MHC-mismatched BMT Research Articles

Vitamin C Stabilizes CD8iTregs and Enhances Their Therapeutic Potential in Controlling GvHD and Leukemia Relapse

Graft-versus-host disease (GvHD) remains a leading cause of non-relapse mortality after allogeneic hematopoietic cell transplantation (allo-HCT). The imbalance between T effector (Teffs) and regulatory cells (Tregs) is a defined characteristic of GvHD. Therefore, promoting Tregs for controlling GvHD has drawn an intense interest in preclinical and clinical studies of allo-HCT. Although CD4 Tregs (CD4+Foxp3+) have been shown to effectively prevent GvHD, they impaired graft-versus-leukemia (GVL) effect. Our lab has previously demonstrated that CD8 iTregs (CD8+Foxp3+) not only suppressed allogeneic T-cell responses, but also possessed the GVL activity themselves. However, instability of Foxp3 in CD8 iTregs is a major obstacle to translate this Treg population into clinic application.Vitamin C has been reported to promote Foxp3 stability by accelerating its demethylation. In the current study, we asked whether vitamin C could stabilize CD8 iTregs and enhance their therapeutic potential in controlling GvHD. Addition of vitamin C in the culture significantly increased the generation of allo-reactive CD8 iTregs in vitro (Fig. 1A). These CD8 iTregs generated with vitamin C had strikingly increased demethylation on Foxp3 CpGs (Fig. 1B) and superior suppressive activity (data not shown) compared to control-treated CD8 iTregs. We further evaluated the capability of vitamin C-treated CD8 iTregs in preventing GvHD in allo-HCT. Using a MHC-mismatched murine BMT model, vitamin C-treated CD8 iTregs could significantly alleviate GvHD reflected by a marked reduction of recipient mortality (Fig. 1C) and clinical scores (data not shown).To determine the Foxp3 stability in vivo, we analyzed the transferred CD8 iTregs and allogeneic Teffs in recipient spleens 7 days after allo-HCT. Vitamin C-treated CD8 iTregs (Ly5.2+) remarkably suppressed Teff (Ly5.1+) expansion and maintained higher Foxp3 expression compared to the control iTregs (Fig. 1D). To address a critical question whether vitamin C-treated CD8 iTregs can preserve the GVL activity, we performed a haploidentical transplant with a leukemia relapse model, and found that vitamin C-treated CD8 iTregs not only attenuated GvHD severity, but also prevented recipients from tumor relapse (Fig. 1E). In conclusion, our studies provide a rationale and mean to use stabilized CD8 iTregs with vitamin C for controlling GvHD and leukemia relapse in the clinic. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

IAPs Protect Host Target Tissues from Graft-Versus-Host Disease (GVHD)

