transplanted mice based on the intensity of conditioning. About 35% of mice in allogeneic group died due to GVHD progression. The number of donor T cell in the recipient skin increased with GVHD progression and was correlated to the level of chimerism in the spleen but not in the BM. CD3+ CD8+ T cells infiltration and apoptosis were found in the skin, intestine and liver of mice suffering from GVHD. None of the syngeneic transplanted mice died or developed any sign of GVHD even in long term follow up, and CD3+CD8+ T cells were absent in their tissue samples. Increasing the dose of Cy in conditioning regimen in allogeneic transplanted mice increased mortality of animals.Conclusion: We have established a new mouse model of acute GVHD using chemotherapy based conditioning. This model can be utilized to study the basic mechanisms underlying GVHD triggered by cytostatics used in the conditioning regimen and affecting different cell sub-populations and cytokine storm. [Display omitted] [Display omitted]