Deficiency of transcription factor RelB perturbs myeloid and DC development by hematopoietic-extrinsic mechanisms

RelB is an NF-κB family transcription factor activated in the noncanonical pathway downstream of NF-κB–inducing kinase (NIK) and TNF receptor family members including lymphotoxin-β receptor (LTβR) and CD40. Early analysis suggested that RelB is required for classical dendritic cell (cDC) development based on a severe reduction of cDCs in Relb−/− mice associated with profound myeloid expansion and perturbations in B and T cells. Subsequent analysis of radiation chimeras generated from wild-type and Relb−/− bone marrow showed that RelB exerts cell-extrinsic actions on some lineages, but it has remained unclear whether the impact of RelB on cDC development is cell-intrinsic or -extrinsic. Here, we reevaluated the role of RelB in cDC and myeloid development using a series of radiation chimeras. We found that there was no cell-intrinsic requirement for RelB for development of most cDC subsets, except for the Notch2- and LTβR-dependent subset of splenic CD4+ cDC2s. These results identify a relatively restricted role of RelB in DC development. Moreover, the myeloid expansion in Relb−/− mice resulted from hematopoietic-extrinsic actions of RelB. This result suggests that there is an unrecognized but critical role for RelB within the nonhematopoietic niche that controls normal myelopoiesis.

Expression Of PD-L1 Is Required To Control Acute GvHD In Allo-HSCT Recipients

Background Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the only curative treatment for patients with both malignant and non-malignant hematologic disorders. But life-threatening graft-vs-host diseases (GvHD) caused by alloreactive donor T cells limits its clinical use. Alloreactive T cells are also required for graft-vs-leukemia (GvL) and to fight opportunistic infections. Hence, a method that modulates donor T cells activity to reduce GvHD but to retain GvL effect is highly desirable. The inhibitory receptor programed cell death-1 (PD-1) reduces T cell activation through binding with its ligand PD-L1 or PD-L2. Interaction between PD-1 and PD-L1 induces cardiac allograft tolerance and expression of PD-L1 is upregulated in presence of inflammatory stimuli. Here, we studied the role of PD-L1 expression on hematologic and non-hematologic tissues and PD-1 - PD-L1 binding in the development of GvHD. Methods Wild type C57BL/6 (WT B6), PD-L1 knock out B6 (KO) and PD-L2 KO B6 mice were transplanted with 2 x106 splenic T cells and bone marrow (BM) cells from H-2K B10.BR donors. The average acute GvHD scores were determined by combining the GvHD scores obtained from the histological tissue sections of small intestine, large intestine and liver, and weight-loss, posture, activity, fur texture and skin integrity data following standard published procedures. The activation status of splenic T cells was analyzed by flow cytometry. Serum cytokines were determined by using 26 plex Luminex assay. The requirement of hematopoietic and or non-hematopoietic tissues expressing PD-L1 to reduce GvHD was investigated by generating radiation chimeras using WT B6 mice engrafted with PD-L1 KO BM and vice versa. Two months later radiation chimeras were transplanted again with 2 x106 splenic T cells along with 2 x106 BM cells from congenic na•ve H-2K donors. The role of PD-L1 expressing donor hematopoietic cells on the development of acute GvHD was tested by transplanting B10.BR mice with donor PD-L1 KO B6 and WT B6 splenocytes. Results PD-L1 KO B6 recipients had significantly increased acute GvHD (scores 1.68 ± 0.07) compared with WT B6 GvHD (0.78 ± 0.024, p<0.0005) and B6 PD-L2 KO B6 (0.86 ± 0.14, p<0.0005) within day 8 after transplant. All PD-L1 KO B6 recipients had severe GvHD with >25% weight loss on day 8 after transplant and were sacrificed. The WT B6 and PD-L2 KO B6 recipients survived 75% and 80%, respectively until 34 days of transplantation with similar levels of chronic GvHD. To test whether excessive activation of donor T cells caused severe acute GvHD in PD-L1 KO B6 recipients, we determined the activation status of donor T cells in the spleen. The numbers of donor CD4+ and CD8+ T cells expressing ICOS-1 and PD-1 in the spleen were significantly higher (p<0.005) in PD-L1 KO B6 recipients compared with the WT B6 and PD-L2 KO B6 recipients. Additionally, significantly increased levels of serum inflammatory cytokines (IFN-g and TNF-a) were also detected in the PD-L1 KO B6 recipients compared with the WT B6 recipients on day 8 post transplant (Figure 1). Using WT B6 or PD-L1KO hematopoietic cell radiation chimeras as allo-HSCT recipients, we further confirmed that both allo-HSCT radiation chimeras having PD-L1 expressing hematopoietic (10% survival, open square) and non-hematopoietic cells (10% survival, closed triangle) were required to protect from GvHD (Figure 2). We next investigate whether PD-L1 KO donor cells cause increased GvHD. The B10.BR recipients transplanted with donor PD-L1 KO B6 splenocytes had 70% survival while the same recipients transplanted with WT B6 donor splenocytes had only 20% survival until 100 days post transplant. These data suggest that only PD-L1 expressed by host tissues is required to inhibit the development of GvHD. In summary, our data suggest that PD-L1 expressed by host tissues are required to control GvHD and method(s) that enhance expression of PD-L1 in allo-HSCT recipients may represent a novel strategy to control GvHD. Disclosures: No relevant conflicts of interest to declare.

