Murine Allogeneic Bone Marrow Transplantation Research Articles

Transfer of Cell-Surface Antigens by Scavenger Receptor CD36 Promotes Thymic Regulatory T Cell Receptor Repertoire Development and Allo-tolerance

The development of Tcell tolerance in the thymus requires the presentation of host proteins by multiple antigen-presenting cell (APC) types. However, the importance of transferring host antigens from transcription factor AIRE-dependent medullary thymic epithelial cells (mTECs) to bone marrow (BM) APCs is unknown. We report that antigen was primarily transferred from mTECs to CD8α+ dendritic cells (DCs) and showed that CD36, a scavenger receptor selectively expressed on CD8α+ DCs, mediated the transfer of cell-surface, but not cytoplasmic, antigens. The absence of CD8α+ DCs or CD36 altered thymic Tcell selection, as evidenced by TCR repertoire analysis and the loss of allo-tolerance in murine allogeneic BM transplantation (allo-BMT) studies. Decreases in these DCs and CD36 expression in peripheral blood of human allo-BMT patients correlated with graft-versus-host disease. Our findings suggest that CD36 facilitates transfer of mTEC-derived cell-surface antigen on CD8α+ DCs to promote tolerance to host antigens during homeostasis and allo-BMT.

Interleukin-22 promotes intestinal-stem-cell-mediated epithelial regeneration.

Epithelial regeneration is critical for barrier maintenance and organ function after intestinal injury. The intestinal stem cell (ISC) niche provides Wnt, Notch, and epidermal growth factor (EGF) signals supporting Lgr5+ crypt base columnar ISCs for normal epithelial maintenance1,2. However, little is known about the regulation of the ISC compartment after tissue damage. Utilizing ex vivo organoid cultures, we provide evidence that innate lymphoid cells (ILCs), potent producers of Interleukin-22 (IL-22) after intestinal injury3,4, increased the growth of murine small intestine (SI) organoids in an IL-22-dependent fashion. Recombinant IL-22 directly targeted ISCs, augmenting the growth of both murine and human intestinal organoids, increasing proliferation, and promoting ISC expansion. IL-22 induced Stat3 phosphorylation in Lgr5+ ISCs, and Stat3 was critical for both organoid formation and IL-22-mediated regeneration. Treatment with IL-22 in vivo after murine allogeneic bone marrow transplantation (BMT) enhanced recovery of ISCs, increased epithelial regeneration, and reduced intestinal pathology and mortality from graft vs. host disease (GVHD). Atoh1-deficient organoid culture demonstrated that IL-22 induced epithelial regeneration independent of the Paneth cell niche. Our findings reveal a fundamental mechanism by which the immune system is able to support intestinal epithelium, activating ISCs to promote regeneration.

In vitro-generated MDSCs prevent murine GVHD by inducing type 2 T cells without disabling antitumor cytotoxicity

AbstractMyeloid-derived suppressor cells (MDSCs) inhibit T-cell expansion and functions by versatile mechanisms such as nutrient depletion, nitrosylation, or apoptosis. Since graft-versus-host disease (GVHD) is characterized by the expansion of donor-derived T cells destroying recipient tissue, we analyzed whether MDSCs can be used for GVHD prevention in murine allogeneic bone marrow transplantation models. Transplantation of MDSCs, generated from bone marrow cells by granulocyte-macrophage colony-stimulating factor (GM-CSF)/G-CSF in vitro, inhibited GVHD-induced death and attenuated histologic GVHD, whereas antitumor cytotoxicity of alloantigen-specific T cells was maintained. MDSCs expanded in vivo and invaded lymphatic and GVHD target organs. Major histocompatibility complex class I expression on MDSCs was dispensable for their suppressive capacity. Inhibition of GVHD required the presence of MDSCs during T-cell priming, whereas allogeneic T-cell numbers and homing in lymphoid and GVHD target organs were not considerably affected in MDSC-treated mice. However, MDSCs skewed allogeneic T cells toward type 2 T cells upregulating T helper 2 (Th2)-specific cytokines. Type 2 T-cell induction was indispensable for GVHD prevention since MDSC treatment failed to prevent GVHD when allogeneic STAT6-deficient T cells, which are unable to differentiate into Th2 cells, were transplanted. MDSC-induced Th2 induction might be applicable for GVHD treatment in clinical settings.

α-GalCer reduces Acute Graft-versus-Host Disease by Altering Donor T Cells Migration

This study was aimed to explore the new mechanism of α-Galactosyleramide (α-GalCer), a synthetic glycolipid, and a well-known activator of natural killer T cells (NKT) for improving acute graft-versus-host disease(aGVHD). Murine allogeneic bone marrow transplantation (allo-BMT) model was established. Recipient mice were injected intraperitoneally with α-GalCer immediately after allo-BMT, whereas mice from the vehicle groups received the diluent (DMSO) only. The severity degree of aGVHD was estimated by survival, aGVHD clinical score and pathology. The mechanism of aGVHD reduced by α-GalCer was explored by detecting T cells migration in vivo and in vitro. Mice in α-GalCer group survived longer than in control group, and their clinical and pathological status of aGVHD were lighter. α-GalCer reduced aGVHD by altering donors T cell migration. After allo-BMT α-GalCer reduces aGVHD by altering donor T cells migration.