The two Inhibitors of apoptosis proteins (IAPs), X-chromosome-liked linked IAP (XIAP) and cellular IAP1 (cIAP1), inhibit apoptosis and are important in leukemogenesis. Recent data suggest that both xIAP and cIAP play critical, non-overlapping roles in regulating innate and adaptive responses to certain stimuli. But the role of IAPs in allo-immunity is not known. We utilized distinct but complementary approaches, namely genetic and small molecule approaches to determine the role of IAPs in allo-immunity.We first utilized AT-406, a small molecule IAP antagonist that is also known as second mitochondria-derived activator of caspase (SMAC) mimetic. This SMAC mimetic, is a pan-IAP inhibitor (inhibits both XIAP and cIAP) and reduces TNFα secretion in vitro. Because of the GVHD potentiating effects of TNFα, we hypothesized that treatment of allogeneic animals with AT-406 will reduce GVHD. We utilized B6→BALB/c MHC-mismatched BMT model. BALB/c recipients were lethally irradiated (8.5Gy) and transplanted withsyngeneic or allogeneic T cells along with bone marrow (BM). Both groups received either AT-406 or its diluent. Surprisingly, allo-recipients receiving AT-406 showed significantly worse GVHD severity and died more rapidly (p<0.05). This observation was also noted in another MHC disparate haploidentical B6→F1 model.To further understand the role of IAPs, we next utilized genetic approach. When donor T cells from B6- cIAP-/- or XIAP-/- animals were compared to T cells from WT-B6, the allo-recipients (BALB/c) showed similar GVHD severity and mortality. Same results were also observed in a second B6→F1 model. Furthermore, in vitro studies showed that XIAP-/- and cIAP-/- T cells had comparable proliferation and cytokine secretion as WT-T cells. These data suggested that increase in GVHD mortality following treatment with AT-406 is not due to its effects on donor T cells.We therefore hypothesized that the absence of IAPs in hosts may impact on GVHD. To test this, cIAP-/-, XIAP-/- and WT-B6 animals were utilized as recipients in BALB/càB6 model. When compared with WT recipients both XIAP-/- and cIAP-/- recipients showed increased mortality (p<0.001) and worse clinical and histopathological GVHD (p<0.05, GI tract). We then determined whether IAPs expression on host hematopoietic derived cells is critical. We generated [B6→B6Ly5.2], [cIAP-/-→B6Ly5.2] and [XIAP-/-→B6Ly5.2] chimeras and utilized them as recipients in 2nd allo-BMT in BALB/càB6 models. All three chimeras, [B6→B6Ly5.2] [cIAP-/-→B6Ly5.2] and [XIAP-/-→B6Ly5.2] chimeras showed equivalent GVHD severity and mortality. Consistently, DCs from XIAP-/- and cIAP-/- animals showed similar functions as WT-B6 in vitro, suggesting that IAP expression in host hematopoietic-derived immune cells is not critical for GVHD. Next, to test the role of IAPs in non-hematopoietic GVHD target tissues, we made the reverse chimeras, namely, [B6Ly5.2→B6], [B6Ly5.2→XIAP-/-] and [B6Ly5.2→cIAP1-/-], where IAPs are absent only in the non-hematopoietic host cells. The allogeneic [B6Ly5.2→XIAP-/-] and [B6Ly5.2→cIAP1-/-] animals demonstrated a significantly worse survival compared to WT [B6Ly5.2→B6] recipient (p<0.01). To determine the potential mechanisms for enhanced mortality, we tested the expression of anti-apoptotic genes (Bcl-2, Bcl-xl) and autophagy (LC3) in the CD326+ intestinal epithelial cells from KO and WT animals harvested after allo-BMT. The cIAP-/- and -XIAP-/- animals showed significantly reduced expression of Bcl-2, Bcl-xl and LC3 than allo-WT animals. These data suggest that enhanced apoptosis and reduced autophagy in the target tissues in the absence of cIAP and XIAP caused greater GVHD. Thus expression of functional IAPs in host target tissues is crucial for reducing the damage from GVHD. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Non-Myeloablative TLI/ATG + Alkylator Conditioning Augments Bidirectional Immune Tolerance Via Regulatory MDSC in a Robust Murine Model of MHC-Mismatched BMT for Beta-Thalassemia

Non-Myeloablative TLI/ATG + Alkylator Conditioning Augments Bidirectional Immune Tolerance Via Regulatory MDSC in a Robust Murine Model of MHC-Mismatched BMT for Beta-Thalassemia

Microrna-17-92 Cluster: Novel Target for Controlling Gvhd While Preserving GVL Effect