Distinct IFN-Gamma Dependent Effect of the TLR5 Agonist, Flagellin On NK and CD4+ T Cell Immunity Against CMV

Abstract 255 Background: Opportunistic CMV infection remains a major clinical complication in allogeneic BMT (allo-BMT) recipients. We previously published that peri-transplant treatment of highly purified flagellin, a TLR5 agonist extracted from S. typhimurium reduced GvHD in lethally irradiated allo-BMT recipients by reducing early activation and proliferation of donor T cells. Flagellin-treated recipients had enhanced lymphoid immune reconstitution and were protected from lethal MCMV infection. CMV initiates innate immunity through the activation of Toll-like receptors (TLR) 9, 3 and 2 but direct enhancement of anti-CMV immunity through TLR5 has not been studied. Methods: To further explore the effects of a TLR5 agonist on anti-CMV immunity, we infected flagellin-treated C57BL/6 (B6), TLR5KO and Myd88KO mice (B6 background) with sub lethal dose (1×105pfu/mouse i.p) of MCMV. Anti-CMV immunity of NK and T-cells were determined by measuring surface activation markers on the NK and T-cells. Flagellin-treated and MCMV infected IL-12KO, IFN-γRKO and IDO KO mice were used to study the role of IL-12, IFN-γ and IDO on anti-CMV immunity. The relationship between the MCMV lethality and flagellin signaling in hematopoietic or epithelial tissues expressing TLR5 was further investigated by generating radiation chimeras, using WT mice engrafted with TLR5 KO bone marrow (BM) and TLR5 KO mice engrafted with WT B6 BM. Chimeric recipients with WT or TLR5KO hematopoietic cells (or the reverse) were then treated with flagellin or PBS and infected with a sub lethal dose of MCMV infection. Results: Without MCMV infection, flagellin treatment significantly increased numbers of NK cells (p= 0.001) and KLRG1+ NK cells (p=0.009) in WT B6 mice compared with the PBS-treated control mice. Following MCMV infection (day 3), there were significantly increased numbers of splenic NK cells (p= 0.01) and KLRG1+ NK cells (p=0.0008) in flagellin-treated mice compared with the control mice. Flagellin-treatment also enhanced viral clearance, tending to decrease liver viral loads in WT B6 mice. In contrast, MCMV-infected and flagellin-treated TLR5 KO or Myd88KO mice had significantly increased liver viral loads (p=00001) compared with the flagellin- and PBS-treated B6 mice on day 3 post MCMV infection. While flagellin-treatment increased the numbers and activation status of NK cells, it decreased the numbers of activated (CD69+, ICOS+ and KLRG1+) CD4+ T cells on day 3 post MCMV infection. On day 10 post MCMV infection, enhanced numbers of splenic CD8+ T cells and CMV-specific tetramer+ CD8+ T cells (p<0.05) were detected in flagellin-treated mice compared with controls. The numbers of splenic CD4+ T cells increased >2 fold (p= 0.008) associated with significantly increased numbers of splenic CD4+CD25+ Foxp3+ regulatory T cells (p<0.05) on day 10 post MCMV infection in flagellin-treated mice compared with the control mice. Flagellin-induced anti-CMV immunity required IFN-γ signaling as flagellin-treated IFN-γRKO mice had markedly increased susceptibility to MCMV infection whereas flagellin-treated IL-12 KO and IDO KO mice had significantly reduced liver viral loads compared to the PBS-treated IL-12 KO and IDO KO mice on day 10 post MCMV infection. To explore interaction of flagellin with TLR5+ host hematopoietic cells and TLR5+host gut epithelium on CMV immunity, we next infected radiation chimeras expressing either TLR5 on hematopoietic cells or on gut epithelium with sub lethal i.p dose of MCMV. Flagellin-treated TLR5 KO BM → B6 radiation chimeras (TLR5+ host epithelium) had increased mortality compared with other groups following MCMV infection indicating that TLR5+ hematopoietic cells are required for protective immunity against CMV infection. In summary, flagellin caused selective early increased activation of NK cells and reduced activation of CD4+ T cells in presence of MCMV infection. The protective effect of flagellin on anti-CMV immune responses are IFN-γ dependent but independent of IL-12 and IDO. The TLR5+ hematopoietic cells are required for the protective immunity against CMV infection. These data suggest the clinical importance of flagellin in modulating both innate and adaptive immunity against CMV infection and also as an alternative GvHD prophylaxis in allo-BMT recipients. Disclosures: No relevant conflicts of interest to declare.