Rapamycin and CTLA4Ig Synergize to Induce Stable Mixed Chimerism Without the Need for CD40 Blockade

The mixed chimerism approach achieves donor-specific tolerance in organ transplantation, but clinical use is inhibited by the toxicities of current bone marrow (BM) transplantation (BMT) protocols. Blocking the CD40:CD154 pathway with anti-CD154 monoclonal antibodies (mAbs) is exceptionally potent in inducing mixed chimerism, but these mAbs are clinically not available. Defining the roles of donor and recipient CD40 in a murine allogeneic BMT model, we show that CD4 or CD8 activation through an intact direct or CD4 T cell activation through the indirect pathway is sufficient to trigger BM rejection despite CTLA4Ig treatment. In the absence of CD4 T cells, CD8 T cell activation via the direct pathway, in contrast, leads to a state of split tolerance. Interruption of the CD40 signals in both the direct and indirect pathway of allorecognition or lack of recipient CD154 is required for the induction of chimerism and tolerance. We developed a novel BMT protocol that induces mixed chimerism and donor-specific tolerance to fully mismatched cardiac allografts relying on CD28 costimulation blockade and mTOR inhibition without targeting the CD40 pathway. Notably, MHC-mismatched/minor antigen-matched skin grafts survive indefinitely whereas fully mismatched grafts are rejected, suggesting that non-MHC antigens cause graft rejection and split tolerance.

Interleukin 21 blockade modulates activated T- and B-cell homeostasis via B-cell activating factor pathway–mediated inhibition in a murine model of acute graft-versus-host disease

Interleukin (IL) 21 plays a key role in the development of acute graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation. Therapeutic manipulation of IL-21 activity may improve acute GVHD during the early-posttransplant period. We investigated the mechanisms regulating T- and B-cells during IL-21 blockade in acute GVHD. Interleukin 21 blockade enhanced regulatory T and T helper (Th) 2 cell differentiation and inhibited Th1- and Th17-derived transcription factors and cytokines as a modulator of activated T-cells. Interleukin 21(-/-) cell recipients showed increased mature B- and marginal-zone B-cells, but decreased memory B-cells, germinal center formation, and plasma cells that did not lead to immunoglobulin production. B-cell activating factor (BAFF) and a proliferation-inducing ligand (APRIL) are involved in the induction and maintenance of T- and B-cell responses. We observed decreased levels of only BAFF during acute GVHD and confirmed that mammalian target of rapamycin complex 1 was reduced by the BAFF/BAFF-receptor pathway. Therefore, this study suggests that IL-21 blockade modulates activated T- and B-cell homeostasis via BAFF-pathway-mediated inhibition in acute GVHD following murine allogeneic bone marrow transplantation.

T-Bet Is Critical for the Development of Acute Graft-Versus-Host Disease Through Controlling T Cell Differentiation and Function

Abstract 452 Background:Allogeneic hematopoietic cell transplantation (HCT) offers great promise for the treatment of hematologic malignancies. However, HCT benefits are frequently offset by graft-versus-host disease (GVHD). Donor T cells can differentiate into Th1 or Th17 subset that contribute to GVHD. The T-box transcription factor T-bet is important for promoting the differentiation of naïve CD4+T cells into Th1 phenotype, while simultaneously inhibiting Th2 and Th17 lineage commitment. Published data indicate that donor T cells deficient for IFN-γ induce exacerbated GVHD. In contrast, our recent study showed that T cells deficient for T-bet were impaired in the induction of GVHD. Given T-bet is a master regulator for the differentiation into Th1 cells that produce IFN-γ, the underlining mechanisms accounted for the distinct outcomes caused by T-bet- versus IFN-γ-deficient donor T cells are not clear. Method:We evaluated the roles of T-bet and IFN-γ in acute GVHD induced by naïve CD4+ T cells or polarized Th17 cells using murine allogeneic bone marrow transplantation (allo-BMT) model. WT, T-bet knockout (KO) and IFN-γ KO mice on C57BL/6 (B6) background were used as donors, and lethally irradiated BALB/c mice were used as recipients. Pathologic analysis and serum cytokine detection were done 14 days after adoptive transfer of WT, T-bet–/–, and IFN-γ–/– CD4+ T cells. Using microarray technology, gene expression profile on donor T cells was analyzed 7 days after adoptive transfer by sorting donor-derived CD4+ T cells from the recipients of WT, T-bet–/– or IFN-γ–/– CD4+ T cells. Results:We compared the ability of WT, T-bet–/–, and IFN-γ–/– CD4 T cells in the induction of acute GVHD. In the comparison with WT cells, IFN-γ–/– CD4 T cells caused similar or even more severe GVHD as expected. In sharp contrast, T-bet–/– CD4 T cells induced much ameliorated GVHD, as significantly higher survival and less body weight loss were observed in the recipients of T-bet–/–T cells. Pathology study on GVHD target organs showed that recipients of T-bet–/– donor T cells had markedly reduced T cell infiltration and tissue damage in liver, gut, and skin, when compared with those of WT or IFN-γ–/– T cells. Reduced GVHD in the recipients of T-bet–/– T cells was consistent with significantly lower levels of pathogenic cytokines IFN-γ, TNF-α, and IL-2 but higher IL-10 (anti-inflammatory), IL-6 (Th17 related) and IL-4 (Th2 related) in serum as compared with those in the recipients of WT T cells. Mechanistic studies in vitro revealed that T-bet–/– CD4 T cells expressed significantly lower levels of IFN-γ, CXCR3 (Th1 specific chemokine receptor) and CD122 (T cell activation marker), but higher levels of IL-17 (Th17 cytokine) and CCR6 (Th17 specific chemokine receptor) compared with WT CD4 T cells, indicating that T-bet–/– T cells impaired in differentiating into Th1 cells and instead into Th17 cells. Given Th17 subset only is capable of causing GVHD and T-bet–/– T cells are prone to Th17-differentiation, we assessed the role of T-bet or IFN-γ in the development of GVHD by comparing the pathogenicity of in vitro polarized WT, T-bet–/– and IFN-γ–/– Th17 cells. While IFN-γ–/– Th17 cells had a comparable ability to cause GVHD compared with WT Th17 cells, T-bet–/– Th17 cells had reduced pathogenicity, and caused ameliorated GVHD. Furthermore, microarray analysis identified genes that are regulated by T-bet but independent of IFN-γ, including Cxcr3, Ccr5, Ccl3, Ccl4, Klrc1, Klrd1, Nkg7 and Pdcd1, which may explain the compromised ability of T-bet−/− not IFN-γ–/–T cells in the induction of GVHD. Conclusions:We conclude that T-bet is required for Th1 differentiation and optimal function of Th17 cells, and it can also control T cell infiltration into GVHD target organs by regulating chemokines and their receptors. Thus, failure in Th1 generation, migration and reduced activity of polarized Th17 cells are likely accounted for impaired ability of T-bet−/− CD4 T cells in the induction of acute GVHD. The current study suggests that targeting T-bet or regulating its downstream effectors independent of IFN-γ may be a promising strategy to control GVHD after allogeneic HCT in clinic. Disclosures:No relevant conflicts of interest to declare.