Graft-versus-host disease (GVHD) remains a life threatening complication after allogeneic hematopoietic stem cell transplantation (HCT). Donor T cells are the key pathogenic effectors in the induction of GVHD. MicroRNAs (miRs) have been shown to play an important role in orchestrating immune response, among which miR-17-92 cluster is one of the best characterized miR clusters that encodes 6 miRs including 17, 18a, 19a, 20a, 19b-1 and 92-1. Although regulatory functions of miR-17-92 cluster have been elaborated in a variety of immune responses including anti-infection, anti-tumor, and autoimmunity, the role of this miR cluster in the modulation of T-cell response to alloantigens and the development of GVHD has not been explored previously. Based on the previous report that miR-17-92 promotes Th1 responses and inhibits induced regulatory T-cell (iTreg) differentiation in vitro, we hypothesized that blockade of miR-17-92 would constrain T-cell alloresponse and attenuate GVHD.To evaluate the function of miR-17-92 on T-cell alloresponse, we utilized the mice with miR-17-92 conditional knock-out (KO) on T cells as donors, and compared the alloresponse of WT and KO T cells after allogeneic bone marrow transplantation (allo-BMT). We observed that KO T cells had substantially reduced ability to proliferate and produce IFNγ as compared to WT counterparts 4 days after cell transfer. Interestingly, CD4 but not CD8 KO T cells had increased cell death in the population of fast-dividing T cells. Thus, miR-17-92 cluster promotes activation and expansion of both CD4 and CD8 T cells, and inhibits activation-induced cell death of CD4 but not CD8 T cells at the early stage of alloresponse in vivo. We further evaluated the role of miR-17-92 on T cells in the development of acute GVHD in a fully MHC-mismatched BMT model. In sharp contrast to WT T cells that caused severe GVHD and resulted in 100% mortality of the recipients, KO T cells were impaired in causing severe GVHD reflected by mild clinical manifestations and no mortality. These observations were extended to MHC-matched but minor antigen-mismatched as well as haploidentical BMT models that are more clinically relevant. We next addressed the critical question whether T cells deficient for miR-17-92 are still capable of mediating graft-versus-leukemia (GVL) effect. Using A20 lymphoma and P815 mastocytoma cell lines, we demonstrated that the KO T cells essentially retained the GVL activity in MHC-mismatched and haploidentical BMT model, respectively.Mechanistic studies revealed that miR-17-92 promoted CD4 T-cell proliferation, survival, migration to target organs, and Th1-differentiation, but reduced Th2-differentiation and iTreg generation. However, miR-17-92 had less impact on CD8 T-cell proliferation, survival, IFNγ production, and cytolytic activity reflected by granzyme B and CD107a expression. Moreover, miR-17-92 negatively regulated TNFα production by both CD4 and CD8 T cells. We therefore conclude that miR-17-92 cluster is required for T cells to induce severe GVHD, but it is dispensable for T cells to mediate the GVL effect.To increase translational potential of our findings, we designed the locked nucleic acid (LNA) antagomirs specific for miR-17 or miR-19, which have been reported to be the key members in this cluster. We observed that the treatment with anti-miR-17 significantly inhibited T-cell expansion and IFNγ production in response to alloantigen in vivo, and anti-miR-19 was more effective. Furthermore, our ongoing experiment showed the treatment with anti-miR-17 or anti-miR-19 was able to considerably attenuate the severity of GVHD as compared to scrambled antagomir in a MHC-mismatched BMT model.Taken together, the current work reveals that miR-17-92 cluster is essential for T-cell alloresponse and GVHD development, and validates miR-17-92 cluster as promising therapeutic target for the control of GVHD while preserving GVL activity in allogeneic HCT. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Activated MHC-mismatched T helper-1 lymphocyte infusion enhances GvL with limited GvHD.

DLI is traditionally used to provide graft-versus-leukemia (GvL) effects when given to patients relapsing post-hematopoietic cell transplantation (HCT). However, it is often associated with significant GvHD and has only modest efficacy against acute leukemias. Therefore, novel cellular therapies are needed to improve the outcome of high-risk or relapsed leukemia patients following HCT. Activated T helper-1 (aTh-1) lymphocytes are CD4(+)CD25(+)CD40L(+)CD62L(lo) effector memory cells that produce large amounts of IFN-γ and TNF-α. We demonstrate that post-transplant adoptive aTh-1 cell therapy enhances GvL with limited GvHD in an MHC-mismatched murine BMT model. aTh-1 infusions result in superior leukemia-free survival when compared with unstimulated splenocytes (SC), purified CD4(+) T-cells and T-cell-enriched SC. aTh-1 cells display cytotoxicity against A20 leukemia cells in vitro and persist in vivo for at least 2 months following adoptive transfer. Furthermore, in contrast to unstimulated SC, aTh-1 cell infusion is associated with only transient, mild suppression of donor-derived hematopoiesis. aTh-1 cell therapy is safe, effective and warrants further investigation as an alternative to DLI.

Targeting STAT1 Separates Graft-Versus-Host Disease (GVHD) and Graft-Versus-Leukemia (GVL) Effects in Mice

