WT B6 Research Articles

Recombinant TLR5 agonist CBLB502 promotes NK cell-mediated anti-CMV immunity in mice.

Prior work using allogeneic bone marrow transplantation (allo-BMT) models showed that peritransplant administration of flagellin, a toll-like receptor 5 (TLR5) agonist protected murine allo-BMT recipients from CMV infection while limiting graft-vs-host disease (GvHD). However, the mechanism by which flagellin-TLR5 interaction promotes anti-CMV immunity was not defined. Here, we investigated the anti-CMV immunity of NK cells in C57BL/6 (B6) mice treated with a highly purified cGMP grade recombinant flagellin variant CBLB502 (rflagellin) followed by murine CMV (mCMV) infection. A single dose of rflagellin administered to mice between 48 to 72 hours prior to MCMV infection resulted in optimal protection from mCMV lethality. Anti-mCMV immunity in rflagellin-treated mice correlated with a significantly reduced liver viral load and increased numbers of Ly49H+ and Ly49D+ activated cytotoxic NK cells. Additionally, the increased anti-mCMV immunity of NK cells was directly correlated with increased numbers of IFN-γ, granzyme B- and CD107a producing NK cells following mCMV infection. rFlagellin-induced anti-mCMV immunity was TLR5-dependent as rflagellin-treated TLR5 KO mice had ∼10-fold increased liver viral load compared with rflagellin-treated WT B6 mice. However, the increased anti-mCMV immunity of NK cells in rflagellin-treated mice is regulated indirectly as mouse NK cells do not express TLR5. Collectively, these data suggest that rflagellin treatment indirectly leads to activation of NK cells, which may be an important adjunct benefit of administering rflagellin in allo-BMT recipients.

Transferred allogenic S1P3 deficient dendritic cells (DCs) interact with recipient splenocytes to prevent kidney ischemic-reperfusion injury (IRI) (IRC5P.459)

Abstract The plasticity of DCs permits phenotypic modulation ex-vivo by gene expression or pharmacological agents. Sphingosine-1-phosphate (S1P), a sphingolipid is the natural ligand for S1Pr1-5s. We tested the ability of S1P3-/-(3KO) DC to suppress kidney IRI. BMDCs from B6 WT (B6DC) or 3KOB6DC were adoptively transferred into (->) Balb/c 1d/7d prior to IRI. Naïve Balb (noDC) had higher plasma creatinine (PCr; mg/dl) compared to sham (1.2±0.17;0.2±0.004,p≤0.01). 3KOB6DCs->Balb/c were protected from IRI (PCr:0.3±0.07,p≤0.01). 3KOB6DCs->Balb/c had higher Treg and less CD4 cells compared to naïve or B6DC mice in spleen. The protection afforded by 3KOB6DCs->Balb/c mice required the recipient mouse to have a spleen; Tregs, CD11c+ DC and T/B cells; Splnx Balb, use of Rag1-/- mice or depletion of recipient Tregs (PC61) or DCs (CD11c-DTR) rendered adoptively transferred 3KODCs ineffective. PKH-26 labeled DCs from WT or 3KO->Balb/c were located in the marginal zone (MZ) of recipient spleen with close proximity to Tregs. WT and 3KO mice injected with latex beads to demonstrate clearance efficiency showed disruption of MZ in 3KO. Adoptive transfer of 3KOB6DCs prior to IRI attenuates injury through interaction with splenocytes, recipient DCs resulting in induction of Tregs through possible disruption of MZ macs. We conclude that genetically induced deficiency or pharmacological blockade of allogenic S1P3DCs could serve as a useful therapeutic approach to IRI or DGF associated with Transplant.

Cross dressing by donor dendritic cells maximizes T cell responses to indirectly presented alloantigen after BMT by promoting immune synapse formation (TRAN3P.903)

Abstract The role of trogocytosis in generating naïve T cell responses is controversial and highly relevant to transplantation. We transplanted B6.CD11cGFP donor bone marrow (BM) grafts (H2Db, IAb, GFP off the CD11c promoter) into irradiated B6D2F1 (H2Db/d, IAb/d,) and tracked donor DC. Using standard and imaging flow cytometry, we noted that all donor cells become “cross-dressed” in high levels of recipient hematopoietic cell-derived MHC early after BMT. To assess the functional consequences of cross-dressed MHC, we transplanted B6.MHCII-/- BM into WT B6 recipients to generate DC cross-dressed with IAb. WT DC induced proliferation in BALB/c CD4 T cells transferred into B6.MHCII-/- recipients but no responses were seen to B6.MHCII-/- DC bearing cross-dressed Ag. Next, DC were generated that were either cross-dressed (B6.MHCII-/-→B6D2F1), bore cross-dressed and indirectly presented Ag (B6→B6D2F1), or could present indirect Ag only (B6→B6.MHCII-/-xD2F1) to the allospecific TEa Tg T cell. T cell proliferation was maximal in response to processed recipient alloAg presented via the indirect pathway in the presence of cross-dressed Ag, markedly reduced to indirect Ag when cross-dressing was absent, and absent to cross-dressed Ag alone. Imaging flow cytometry confirmed that cross-dressing promotes formation of the immunologic synapse. Thus, cross-dressing serves to maximize naïve T cell responses to indirectly presented alloAg and exerts this effect after BMT via the immunologic synapse.