A Critical Role for the Retinoic Acid Signaling Pathway in the Pathophysiology of Gastrointestinal Graft-Versus-Host Disease

Abstract 341Damage to the gastrointestinal tract during graft-versus-host disease (GVHD) is one of the major causes of morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. However, the molecular mechanism(s) responsible for colonic GVHD induction remain obscure. All-trans retinoic acid (RA) is an active metabolite of vitamin A and has recently emerged as a critical regulator of gut immunity. We hypothesized that administration of RA might be a strategy to reduce inflammatory responses during GVHD, particularly within the colon microenvironment. In a B6→Balb/c MHC-mismatched murine allogeneic bone marrow transplantation (BMT) model, we found unexpectedly that exposure of recipient mice to exogenous RA resulted in significantly accelerated GVHD-associated mortality compared with DMSO-treated animals (p<0.0001). Histological examination of GVHD target organs 5 weeks after transplantation revealed a significant increase in pathologic damage in the colons of RA-treated mice compared with DMSO-treated animals (9.7 ± 0.8 versus 3.3 ± 0.9, p<0.001), whereas there was no significant difference in pathology scores in either the lung or liver. RA treatment resulted in significantly increased expression of the gut-homing molecules, CCR9 and α4β7, on donor T cells in mesenteric lymph nodes early after BMT. Notably, expression of other chemokine receptors that have been implicated in GVHD pathogenesis, such as CCR5, CXCR3, and CCR10, was not affected by RA treatment, demonstrating a selective role of RA in imprinting gut-homing donor T cells. Examination of GVHD target organs three weeks after transplantation revealed a significant increase (eight-fold) in the absolute numbers of both CD4+IFN-γ+ (TH1) and CD4+IL-17+ (TH17) cells in the colon of RA-treated mice versus DMSO-treated animals, whereas there were no statistical differences in TH1 or TH17 cells in the spleen, liver and lung. There was also a significant increase in the absolute number of regulatory T cells (Tregs) in the colon of RA-treated mice. However, the increase in Tregs was noted to be more modest (three-fold) than was observed for TH1 and TH17 cells, suggesting a pro-inflammatory role of RA. To further confirm a role for RA in imprinting gut-homing donor T cells during GVHD, we generated vitamin A-deficient mice and employed them as recipients. Depletion of RA in recipient mice by vitamin A deprivation resulted in a dramatic reduction of gut homing molecule expression on donor T cells after allogeneic BMT. Retinoic acid receptor-α (RAR-α) signaling appears to be critical in mediating the effects of RA on alloreactive T cells. In mixed lymphocyte culture, when B6 T cells were stimulated with Balb/c dendritic cells, the presence of an RAR-α agonist was able to significantly enhance the expression of gut-homing molecules on T cells. In contrast, the addition of a selective RAR-α antagonist abolished the effects of RA on T cells. We then measured endogenous RA levels in the colons of mice undergoing either syngeneic or allogeneic BMT by mass spectrometry. These studies revealed that RA was significantly increased in the colon tissue of allogeneic transplant recipients compared with syngeneic transplant recipients early after BMT (day 7). In addition, Real-Time q-PCR analysis revealed a mean 3.5-fold increase in RAR-α expression in purified donor splenic CD4+ T cells 4 days post transplantation when compared to purified naïve CD4+ T cells obtained from original donor animals. Finally, we examined the ability of RAR-α-deficient T cells to cause GVHD. We found that recipients of RAR-α-deficient marrow grafts had significantly decreased GVHD-associated mortality when compared with the recipients of wild type T cells (93% versus 36% at day 90, p=0.002). Histological examination of GVHD target organs on days 10–12 after transplantation revealed a significant reduction in pathologic damage in the colon of mice that received RAR-α-deficient T cells compared with control mice (7.3 ± 2.5 versus 0.8 ± 1.0, p<0.01). In summary, we have identified RA as a novel and critical molecule in the induction of gastrointestinal GVHD. These findings provide evidence that blockade of the RA signaling pathway may represent a novel strategy to mitigate the severity of colonic GVHD. Disclosures:No relevant conflicts of interest to declare.