Flagellin, a TLR5 Agonist, Reduces GvHD in Allogeneic HSCT Recipients While Enhancing Anti-Viral Immunity: A Novel Therapeutic Approach

Abstract 144 In MHC-mismatched allogeneic hematopoietic stem cell transplantation (allo-HSCT), host antigen specific donor T cells mediate acute and chronic graft-versus-host disease (GvHD). Based upon the radio-protective effects of flagellin, a TLR5 agonist protein (∼50 kDa) extracted from bacterial flagella, we reasoned that flagellin might modulate donor T cells immune responses toward host antigens, reduce GvHD, and improve immune responses to CMV infection in experimental models of allogeneic HSCT. Two 50mg/mouse i.p doses of highly purified flagellin were administered 3 hrs before irradiation and 24 hrs after allo-HSCT in H-2b ^ CB6F1 and H-2k ^ B6 models. GvHD scores were obtained with weekly clinical examination and with histological scoring of intestine, colon, liver and skin at necropsy. Flagellin treatment successfully protected allo-HSCT recipients from acute and chronic GvHDs after transplantation of 5×106 splenocytes and 5×106 T cell depleted (TCD) BM, and significantly increased survival compared to PBS-treated control recipients. Reduced acute GvHD was associated with significant reduction of a) early post-transplant proliferation of donor CD4+ and CD8+ T cells measured by Ki67 and CFSE staining, b) fewer CD62L+, CD69+, CD25+, ICOS-1+ and PD-1+ donor CD4+ and CD8+ T cells compared with the PBS-treated control recipients. Decreased numbers of activated and proliferating donor T cells were associated with significantly reduced pro-inflammatory serum IFN-g, TNF-a, and IL-6 on days 4–10 post transplant in flagellin-treated recipients compared with the PBS-treated recipients. Interestingly, both flagellin-treated recipients and PBS-treated recipients had over 99% donor T cell chimerism at 2 months post transplant. Moreover, MCMV infection on 100+ days post-transplant flagellin-treated mice significantly enhanced anti-viral immunity, including more donor MCMV-peptide-tetramer+ CD8+ T cells in the blood (p<0.05), and less MCMV in the liver on day 10 post infection (p<0.02) compared with the PBS-treated control recipients. Overall immune reconstitution after flagellin-treatment was robust and associated with larger numbers of CD4+CD25+foxp3+ regulatory T cells in the thymus. To further define the role of flagellin-TLR5 agonistic interactions in the reduction of GvHD, we next generated B6 ^ TLR5 KO (KO) and KOB^6 radiation chimeras by transplanting 10 × 106 BM cells from wild-type (WT) B6 or TLR5 KO donors into the congenic CD45.1+ B6 or KO recipients conditioned with 11Gy (5.5Gyx2) TBI. The radiation chimeras were irradiated again with 9.0Gy (4.5Gy × 2) on 60 days after the first transplant and transplanted with 3 × 106 splenocytes and 5 × 106 TCD BM from H-2K congenic donors. Two 50mg doses of flagellin were administered 3 hrs before irradiation and 24 hrs after HSCT. All flagellin-treated B6 ^ B6 radiation chimeras survived with only 12% weight-loss by 80 days post transplant compared with 50% survival among recipients of flagellin-treated B6 ^ KO and 40% survival among KO ^ B6 radiation chimeras. All flagellin-treated KO^ KO and PBS-treated radiation chimeras died within 65 days post transplant. These data suggested that interaction of flagellin with the TLR5 expressing host gut epithelium and donor hematopoietic cells are both required for the maximum protective effect of this TLR5 agonist on GvHD in allogeneic HSCT recipients. Together our data demonstrate that peritransplant administration of flagellin effectively controls acute and chronic GvHD while preserving enhanced post-transplant donor anti-opportunistic immunity. Since flagellin has been found to be safe for use in humans as vaccine adjuvant in a number of clinical trials, the clinical use of flagellin in the setting of allogeneic HSCT is of interest. Disclosures: No relevant conflicts of interest to declare.