Abstract 2973We have recently shown that loss of STAT1 signaling in donor T cells significantly reduces acute GVHD in both MHC-mismatched and mHA-mismatched BMT and is associated with increased expansion of Treg cells. In this study we further addressed the role of STAT1 in GVL reactions and the underlying mechanisms. We first sought to delineate the role of STAT1 in the generation of CD8+ CTL against host-type spleen cells and A20 lymphoma targets using standard CML assays. Whole spleen cells (SPCs) or CD8+ selected STAT1+/+ or STAT1−/− cells from 129Sv mice (H2b) were stimulated for 5 days with irradiated BALB/c (H2d) spleen cells or A20 cells and then assessed for CTL activity against 51Cr-labelled target cells. STAT1-deficient SPCs appeared to have reduced CTL activity against host SPCs and host type A20 lymphoma cells compared to STAT1+/+ splenocytes. In contrast, isolated STAT1-deficient CD8+ cells had significantly increased CTL activity against both host-type targets and A20 cells. We then assessed the role of STAT1 in mediating GVL effects in vivo using a fully MHC-mismatched (129 [H2b]→BALB/c [H2d]) strain combination and the A20 lymphoma model. Lethally irradiated (800rad) BALB/c mice received T cell-depleted (TCD) wildtype 129 bone marrow cells (BMCs, 5×10E6) plus pan-T cells (5×10E5) from either 129.STAT1−/− or 129.STAT1+/+ donors. In addition, recipients were injected with 5×10E5 luciferase-expressing A20 lymphoma cells (A20-luc) on day 0. As expected animals receiving STAT1-deficient T cells showed significantly reduced GVHD-induced mortality (mean survival time [MST] not reached versus 11 days, p<0.001). Mice receiving TCD BMCs alone plus A20-luc had a MST of 25 days after BMT with all animals succumbing to death by tumor. Mice receiving TCD BMCs plus wild type pan-T cells and challenged with A20-luc died before day 20 due to severe GVHD (MST 10 days post-BMT). In contrast, mice receiving STAT1−/− pan-T and A20-luc cells showed significantly prolonged survival (MST 43 days post-BMT, p=0.0027, Log-rank test). Sustained GVL effects of STAT1-deficient splenocytes against A20-luc cells were further confirmed by bioluminescence imaging on days +10 and days +20 post-BMT. As we recently reported that absence of STAT1 in donor lymphocytes impairs Th1 differentiation and enhances Treg generation in CD4+ lymphocytes, we further investigated wether absence of STAT1 would influence differentiation of CD8+ cells. Our data revealed that CD8+ cells lacking STAT1 failed to be activated into IFN- γ-producing cells upon anti-CD3/CD28 stimulation. However, when cultured under Th1 conditions (i.e. in the presence of IL-12 and neutralizing anti-IL4 antibodies), IFN- γ secretion could be restored. This intact CD8+ cell differentiation was also observed in vivo on day +6 post-BMT in recipients of STAT1−/− BMCs plus splenocytes where we were able to detect comparable levels of IFN- γ-expressing CD8+ cells compared to recipients of STAT1+/+ donor grafts in both MHC- and mHA-mismatched BMT settings. Furthermore, we could demonstrate preserved CTL function of STAT1-deficient CD8+ in vivo. For that purpose BALB/c mice were reconstituted with 129 wildtype BMC together with either STAT1+/+ or STAT1−/− CD8 T cells. On day+37 post-BMT mice were infused with CFSE-labelled host splenocytes as targets cells. Similar clearance of CFSE-labelled target cells was observed between STAT1-deficient and STAT1 wildtype cells. These results suggest that STAT1 in donor T cells may serve as a potential target to separate GVHD and GVL through promoting Treg generation from CD4+ cells while conserving or even promoting Tc1 differentiation of CD8+ cells. Disclosures:Lentzsch:Celgene: Consultancy, Research Funding; Onyx: Consultancy; Genzyme: Consultancy; Centocor Ortho Biotech: Research Funding.

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.

In Vitro expanded STAT1−/− Regulatory T Cells Prevent Acute Graft Versus Host Disease (GVHD) and Promote Donor Cell Engraftment In Allogeneic Fully MHC-Mismatched Bone Marrow Transplant