Blocking Vasoactive Intestinal Peptide Signaling Enhances Anti-Viral Immunity without Increased Graft Versus Host Disease in Murine Allogeneic Bone Marrow Transplantation

Abstract 1002 Background:Vasoactive intestinal peptide (VIP) is a neuropeptide hormone and type 2 cytokine that inhibits Th1 immunity and induces the generation of regulatory T-cells. We have recently reported that non-transplanted mice “knocked-out” for VIP and syngeneic transplant recipients of VIP-knockout (KO) BM had dramatically improved survival, viral clearance, and increased numbers of specific antiviral CD8+ T-cells following murine cytomegalovirus (mCMV) infection (JI 2011. 187:1057–65). In this study, we used a small molecule VIP antagonist as well as VIP-KO mice to further investigate effects and mechanisms of VIP-signaling on antiviral immune responses in wild type (WT) non-transplanted mice and following allogeneic BMT. Methods:B10BR (CD45.2, H-2Kk) and CB6/J F1 (CD45.2, H-2Kb/d) mice were transplanted with 3 × 103 FACS-sorted hematopoietic stem cells (HSC), 5 × 104 dendritic cells (DC), and 0.3, 1, or 3 × 106 splenic T-cells either from VIP-KO (CD45.2, H-2Kb) or WT donors after myeloablative conditioning (11Gy). WT mice and BMT recipients transplanted with WT grafts were treated with daily subcutaneous injection of VIP antagonist (10 μg/100μL per mouse) or PBS for 7 days (from one day prior to infection to 6 days post-infection). BALB/C mice, B6 VIP-KO and WT littermates, as well as CB6/J F1 BMT recipients, were infected with graded doses (LD10, LD50 and LD90) of mCMV by intraperitoneal injection. Survival, viral load, antigen specific T-cells, and clinical scores of graft versus host disease (GvHD) were assessed at distinct time-points post-BMT or after mCMV infection. The expression of co-stimulatory or co-inhibitory markers (CD25, CD62L, CD69, PD-1, FoxP3, PD-L1, CD80, CD86, and MHC-II) and intracellular expression of cytokines (IL-10, IFN-γ, TNF-α, and IL-12) on T-cells and DC from the mice were measured by flow cytometry. Results:Improved survival was seen in mCMV-infected allogeneic B6→CB6/J F1 transplant recipients of VIP-KO grafts (100%) compared with recipients of WT grafts treated with PBS (40%). Allogeneic recipients of VIP-KO grafts and allogeneic recipients of WT grafts treated with VIP antagonist had increased viral clearance and enhanced in vivo killing of viral-peptide-pulsed targets compared with PBS-treated recipients of WT grafts. No difference in the incidence or severity of acute GvHD was seen in allogeneic BMT recipients of graded doses of VIP-KO versus WT splenic T-cells (0.3, 1, and 3 × 106) in murine MHC mis-matched BMT models. Allogeneic transplant recipients of VIP-KO grafts and WT grafts treated with VIP antagonist, infected with low dose mCMV, had lower levels of PD-L1 and PD-1 expression on DC and T-cells, respectively, and higher levels of CD80, CD86 and MHC-II expression on conventional DC (cDC) and plasmacytoid DC (pDC) compared with recipients of WT allografts treated with PBS. Recipients of VIP-KO grafts and recipients treated with VIP antagonist had higher-levels of IL-12+ cDC, activated CD25+/CD69+ CD4 and CD8 T-cells, and more mCMV-M45-peptide MHC-I tetramer+ CD8+ T-cells compared with recipients of WT grafts treated with PBS. Absence of VIP-signaling led to enhanced intracellular expression of IFN-γ and less IL-10 expression in T-cells from mCMV-infected recipients of VIP-KO B6→CB6/J F1 allogeneic transplants, and mCMV-infected, VIP antagonist-treated recipients of WT allogeneic transplants. In the absence of mCMV infection, the numbers of regulatory T cells (Treg) were similar among VIP-KO mice, WT mice treated with VIP antagonist, and PBS-treated WT controls. In contrast, mCMV-infected VIP-KO mice had significantly fewer Treg compared with mCMV- infected WT mice, non-infected WT mice and non-infected VIP-KO mice. Conclusion:Genetic or pharmacological blockade of VIP-signaling enhanced both innate and adaptive antiviral immune responses in allogeneic BMT recipients without significantly elevating GvHD. Selective targeting of VIP-signaling represents a novel therapeutic approach to enhance antiviral immunity in the setting of immunodeficiency and allogeneic BMT. Disclosures:No relevant conflicts of interest to declare.

Xenogeneic immunosuppression of human umbilical cord mesenchymal stem cells in a major histocompatibility complex-mismatched allogeneic acute graft-versus-host disease murine model

Mesenchymal stem cells (MSCs) hold great promise for treating immune disorders owing to their immunoregulatory capacity, but the mechanism remains controversial. As we show here, the mechanism of human umbilical cord mesenchymal stem cell (HUCMSC)-mediated immunosuppression involves TGF-β and indoleamine 2,3-dioxygenase (IDO). In this study, we investigated the influence of xenogeneic HUCMSCs on acute graft-versus-host disease (aGVHD) in murine allogeneic bone marrow transplantation (BMT). In the HUCMSC-treated group, lethally irradiated DBA/2(H-2Kd) mice were adoptively transferred with expanded HUCMSCs, bone marrow (BM), and splenocytes (SCs) from C57BL/6 (H-2Kb) mice. Recipients in the control group were transferred only BM and SCs. The two groups were compared in survival, weight, histopathologic specimens, and aGVHD scoring. In the HUCMSC-treated group, 60% of the mice survived past day 30 after BMT, but in the control group, all mice died within 18 d. The mice treated with HUCMSCs exhibited light symptoms of aGVHD after day 30. The results suggest that xenogeneic HUCMSCs could alleviate aGVHD symptoms and prolong survival after allogeneic BMT. Our study suggests that in vitro expanded HUCMSCs might be used to inhibit severe aGVHD effectively in allogeneic hematopoietic cell transplantation clinically.