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

BackgroundAllogeneic 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. MethodsWild 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. ResultsPD-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. [Display omitted] [Display omitted] 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.

Atypical E2F Dependent Dysregulation Of Cell Cycling By Microrna-142 Regulates T-Cell Responses and Experimental Graft-Versus-Host Disease

Noncoding microRNAs (miRs) have recently been linked to immune system function. We investigated the role of miR-142, a hematopoietic specific miR, in regulating T cell responses. To understand its specific function in T cell immunity we utilized homologous recombination technology and generated mutant mice bearing a targeted deletion of the miR-142 gene on the B6 background. The homozygous miR-142 knockout (KO) animals were viable, fertile and showed no apparent developmental anomalies. Thymic analyses of the miR142-/- animals demonstrated no significant differences when compared with WT littermate controls in total thymocytes, early thymic progenitors, DP and DN cells. Bone marrow studies demonstrated similar numbers of LSK+ HSCs while analyses of secondary lymphoid organs (spleen and popliteal lymph nodes) demonstrated similar absolute numbers of naïve T cells (CD44low62L+), memory like T cells (CD44hi62L+CCR7- and CD44hi62L-CCR7- cells), CD4+25+Foxp3+ cells, CD69+VLA4+CD3+ cells and weekly peripheral blood examination demonstrated similar lymphocyte counts as the WT littermates.Functional studies, however, demonstrated that when compared with WT T-cells, the KO T-cells showed significantly slower rate of proliferation by CFSE analyses upon stimulation anti-CD3+ and 28+ antibodies (P<0.003). They showed lower IL-2, IFNγ and IL-17 but greater amount of IL-6 production (P<0.001) and demonstrated greater apoptosis (P<0.02). Cell cycling analyses with flow cytometry demonstrated that a significantly greater percent of the miR142-/- T-cells were in the S and G2 phase (P<0.01) when compared with WT T-cells suggesting altered cell cycling. Similar reduction in proliferation, cytokine secretion by the miR142-/- T-cells was also observed upon in vitro stimulation with allogeneic BALB/c DCs.To determine the in vivo relevance of miR142 deficiency in T cells, we next utilized MHC mismatched B6àBALB/c model of allogeneic BMT. BALB/c animals were lethally irradiated (9Gy) and transplanted on day 0 with 5x106 BM from WT B6 animals along with 2x106 splenic CD90+T cells from the WT or miR142-/- donors. The allogeneic animals that received KO T-cells showed significantly less severe clinical, histopathological GVHD (GI tract on days 7 and 21) and mortality (P<0.02). Analyses of donor T cells on day 7 post-BMT demonstrated reduced expansion and IFNγ secretion (P<0.04) but showed no significant differences in the ratio of Treg:conventional T-cells between the WT and KO T-cell allogeneic recipients. To further confirm the specific role of miR142 deficiency, we next treated the WT animals with miR-142 anatgomir (days 1, 3 and 7) and found that it significantly reduced GVHD mortality (P<0.003). The KO T-cells also reduced GVHD mortality in a MHC matched minor mismatched B6→C3H.sw BMT model demonstrating strain independent effects.To further determine the miR142 specific molecular mechanisms we performed extensive bioinformatic analyses. In light of a defect in T cell cycling in miR142-/- T-cells, we focused on the putative miR142 targets that are known to regulate cell cycling. Two of the three bioinformatic programs suggested the following known regulators of cell cycling, EGR2, DAG, all eight E2F transcription factor family (the typical E2F1-6 and atypical E2F7-8) members as putative targets. We next performed DNA microarray analyses to determine differential gene expression patterns in miR142-/- and WT T cells, which demonstrated an increase in the expression (>15x) of only the atypical E2Fs, namely E2F7 and E2F8, but not in any of the other above predicted cell cycle regulating molecules. The increase in the expression of the atypical E2Fs in the miR142-/- T-cells was next confirmed by PCR analyses at baseline (unstimulated) and also sequentially at 6, 12, 24 and 48 hours following in vitro stimulation. Knockdown of E2F7 and E2F8 in miR142-/- T cells with sh-RNA rescued their proliferative responses and corrected the cell cycling defects to the levels comparable to WT T-cells, thus demonstrating that the atypical E2F7 and 8 are critical for miR-142 mediated regulation of T cells.Thus our data show (a) generation of a novel miR142 knockout mouse (b) demonstrate that miR142 regulates T cell responses in vitro and in vivo by targeting atypical E2Fs and (c) suggest that targeting miR142 in vivo with its antagomir might be a novel therapeutic strategy for regulating GVHD. Disclosures:No relevant conflicts of interest to declare.