Lack of Host Bone Marrow-Derived Interleukin-12 Increased the Incidence of Allograft Rejection in Allogeneic Bone Marrow Transplantation

Abstract 2960 Background: Donor cell engraftment following allogeneic bone marrow transplantation (BMT) is affected by several factors, including immunological major histocompatibility complex (MHC) barriers, the intensity of the conditioning regimen, and the content of T-cells in the graft. The current model for engraftment in allogeneic BMT is that host dendritic cells (DCs) activate donor T-cells which promote engraftment by eliminating radio-resistant cytotoxic host immune cells, especially natural killer (NK) cells and T-cells. To explore the interaction between donor T-cell and host antigen-presenting cells (APC) in engraftment in allogeneic BMT, we focused on the role of interleukin-12 (IL-12), a key cytokine produced mainly by DCs that drives the development of donor type 1 helper T cells (Th1) and type 1 cytotoxic T lymphocytes (Tc1). Methods: Radiation chimeras with >95% donor chimerism were created by transplanting 5 × 106 bone marrow (BM) cells from IL-12 knock out (IL-12 KO) or wild type (WT) B6 (H-2Kb, CD45.2) donors into congenic BL6 Pepboy (B6.SJL-PtprcaPep3b/BoyJ, H-2Kb, CD45.1) mice following lethal 11 Gy irradiation. A second allogeneic BMT was conducted 2 months later using MHC mismatched FVB (H-2q, CD45.1), BA.B10 (H-2Kk, CD45.2, CD90.1) or B10.BR (H-2Kk, CD45.2, CD90.2) donor cells. In vivo bioluminescent imaging (BLI) was performed to analyze the number and in vivo distribution of luciferase+ donor T-cells. The whole-body bioluminescent signal was used as a marker of the donor T cell expansion. Engraftment of donor myeloid cells was determined by flow cytometry using mAbs for specific leukocyte markers expressed on donors and recipients (CD45.1, CD45.2, H-2Kb). Intracellular cytokine expression (IL-4, IL-10, IFN-g) by donor CD4+ and CD8+ T cells was analyzed by flow cytometry. Results: WT BL6→BL6 radiation chimeras recipients showed greater expansion of luciferase+ donor T-cells compared with IL-12 KO BL6→BL6 radiation chimeras recipients and FVB→FVB syngeneic recipients at early time point (2 wks) following 9 Gy re-irradiation and transplantation of 3 × 105 luciferase+ FVB-L2G85 T-cells in combination with 5 × 106 T cell depleted (TCD) BM cells from FVB mice following (Fig 1). At 4 weeks post transplant, more WT BL6→BL6 radiation chimeras achieved myeloid engraftment than IL-12 KO BL6→BL6 radiation chimera recipients(75.0% versus 33.3% respectively, p = 0.086), and the former group had better erythroid engraftment than the latter group (RBC 8.65 ± 1.88 × 1012/L versus 5.67 ± 2.22 × 1012/L respectively, p = 0.011). However, when FVB, WT BL6→BL6 or IL-12 KO BL6→BL6 radiation chimeras recipients were conditioned with a larger dose of irradiation prior to the second transplantation (10 Gy) and received a larger dose of donor T-cells (5 × 105), both the WT BL6→BL6 and IL-12 KO BL6→BL6 radiation chimeras recipients achieved full donor engraftments (85.7% versus 