Control of mouse graft-versus-host disease following allogeneic bone marrow transplantation by blocking the CD28/B7 signaling pathway with lentiviral vector-mediated RNA interference

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is an effective way to cure hematological malignancies. However, graft-versus-host disease (GVHD) following transplantation limits the clinical application to some extent. The donor T lymphocytes play a central role in the occurrence and development of GVHD. Control of GVHD by inhibition of T cell proliferation by blocking the CD28/B7 signaling pathway with RNA interference has not been examined. In this study, we constructed a lentiviral vector carrying CD28 shRNA and generated genetically engineered splenocytes through transduction in a murine allogeneic bone marrow transplantation model. The survival and the occurrence of GVHD in transplanted mice were monitored every day. Liver, intestine, skin, and other tissues from the mice in each group were used for histological examination. We also determined plasma concentrations of interleukin (IL)-2, IL-4, IL-5, IL-10, IL-13, and interferon gamma (IFN-γ). Recipient bone marrow from mice that had survived for an extended period was examined to detect chimerism. We succeeded in suppressing the expression of CD28 gene and controlling mouse GVHD following allogeneic bone marrow transplantation in the engineered spleen cell group. These suggest that blocking the CD28/B7 signal transduction pathway with lentiviral vector-mediated RNA interference effectively controlled the occurrence of mouse GVHD following allogeneic bone marrow transplantation. Its mechanism could be due to the inhibition of T cell proliferation and, simultaneously, the promotion of the differentiation of TH0 to TH2 cells, thereby reducing GVHD in the mouse transplantation model.

Organ-Specific Protection from CD8+ T Cell-Mediated Acute Skin GVHD by Bortezomib Administration Correlates with Decreased IL-6.

AbstractAbstract 3735We have previously demonstrated that while early administration of bortezomib after murine allogeneic bone marrow transplantation (allo-BMT) can prevent acute graft versus host disease (GVHD), delayed administration of bortezomib results in increased GVHD-dependent gastrointestinal toxicity (PNAS 2004, Blood 2005). This was primarily mediated by donor CD4+ T cells in fully MHC-mismatched allo-BMT (Blood 2008). In the current study, we assessed continuous bortezomib administration on CD8+ T cell-mediated acute GVHD. Using an MHC-matched, mHAg-mismatched model (C3.SW donor cells into lethally irradiated C57BL/6 recipients) in which acute GVHD is primarily induced by CD8+ T cells, we confirmed that significant skin and liver GVHD was observed after CD8+ T cell transfer. Histological evaluation of acute liver GVHD displayed severe lymphocyte infiltrates and severe subacute bile duct hepatitis consistent with hepatic GVHD. Severe acute skin GVHD was noted by persistent fur loss and skin ulcerations. Cutaneous histopathological assessment exhibited severe inflammatory infiltrates, epidermal hypertrophy, loss of hair follicles, satellite cell necrosis and dermal fibrosis. Notably, after allo-BMT, 6 weeks or thereafter, a significant increase in serum IL-6 levels was observed in the mice exhibiting marked skin damage, suggesting organ-specific correlation of higher serum IL-6 expression with acute cutaneous GVHD associated injury. We next investigated the effects of continuous treatment with bortezomib in this GVHD model. Surprisingly, we found that continuous bortezomib administration at the conventional dose of 0.75–1 mg/kg twice per week resulted in fractional GVHD- dependent lethal toxicity, exacerbating lymphocyte infiltration in liver, gut and lung as evidenced by pathological examination. However, skin GVHD pathology was markedly improved. Due to the limitations of conventional doses of bortezomib for treatment of acute GVHD, we then performed a low-dose bortezomib treatment at 0.125 mg/kg, twice per week. We found that mice receiving low-dose bortezomib were protected from lethal toxicity and displayed significant amelioration of skin GVHD pathology. Significant decreases in serum IL-6 levels were also consistently observed consistent with the amelioration of skin GVHD. Interestingly, while skin GVHD was markedly improved, there was no difference in liver, gut and lung pathology compared to control GVHD recipients indicating an organ-specific protection. These results demonstrate that continuous bortezomib can be applied in CD8+ T cell-mediated acute GVHD but necessitates lower dosing and organ-specific protection is observed. Disclosures:No relevant conflicts of interest to declare.

Epigenetic Control Of GvHD And GvL Using The Hypomethylating Agent Azacitidine

Allogeneic bone marrow transplantation (BMT) represents the most effective treatment for patients with high risk and relapsed hematologic malignancies because allogeneic donor T cells provide a graft-versus-leukemia (GvL) effect. However, the same cells can cause graft-versus-host disease (GvHD), one of the major complications. Regulatory T cells (Tregs) have been shown to suppress GvHD while preserving GvL, their use provides a promising strategy in the allogeneic transplant setting if three major obstacles will be overcome: 1) the low numbers of Tregs, 2) loss of suppressor activity following ex vivo expansion and 3) the lack of Treg-specific markers to purify ex vivo expanded Tregs. The FOXP3 transcription factor, which is exclusively expressed in Tregs, can convert effector T cells (Teff) into Tregs when ectopically overexpressed. The Foxp3 locus is unmethylated in Tregs while highly methylated and silenced in all other T cells. The hypomethylating agent azacitidine (AzaC) can modulate this methylation status and induce stable FOXP3 expression (>7 days) in Teff. Furthermore, we have shown that these AzaC-induced FOXP3+T cells are suppressive phenotype in vitro. Thus, we hypothesize that AzaC treatment of mice after allogeneic BMT will dramatically mitigate GvHD while preserving GvL via upregulation of Foxp3 in alloreactive Teff. In murine T cell depleted (TCD) BMT model (B6→Balb/c) with delayed infusion of conventional T cells (Tconv) (2X106) at day 11 post BMT, followed by subcutaneous treatment of AzaC (2 mg/kg at days 15, 17, 19, and 21 post BMT), we found that AzaC dramatically suppressed GvHD caused by allogeneic donor T cells while maintaining donor engraftment of all lineages. The AzaC group had significantly higher FOXP3+Tregs than in PBS control group and that these Tregs were derived from donor T cells, suggesting that the suppression of GvHD was mediated by AzaC-induced Tregs. We further tested whether AzaC treatment of mice transplanted with allogeneic T cells preserve GvL while mitigating GvHD. Using the same murine allogeneic BMT model, Click Beetle Red luciferase-expressing A20 leukemia cells (Balb/c derived; 1X104) were injected with TCD BM and 10×106 Tconv and in vivo bioluminescence imaging was performed to assess tumor burden. We found that AzaC treatment mitigated GvHD without abrogating GvL or donor engraftment. Thus, the adminstration of hypomethylating agents like AzaC might be a promising strategy to treat GvHD.