AbstractAbstract 732Treg cells have been recognized as critical regulators of the immune response and shown to prevent the development of GVHD. However, little is known about of the role of STAT1 signaling in Treg cells during the development of GVHD. In this study, we tried to investigate how STAT1 signaling controls donor Treg development and function in the setting of GVHD. For this purpose we studied the role of STAT1 in natural and inducible Treg (nTreg and iTreg, respectively). To better understand the influence of STAT1-deficiency on the proliferation of nTreg cells, purified splenic STAT1−/− or STAT1+/+ CD4+CD25+ cells were labeled with Carboxyfluorescein succinimidyl ester (CFSE) and cultured on anti-CD3 coated plates in the presence of anti-CD28 and IL-2 for 3 days and analyzed for proliferation and viability. After 72h of in vitro culture 50% of the STAT1+/+ starting population were no longer viable compared to only 10% of STAT1−/− cells. Furthermore, we noted a significantly increased expansion of STAT1-deficient CD4+CD25+Foxp3+ Treg cells compared to STAT1+/+ Treg cells (p<0.001). In line with these findings, STAT1-deficiency resulted in a significantly higher proportion of CFSElo cells indicating vigorous proliferation (85% Foxp3+CFSElo in STAT1−/− compared to only 65% Foxp3+CFSElo in STAT1+/+ Treg cells. Furthermore, at the end of the culture 30% of the STAT1+/+ CD4+CD25+ population were Foxp3-negative compared to only 10% of the STAT1−/− cells. We next determined the impact of STAT1 on the generation of iTreg cells in vitro. For this purpose CD4+CD25− cells from STAT1−/− or STAT1+/+ mice were cultured for 3 days on anti-CD3 coated plates in the presence of anti-CD28 antibodies, hTGF-β, mIL-2, anti-IFN-γ and anti-IL-4 for 3 days. Compared to STAT1+/+, we observed significantly enhanced generation of iTregs from STAT1−/− splenocytes (19.9%±3.0% vs. 10.6%±1.3%, p=0.008). We then performed studies to assess the in vivo generation of iTreg. For that purpose BALB/c mice were reconstituted with T Cell Depleted (TCD) 129.STAT1+/+Bone Marrow Cells (BMC) following lethal irradiation and recipients were co-injected with CD4+CD25− cells purified from either 129.STAT1+/+ or 129.STAT1−/− splenocytes. We again noted a significantly higher proportion of CD4+CD25+ Foxp3+ cells in recipients of CD4+CD25−STAT1−/− cells compared to recipients of STAT1+/+ T cells indicating a significantly increased conversion of CD4+CD25- cells into Treg cells. To confirm the in vitro results we tested the functional ability of in vitro expanded (using anti-CD3, anti-CD28, IL-2 and TGF-β) STAT1+/+ or STAT1−/− Treg cells to block induction of GVHD. GVHD was induced in BALB/c mice following lethal irradiation (800rad) and fully MHC-mismatched BMT using 129.STAT1+/+ bone marrow cells plus 129.STAT+/+ conventional T cells (Tcon). Animals were co-injected with expanded Treg cells from either 129.STAT1+/+ or 129.STAT1−/− donors at a ratio of 1:1 or 1:4 (Treg:Tcon). STAT1−/− or STAT1+/+ Treg cells were equipotent in completely preventing GVHD mortality. However, compared to recipients of STAT1+/+ Treg recipients of STAT1−/− Treg showed reduced signs of GVHD morbidity as determined by a significantly improved weight development. Furthermore, recipients of STAT1−/− Treg showed significantly increased donor cell engraftment compared to recipients of STAT1+/+Treg (donor CD4+ [87% vs. 60%, p=0.03], CD8+[99% vs. 96%, p=0.04], Mac1+[96% vs. 77%, p=0.02] and B220+[100% vs. 96%, p=0.007]) cells in the recipient spleen. These observations clearly demonstrate that STAT1 is a critical regulator of Treg cell development and expansion and that targeting STAT1 in CD4+ T cells may facilitate in vitro and in vivo generation/expansion of Treg cells for therapeutic use in GVHD while also promoting donor cell engraftment. Disclosures:Lentzsch:Celgene Corp: Research Funding. Mapara:Resolvyx: Research Funding; Gentium: stocks.

Inhibition of Graft-Versus-Host Disease (GVHD) by Histone Deacetylase Inhibitor (HDAC) SAHA Leads to Downregulation of STAT1 Activation.