87.5% respectively, p = NS). Donor T cells in allogeneic BMT recipients were Th1/Tc1 polarized, there were no differences in frequencies and total numbers of Th1/Tc1 donor CD4+ and CD8+ T cells comparing recipients of WT BL6→BL6 and IL-12 KO BL6→BL6 radiation chimeras. In spite of an increased irradiation dose and larger number of donor T-cells in the second transplant regimen, no increase in graft versus host disease (GVHD) clinical scores and GVHD-mortality were observed in the recipients of WT BL6→BL6 radiation chimeras compared with recipients of IL-12 KO BL6→BL6 radiation chimeras. Conclusion: These data support a role for host BM-derived IL-12 in facilitating engraftment in allogeneic BMT following a reduced dose (9 Gy) radiation. The lack of host BM-derived IL-12 expression led to allograft rejection. Rejection could be overcome by increasing the dose of pre-transplant irradiation and the content of donor T-cells without causing lethal GVHD. As the main source of host BM-derived IL-12, recipient APC thus play an important role in donor T-cell activation. As has been previously demonstrated in a murine BMT model, the addition of IL-12 in the peri-transplant period helped to separate graft versus leukemia effects from the GVHD-promoting activity of donor T-cells (Yang, 1997). Patients predicted to be high risk of graft failure may benefit from treatment strategies that contribute to production of IL-12 during the early phases of hematopoietic engraftment. Disclosures: No relevant conflicts of interest to declare.

Adult hematopoietic stem cells require NKAP for maintenance and survival

Steady-state hematopoiesis is sustained through differentiation balanced with proliferation and self-renewal of hematopoietic stem cells (HSCs). Disruption of this balance can lead to hematopoietic failure, as hematopoietic differentiation without self-renewal leads to loss of the HSC pool. We find that conditional knockout mice that delete the transcriptional repressor NKAP in HSCs and all hematopoietic lineages during embryonic development exhibit perinatal lethality and abrogation of hematopoiesis as demonstrated by multilineage defects in lymphocyte, granulocyte, erythrocyte and megakaryocyte development. Inducible deletion of NKAP in adult mice leads to lethality within 2 weeks, at which point hematopoiesis in the bone marrow has halted and HSCs have disappeared. This hematopoietic failure and lethality is cell intrinsic, as radiation chimeras reconstituted with inducible Mx1-cre NKAP conditional knockout bone marrow also succumb with a similar time course. Even in the context of a completely normal bone marrow environment using mixed radiation chimeras, NKAP deletion results in HSC failure. NKAP deletion leads to decreased proliferation and increased apoptosis of HSCs, which is likely due to increased expression of the cyclin-dependent kinase inhibitors p21Cip1/Waf1 and p19Ink4d. These data establish NKAP as one of a very small number of transcriptional regulators that is absolutely required for adult HSC maintenance and survival.