Epigenetic Control of GvHD and Gvl Using the Hypomethylating Agent Azacitidine.

Abstract 2447Poster Board II-424Allogeneic bone marrow transplantation (BMT) represents the most effective treatment for patients with high risk and relapsed hematologic malignancies. One of the major complications of allogeneic BMT is graft-versus-host disease (GvHD), which is caused by donor T cells reacting against host antigens. These same alloreactive donor T cells can provide a beneficial ¡°graft-versus-leukemia¡± (GvL) effect as well resulting in reduction in leukemia relapse. Because regulatory T cells (Tregs) have been shown to suppress GvHD while preserving GvL, their use in the allogeneic transplant setting provides a promising strategy to treat GvHD. However, three major obstacles prevent their routine use in human clinical trials: 1) the low circulating numbers of Tregs in peripheral blood, 2) loss of suppressor activity following ex vivo expansion and 3) the lack of Treg-specific markers to purify ex vivo expanded Tregs. Foxp3 is a forkhead transcription factor which is both exclusively expressed in Tregs and, when overexpressed in conventional effector T cells (Teff), can convert these Teff into functionally suppressive Treg-like T cells. The Foxp3 locus is unmethylated in Tregs while highly methylated and silenced in all other T cells. Several groups have shown that the hypomethylating agent azacitidine (AzaC) induces FOXP3 expression in non-Tregs. Furthermore, we have shown that treatment of anti-CD3/CD28 antibody-coated bead-activated CD4+CD25- T cells with AzaC results in robust and prolonged (>7 days) expression of FOXP3. AzaC-induced FOXP3 expression is also associated with a potent Treg-like suppressive phenotype in vitro. Thus, we hypothesize that AzaC treatment of mice after allogeneic BMT will dramatically mitigate GvHD while preserving GvL via transcriptional regulation of Foxp3 in alloreactive Tconv.In murine T-cell depleted BMT model (B6¡æBalb/c; 900 cGy TBI) with delayed infusion of conventional T cells (Tconv) (2 ×106) at day 11 post BMT, followed by subcutaneous treatment of AzaC (2 mg/kg at days 15, 17, 19, and 21 post BMT), we found that AzaC dramatically suppressed GvHD caused by allogeneic donor T cells while maintaining donor (H2Kb) engraftment of all lineages. We found that the AzaC group had significantly higher FOXP3+ Tregs than in PBS control group and that these Tregs were derived from donor T cells, suggesting that the suppression of GvHD was mediated by AzaC-induced Tregs. We further tested whether AzaC treatment of mice transplanted with allogeneic T cells preserve GvL while mitigating GvHD. Using the same murine allogeneic BMT model, Click Beetle Red luciferase-expressing A20 leukemia cells (BALB/C derived) were injected with T-cell depleted BM and 10 × 106 Tconv and in vivo bioluminescence imaging was performed to assess tumor burden in vivo post transplant. We found that AzaC treatment mitigated GvHD without abrogating GvL (Fig. 1) or donor engraftment. Thus, the adminstration of hypomethlating agents like AzaC in vivo after allogeneic stem cell transplant dramatically reduces GvHD while maintaining both donor engraftment and a potent GvL effect providing the foundations for future clinical trials. [Display omitted] Disclosures:DiPersio:Genzyme Corporation: Honoraria.

NK Cells Suppress Gvhd by Directly Inhibiting Activated Alloreactive T Cells through An NKG2D-Mediated Mechanism