Histone deacetylase (HDAC) inhibitors have been shown to reduce development of graft versus host disease [GVHD] following allogeneic bone marrow transplantation [BMT]. Administration of the HDAC inhibitor suberonylanilide hydroxamic acid [SAHA] resulted in a significantly reduced GVHD-dependent mortality following fully MHC-mismatched allogeneic BMT. Median Survival Time (MST) for vehicle and SAHA-treated mice were 7.5 days and 38 days respectively. However, SAHA treatment did not affect T cell activation nor T cell expansion in vitro and in vivo as determined by MLR assays, phenotypic analysis of donor T cells with regard to expression of the CD25 activation antigen and calculation of donor CD4+ and CD8+ T cell numbers on days +3 and +6 post-BMT. Thus, SAHA treatment was not able to inhibit the strong upregulation of CD25 antigen on CD8+ T cells observed during induction of GVHD on days +3 and +6 post-BMT. We therefore focused on the effects of SAHA treatment on efferent immune effects including cytokine secretion and intracellular signaling events in vitro and in vivo following GVHD induction. SAHA treatment broadly inhibited lipopolysaccharide [LPS] and allo-antigen-induced cytokine/chemokine secretion in vitro like MIP-1-α, IP-10, IFN-γ, TNF-α and IL-6 and led also to a significant decrease in IFN-γ and TNF-α levels in vivo following induction of GVHD. Concomitantly, SAHA treatment inhibited phosphorylation of STAT1 and STAT3 in response to LPS and allo-activation in vitro. Furthermore, analysis of liver tissue and spleens from SAHA-treated animals with GVHD showed a significant decrease in phosphorylated STAT1. In contrast SAHA treatment had only moderate effects on p38 or ERK1,2 Mitogen-activated Protein Kinase (MAPK) pathway underscoring the relevance of the inhibition of the STAT1 pathway. In conclusion, GVHD is associated with a strong induction of phosphorylation of STAT1 in the liver and spleen and SAHA-dependent reduction of GVHD is associated with systemic and local inhibition of pSTAT1 and modulation of the inflammatory cytokine milieu during the efferent immune response.

Separation of GvH- and GvL-Immune Responses in Allogeneic Stem Cell Transplantation by CD95-Mediated Depletion of Alloreactive T-Cells Using CD178 Based Proapoptotic Chimeric Molecules.

Depletion of donor T cells (TCD) from the graft effectively ameliorates graft-vs-host (GvH) disease (GvHD) in allogeneic stem cell transplantation (SCT). TCD, however, is associated with impaired engraftment, immunosuppression, and abrogation of the beneficial graft-vs-leukemia (GvL) effect. Thus, based on previous results (Hartwig et al., Blood 2002) to eliminate alloreactive specificities but preserve oligoclonal antiviral- and GvL- immunity we explored proapoptotic chimeric molecules composed of the extracellular domains of human CD178 (FasL) and CD28 or RANK to selectively deplete GvH-reactive T cells by AICD in vitro and in vivo. As these fusion proteins are expressed as soluble Flag-tagged trimers and reconstitute bioactivity only upon binding to well defined cell-surface molecules (Samel et al., J. Biol. Chem., 2003), they can be specifically targeted onto e.g. recipient derived DC or donor T cells via the molecules CD80/86 or RANKL/TRANCE, respectively.Using an MHC-mismatched murine BMT model proliferative responses of purified CD90+ alloreactive T cells from C57BL/6 (H-2b) mice were strongly reduced following stimulation with DC from F1 (C57BL/6xBalb/c; H-2bxd) mice in vitro in the presence of CD178-CD28 or CD178-RANK to levels comparably obtained with agonistic anti-CD95 mAb (Jo2). Depletion was specific to alloantigen since H-2d restricted Hemagglutinin (HA)-peptide specific T cells titrated into the system persisted among retained T cells.In addition, adoptive transfer experiments of H-2b derived CD90+ T cells depleted of alloreactivity in vitro prior to injection into irradiated F1 recipients confirmed the in vitro results as these mice did not develop clinical signs of GvHD as compared to controls.Moreover, in current studies the CD178-fusion proteins are applied in vivo in our BMT model by injecting 2.5 to 5.0 x 106 syngeneic (H-2bxd) DC preloaded with CD178-CD28 into lethally irradiated F1 mice reconstituted with 1 x 107 TCD H-2b donor marrow cells. Alternatively, soluble CD178-fusion proteins are applied i.v. following BMT. Preliminary results obtained in first experiments suggest that host DC expressing cell surface bound CD178 fusion proteins confer protection to acute GvH-reactions in recipients challenged with purified CD90+ T cells of H-2b origin as these mice show prolonged survival compared to controls. Furthermore, studies on evaluating GvL-reactivity in this model using donor T cells from HA-primed Balb/c mice and A20 cells expressing HA as a defined tumor surrogate antigen are in progress, and their results will be discussed.Taken together, these results suggest that CD178-based proapoptotic fusion proteins devoid of systemic toxicity due to cell-surface antigen-restricted activation might provide a promising tool to separate GvH- and GvL-responses in vivo without inducing severe side effects on CD95-mediated apoptosis in sensitive tissues.

ICSBP Gene Deficient Mice Are Highly Susceptible to the Development of Graft-versus-Host Disease (GVHD) Following Administration of Delayed Donor Lymphocyte Infusions (DLI).

Delayed administration of DLI to mixed hematopoietic chimeras following allogeneic bone marrow transplantation (alloBMT) leads to conversion from mixed to full donor chimerism without GVHD. However, in the clinical setting development of GVHD is still the most common complication. Donor T cells are the main mediators of GVHD in alloBMT. However, the precise role of host dendritic cells (DC) and their different subsets needs still to be elucidated. CD8a+ DCs seem to have a regulatory function and to reduce experimental GVHD. Recently, it has been shown that the ICSBP (Interferon Consensus Sequence Binding Protein) gene is essential for the development and activation of CD8a+ DCs. ICSBP-gene deficient mice (ICSBP KO) are characterized by several immune defects and systemic expansion of myeloid cells mimicking human CML and may be therefore an attractive in vivo model to evaluate the role of DCs in alloBMT. The aim of the study was to evaluate the 1. feasibility of inducing mixed hematopoietic chimerism following nonmyeloablative conditioning in ICSBP KO mice using fully MHC-mismatched BMT (Balb/c [H2Dd] to B6 x 129 [H2b]), and 2. susceptibility of ICSBP KO mice to the development of GVHD following administration of DLI. Nonmyeloablative conditioning consisted of in-vivo T cell depletion (anti-CD4-[GK1.5], anti-CD8-[2,43] mAb, d-5), fludarabine (30 mg/kg, d-4 to d-2), cyclophosphamid (200 mg/kg, d-1) and 3 Gy TBI (d0) followed by i.v. injection of 1,5 x 10E7 Balb/c bone marrow cells. Stable mixed chimerism was observed in ICSBP wildtype (wt) mice through week 35 post-BMT without signs of clinical GVHD. In contrast, we observed increasing donor CD4 and CD8 T cell chimerism over time in ICSBP KO mice suggesting a lack of donor T cell tolerance. Furthermore, chimerism was higher in granulocyte, B cells and monocyte compared to ICSBP wt mice. In a second experiment we were able to observe spontaneous conversion to full donor hematopoietic chimerism in 2 of 9 mice after nonmyeloablative BMT. In two separate experiments ICSBP wt or KO mice with mixed chimerism received (DLI) on day 35 post BMT. After injection of DLI all wt mice (n = 10) showed a rapid conversion to full donor chimerism and remained healthy until the end of the observation period without signs of GVHD. Despite myeloid hyperproliferation all ICSBP KO mice showed conversion to full donor chimerism. Furthermore, in ICSBP KO mice conversion to full donor chimerism was associated with development of severe GVHD (n = 9). In vitro studies using mixed lymphocyte reactions showed that splenic stimulator cells from ICSBP KO mice had a higher stimulatory capacity compared to wt stimulators (SI 1,5 versus 2,3). Using the same mixed lymphocyte culture (MLC) conditions, supernatants collected from BALB/c responders with ICSBP KO stimulators had higher levels of TNF-alpha and IFN-g compared to MLC using ICSBP wt stimulators. In conclusion, following alloBMT a defective development of host-specific tolerance and high susceptibility for GVHD in ICSBP KO mice is suggested. Further studies are warranted to delineate the precise underlying mechanism by addressing the potential contribution of defective regulatory host T cells and the lack of host CD8a+ DCs in ICSBP KO mice.

IFN-gamma-mediated prevention of graft-versus-host disease: pharmacodynamic studies and influence on proliferative capacity of chimeric spleen cells.