Natural Killer (NK) cells have the ability to suppress graft-versus-host disease (GVHD) while inducing a graft-versus-tumor response (GVT) following murine allogeneic bone marrow transplantation (BMT). Prior studies have shown that NK cells suppress GVHD by eliminating recipient dendritic cells. To assess additional potential mechanisms of GVHD suppression we evaluated the impact of donor NK cells on GVHD-inducing donor T cells. Interleukin-2 activated allogeneic NK cells isolated from C57Bl6 (H-2b) or FVB (H-2q) animals were transplanted along with T cell-depleted bone marrow (TCD-BM) into lethally irradiated BALB/c (H-2d) mice, followed 2 days later by luciferase-expressing CD4+ and CD8+ conventional T cells (Tcon) from the same donor strain (Tcon+NK group). Control mice received TCD-BM on day 0, and luciferase-expressing T cells on day 2 after transplant (Tcon group). Bioluminescence imaging of Tcon+NK mice revealed a significantly lower T cell bioluminescent signal compared to Tcon mice (p=0.01 on day 5 post T cell transplant). We assessed the impact of NK cells on donor T cell activation and proliferation. CFSE proliferation analysis of alloreactive CD4 and CD8 T cells reisolated on day 4 post transplant showed a decreased percentage of dividing donor T cells in the Tcon+NK group. A reduced percentage of T cells in the Tcon+NK group as compared to the Tcon group expressed the T cell activation marker CD25 (11% and 49%, respectively, among donor CD4) and a reduced percentage of T cells from the Tcon+NK group down-regulated CD62L. Reisolated donor T cell numbers were reduced in the Tcon+NK mice compared to Tcon control mice. The impact of donor NK cells on donor Tcon function was addressed by intracellular cytokine staining. Fewer donor T cells reisolated from the spleen and lymph nodes of Tcon+NK mice produced the proinflammatory cytokines IFN-γ and IL-2 on day 3 after transplant. These observations can be explained by an NK cell-mediated induction of apoptosis in the donor Tcon. T cells reisolated from the peripheral lymph nodes of Tcon+NK animals at day 4 post transplant stained higher for the TUNEL apoptosis marker than those from Tcon mice (p<0.0001 for CD4 and CD8). To determine if this increase in apoptosis was due to a direct interaction between the donor T cells and NK cells, donor Tcon were reisolated from transplanted mice and used as targets in a killing assay. We demonstrated direct, specific lysis of these reisolated T cells by activated NK cells, both of which are from the donor strain and thus syngeneic to each other. Donor T cells reisolated from the lymph nodes of transplanted mice upregulated the NKG2D ligand Rae1γ as compared to naïve T cells, as shown by FACS. Further, use of an NKG2D-blocking antibody decreased the specific lysis of donor Tcon reisolated from the lymph nodes by activated NK cells in the in vitro killing assay, compared to an isotype control antibody (p=0.004). These data indicate that NK cells are causing direct, NKG2D-dependent lysis of alloreactive donor T cells in vivo during GVHD induction. Recent data from our laboratory has shown a lack of NKG2D ligand expression on GVHD target tissues in irradiated recipient mice. The tissue-specific expression of NKG2D ligands may explain why allogeneic NK cells do not cause GVHD but do impact donor T cells. We further investigated the ability of T cells in this model to elicit a GVT effect in the presence or absence of NK cells. Using a luciferase-expressing A20 lymphoma cell line, we demonstrated tumor clearance in groups receiving A20+Tcon and A20+Tcon+NK, as measured by A20 bioluminescence signal. Animals in the A20+Tcon+NK group had a lower peak bioluminescent signal than animals in the A20+Tcon group (p=0.03 on day 4 post T cell transplant), indicating an additive GVT effect of the T cells and NK cells. Thus, the T cells in this model are capable of mounting an effective GVT response. In addition to the established mechanism of NK cell-mediated elimination of recipient dendritic cells, we have demonstrated a novel mechanism of NK cell action in murine models of GVHD, whereby the donor NK cells inhibit T cell proliferation and activation and cause direct, NKG2D-mediated lysis of alloreactive donor T cells.

382: β2 Intergrins Separates Graft-versus-host Disease and Graft-versus-leukemia Effect

Graft-versus-host disease (GVHD) remains a major cause of morbidity and mortality in allogeneic hematopoietic stem cell transplantation. Migration of donor-derived T cells into GVHD target organs plays an essential role in the development of GVHD. β2 integrins are critically important for leukocyte extravasation through vascular endothelia and for T cell activation. We asked whether CD18-deficient T cells would induce less GVHD while sparing graft-versus-leukemia (GVL) effect. In murine allogeneic bone marrow transplantation models, we found that recipients of CD18-/- donor T cells had significantly less GVHD morbidity and mortality compared with recipients of WT donor T cells.

β2 integrins separate graft-versus-host disease and graft-versus-leukemia effects

Graft-versus-host disease (GVHD) remains a major cause of morbidity and mortality in allogeneic hematopoietic stem cell transplantation. Migration of donor-derived T cells into GVHD target organs plays an essential role in the development of GVHD. beta2 integrins are critically important for leukocyte extravasation through vascular endothelia and for T-cell activation. We asked whether CD18-deficient T cells would induce less GVHD while sparing the graft-versus-leukemia (GVL) effect. In murine allogeneic bone marrow transplantation models, we found that recipients of CD18-/- donor T cells had significantly less GVHD morbidity and mortality compared with recipients of wild-type (WT) donor T cells. Analysis of alloreactivity showed that CD18-/- and WT T cells had comparable activation, expansion, and cytokine production in vivo. Reduced GVHD was associated with a significant decrease in donor T-cell infiltration of recipient intestine and with an overall decrease in pathologic scores in intestine and liver. Finally, we found that the in vivo GVL effect of CD18-/- donor T cells was largely preserved, because mortality of the recipients who received transplants of CD18-/- T cells plus tumor cells was greatly delayed or prevented. Our data suggest that strategies to target beta2 integrin have clinical potential to alleviate or prevent GVHD while sparing GVL activity.