Recently we demonstrated that prolonged administration of IFN-gamma prevented the development of GVHD in a MHC-mismatched murine BMT model. Treatment with IFN-gamma allowed the development of mature donor-derived allo-tolerant immunocompetent cells in complete chimeric recipients. Here we present data on the pharmacodynamics of this cytokine-mediated protection against GVHD. Treatment with 50000 U IFN-gamma twice weekly for a period of 5 weeks, starting at the day of BMT, was shown to be the optimal treatment protocol, resulting in complete prevention of GVHD-related mortality. Treatment during 1 week with a three-fold higher weekly dose of IFN-gamma (50000 U six times) did not result in significantly improved survival. The start of IFN-gamma administration was a critical factor since a delay of 3 days from the time of BMT resulted in substantial GVHD-induced mortality. Furthermore, it was shown that IFN-gamma treatment inhibited the spontaneous and Con-A-induced proliferation of T cells at 7-14 days after BMT, which is the critical period for the initiation of acute GVHD. However, long-term survivors after IFN-gamma treatment showed a recovery of immunity in contrast to long-term survivors of saline-injected animals, as tested by Con-A responsiveness. It seems that injection of high dose IFN-gamma suppresses the response of potentially alloreactive donor T cells during what normally is the initiation phase of the GVH reaction (GVHR), resulting in the abrogation of GVHD.

A decrease in graft-vs.-host disease without loss of graft-vs.-leukemia reactivity after MHC-matched bone marrow transplantation by selective depletion of donor NK cells in vivo.

It is thought that natural killer cells may play a role in graft-vs.-host reactions after allogeneic bone marrow transplantation, but the use of NK cell-specific reagents has been limited. In this report, an NK allele-specific monoclonal antibody, anti-NK 1.1, was used to study the impact of in vivo donor NK cell depletion on GVH disease, graft-vs.-leukemia (GVL) reactivity and donor T cell chimerism after allogeneic murine BMT. AKR/J (H-2k) recipient mice were preconditioned with suboptimal irradiation (9 Gy = LD50) and transplanted with major histocompatibility complex-matched B10.BR (H-2k) BM cells with or without added spleen cells as a source of T cells. The addition of increasing numbers of spleen cells to the BM inoculum produced GVHD of varying intensities. The beneficial effect of NK depletion on GVHD was dependent on the intensity of the GVH reaction. Donor NK cell depletion had no effect on the survival of mice with severe GVHD after MHC-matched BMT (B10.BR into AKR) or after MHC-mismatched BMT (B10.BR into DBA/2; H-2k into H-2d). However, donor NK depletion increased survival of AKR hosts given sufficient B10.BR splenic T cells to induce mild-to-moderate GVHD. Ex vivo depletion of donor CD8+ T cells also reduced GVH-associated mortality, but the use of both CD8 and NK depletion offered no improvement over either alone, suggesting an interaction between CD8+ and NK 1.1+ cells. In contrast to CD8 depletion, donor NK depletion did not compromise the rapid and complete establishment of donor T cell chimerism nor the ability of chimeras to mount an effective GVL reaction. Thus, elimination of donor NK cells provides an alternate strategy for reducing GVHD without loss of GVL reactivity following MHC-matched allogeneic BMT.

INDUCTION OF KIDNEY TRANSPLANTATION TOLERANCE ACROSS MAJOR HISTOCOMPATIBILITY COMPLEX BARRIERS BY BONE MARROW TRANSPLANTATION IN MINIATURE SWINE

Previous studies from our laboratory have shown that permanent lymphohematopoietic chimerism can be induced in MHC-disparate miniature swine by bone marrow transplantation after lethal total-body irradiation. The purpose of the present study was to determine in this large animal model whether such chimerism would lead to permanent tolerance to a vascularized allograft without a requirement for exogenous immunosuppression. Eight miniature swine that had received MHC-mismatched BMT more than five months earlier underwent kidney transplantation (KTx) from a donor MHC matched (n = 5) or MHC mismatched (n = 3) with the BMT donor. All animals had regained in vitro responsiveness to third-party MHC antigens, as measured by mixed lymphocyte reaction (MLR), before KTx but remained nonresponsive to MHC antigens of the BMT donor and self. All three animals that received KTx mismatched for BMT donor MHC rejected promptly (mean survival time 7.0 days). Of the five animals that received KTx matched for BMT donor MHC, four showed no evidence of rejection and have functioning KTx greater than 200 days after KTx. The fifth animal had excellent renal function for 60 days but then developed a slowly rising BUN and serum creatinine, and died 75 days after KTx. The course of this animal's rejection is consistent with that previously described for rejection due to minor antigen disparities. The difference in survival of KTx matched or mismatched for the MHC of the BMT donor was statistically significant (P = 0.0062). The survival of KTx matched for the MHC of the BMT donor was significantly different from that of control animals without BMT receiving KTx mismatched for MHC (P = 0.0018). We therefore conclude that BMT is an effective means for induction of tolerance to an MHC mismatched KTx in this large animal model.