T-cell signaling in allogeneic bone marrow transplantation

We examined the signaling profiles of T cells in murine allogeneic bone marrow transplantation with a novel high-throughput technique to analyze activation of signaling pathways by flow cytometry. We first studied normal splenic, liver, and peripheral lymph node (PLN) T cells. Relative to naive CD4 and CD8 cells, effector and memory cells displayed nearly global increases in activation of signaling pathways including MEK1/2 and PKC (both up ∼10-fold), and S6 ribosomal protein (∼6 fold) (Table 1). In the thymus, there was a general lack of signaling activity in CD4+CD8+ cells, and these did not respond to cytokines. By contrast, CD4−CD8− (DN) cells of the DN1 and DN2 stage had the greatest signaling activity of all subsets. CD4+, CD8+, and DN cells responded to the cytokines IL-7 and IL-15 by phosphorylating STAT-5A (∼10 fold). We then studied alloreactive and homeostatically proliferating T cells by infusion of CFSE labeled cells into irradiated hosts. We noted a gradual and global increase in signaling activity with each cell division in the spleen, liver, and PLN. Molecules that were phosphorylated included c-Raf, MEK1/2, ERK1/2, Akt, STAT-1, STAT-3, STAT-4, and S6 ribosomal protein. Tacrolimus and cyclosporin inhibited both cell cycling and increases in signaling. We defined slow-cycling cells as homeostatically expanding and fast-cycling cells as alloreactive, and found a distinct signaling pattern in homeostatically expanding T cells, with lower levels of MAP kinase pathway activity and STAT pathway activity. We hypothesize that this difference can be attributed to strong IL-2 and TCR signaling in alloreactive cells through the MAP kinases, Akt and STAT-3, by IFNγ for STAT-1, and by IL-12 for STAT-4. In the setting of T-cell depleted bone marrow transplantation, we found in several models that bone marrow-derived splenic T cells had signaling levels generally comparable with that of the naive populations in a normal spleen, but with a deficiency in STAT-3 phosphorylation. In the thymus, we observed a defect in Akt signaling in all subsets relative to signaling levels in the normal thymus, with up to 100-fold decreases. The defect persisted up to day 28 post-transplant. We conclude that the high-throughput analysis of signaling activity in T cells during allogeneic BMT allows for the definition of distinct signaling profiles. These signaling signatures can be used for the development of targeted therapies to enhance or inhibit specific T cell subsets.Table 1Signaling in the C57BL/6 SpleenCD4CD8Naive(CD44lo CD62Lhi)Effector(CD44hi CD62Llo)Memory(CD44hi CD62Lhi)Naive(CD44lo)Effector/Memory (CD44hi)c-Raf (pS259)12.21.811.2MEK 1/2 (pS217/pS221)12.79.712.2ERK 1/2 (pT202/pY204)11.61.811.2JNK (pT180/pY182)11.16.512.3pan-p38 (pT180/pY182)12.13.011.8pan-Akt (pT308)12.13.811.4pan-Akt (pS473)12.63.911.7Pan-PKC (βII pS660)12.710.111.4STAT-1 (pY701)10.96.312.4STAT-3 (pY705)11.41.811.3STAT-3 (pS727)11.21.311.3STAT-4 (pY693)12.02.311.2STAT-5A (pY694)11.31.711.3S6 ribosomal protein (pS235/pS236)13.06.311.8Histone H2B (pS14)12.64.211.8PIP211.30.410.6Ratio of activation (phosphorylation) of signaling molecules in effector or memory CD4 or CD8 cells compared with corresponding naive CD4 or CD8 cells. Open table in a new tab Ratio of activation (phosphorylation) of signaling molecules in effector or memory CD4 or CD8 cells compared with corresponding naive CD4 or CD8 cells.

Peyer patches are not required for acute graft-versus-host disease after myeloablative conditioning and murine allogeneic bone marrow transplantation

Graft-versus-host disease (GVHD) is a multistep disease process following allogeneic bone marrow transplantation (BMT). It has been postulated that the induction of acute GVHD requires the presence of Peyer patches (PPs). A new tumor necrosis factor (TNF)-deficient strain has been developed that totally lacks PPs and displays the defects characteristic of TNF ablation but not lymphotoxin-associated defects characterized by lack of both PPs and lymph nodes. To determine the necessity of PPs in acute lethal GVHD induction, we transplanted full major histocompatibility complex (MHC)-mismatched grafts into myeloablated TNF knockout recipients. No differences in the survival or GVHD-associated histopathologic lesions were observed between the recipients. We conclude that neither PPs nor host TNF-alpha is required for the development of acute lethal GVHD in mice that undergo myeloablative conditioning and allogeneic BMT.

Influence of Valproic Acid on Cytokine Expression of Spleen Cells and Acute Graft-Versus-Host-Disease in a Murine Model.

Inhibition of histone deacetylase, one of the mechanisms for the immunologic changes by antiepileptic drugs, induces gene expression which, in turn, alters cytokine secretion. Valproic acid (VPA), the most commonly used antiepileptic drug, inhibits TNF-α and IL-6 secretion induced by PHA in THP-1 cells, a human monocytic leukemia cell line. We examined the changes in cytokine secretion induced by differing concentrations of VPA and whether VPA could prevent lethal GVHD in murine allogeneic bone marrow transplantation (BMT).For the evaluation of inhibitory effect of VPA, C3H/He (H-2k) splenocytes, irradiated BALB/c (H-2d) splenocytes, and PHA were cocultured with 0.1 mM, 1 mM, and 10 mM VPA. Concentrations of 0.1 mM and 1 mM VPA did not inhibit cell proliferation but 95% was inhibited at 10 mM. Apoptosis was induced more than 99% cells with 10 mM VPA but less than 5% of cells with 0.1 mM, and 1 mM. RT-PCR was performed to evaluate the changes and concentrations of each cytokine. TNF-α and IL-1β secretion, determined by the RNA quantity, was inhibited significantly more at 1 mM VPA than 0.1 mM (P<0.05). Decrease in IFN-γ concentration and increase in TGF-β concentration were significantly more at 1 mM VPA than 0.1 mM (P<0.05). However, differences in IFN-γ and TGF-β secretion, determined by the RNA quantity, were not statistically significant. Lethally irradiated BALB/c mice were transplanted with C3H/He bone marrow cells and splenocytes. Recipients were given 30 mg/kg and 100 mg/kg VPA intraperitoneally once daily from day −3 to day +14, respectively, whereas controls received phosphate buffered saline. There were no significant differences in the GVHD severity and mortality.These results show that VPA inhibits the secretion of inflammatory cytokines and stimulates the secretion of an antiinflammtory cytokine without inhibition of cell proliferation. Although lethal GVHD was not prevented or controlled in a major and minor histocompatibility complex-mismatched murine allogeneic BMT model, further investigations concerning the effects of VPA on GVHD prevention using a congeneic murine model should be performed.