Enhanced pro-inflammatory profile and Th17 alloresponse by targeting mTORC2 in DC (P2138)

Abstract Mammalian target of rapamycin (mTOR) is an important integrative kinase that regulates immune cell function. mTOR functions in two independent complexes: mTOR complex (mTORC) 1 and 2. The immunosuppressant Rapamycin inhibits mTORC1 but not mTORC2. Our aim was to study the immune regulatory role and underlying mechanistic properties of mTORC2 in DC. We generated a conditional mTORC2 knockout, targeting Rictor. Bone marrow cells were obtained from KO and WT B6 controls, and DC propagated in the presence of IL-4 and GM-CSF, with or without LPS. The phenotype, cytokine production, STAT3 activation and ability of DC to induce allogeneic T cell proliferation were evaluated, and also the responder T cell phenotype and cytokine production. DC differentiation was not affected, while DC yield was slightly reduced in mTORC2-deficient BM cultures compared with WT controls. DC lacking mTORC2 activity displayed similar levels of MHC and co-stimulatory molecules, but diminished B7-H1 expression, increased levels of IL-12p40, diminished STAT3 activation and higher T cell stimulatory ability following LPS stimulation, compared with WT. Staining of allogeneic T cells stimulated by Rictor-KO DC revealed more CD4+IL-17+ cells than those stimulated with WT DC. These novel data indicate that DC lacking mTORC2 activity exhibit an enhanced pro-inflammatory profile with ability to promote Th17 responses. Thus, targeting mTORC2 provides new insight into molecular regulation of DC function.

Obesity induced fibrinogen formation and its effect on adipocyte inflammation and glucose uptake

Obesity and insulin resistance are associated with increased fibrinogen production and inflammation, yet the effect of fibrin degradation products (FDP‐E and FDP‐D) on glucose disposal is completely unknown. We hypothesize that FDP‐E will decrease glucose disposal by increasing inflammation and cytokine production in macrophages.First, we determined the effect of obesity on fibrinogen expression and fibrin deposition. WT B6 mice fed a high fat diet for 15 weeks increased plasma fibrinogen content along with hepatic fibrinogen expression and FDP‐E content increased 4‐fold. Next, we injected FDP‐E directly into S.C fat which induced inflammation and apoptosis. In fibroblasts, FDP‐E reduced cell viability and induced pro‐fibrotic gene expression. 3T3‐L1 adipocytes were treated with FDP‐E or conditioned media (CM) from FDP‐E treated peritoneal macrophages. We found that FDP‐E and CM suppressed glucose uptake in adipocytes and caused a significant increase in M1 proinflammatory biomarkers such as TNF‐á and IL‐6 in macrophages and mature adipocytes with no appreciable effect on M2 antiinflammatory gene expression.Fibrinogen expression and deposition is increased with obesity. The increase in fibrinogen expression and fibrin deposition leads to increased adipocyte inflammation and macrophage infiltration which suppresses glucose uptake and may promote adipose tissue fibrosis.

Neutrophils are responsible for impaired medial smooth muscle cell recovery and exaggerated allograft vasculopathy in aortic allografts exposed to prolonged cold ischemia

Ischemia and reperfusion injury is critical in allograft vasculopathy (AV) development. We have shown that neutrophil-mediated medial smooth muscle cell (SMC) loss precedes AV and that prolonged cold ischemia (CI) impairs medial SMC recovery and accelerates AV development. We hypothesize that neutrophils (NØs) are responsible for failed medial SMC recovery that precedes AV. Aortic transplants were performed between fully disparate C3H/HeJ murine donors and wild-type C57BL/6 (WT B6), B6.129S7-Rag1 (Rag1(-/-); intact innate but no adaptive immunity), and B6.129S-Cybb (NOX2(-/-); NØ loss-of-function) recipients under cyclosporine A immunosuppression. Grafts were exposed to 20 or 60 minutes CI before transplant and harvested at 1 day, 2 weeks, and 8 weeks after transplant. Some WT B6 recipients were treated with remote ischemic pre-conditioning (rIPC). Grafts were assessed for medial SMCs, NØs, and lesion area. The 60-minute vs 20-minute CI grafts exhibited reduced SMC recovery at 2 weeks in WT B6 and Rag1(-/-) recipients (WT B6: p = 0.0009; Rag1(-/-): p = 0.0006). NØ influx was greater in Rag1(-/-) recipients of 60-minute vs 20-minute CI grafts at 1 day (p = 0.0002). The difference in 2-week medial SMC recovery between ischemia groups was abrogated in NOX2(-/-) recipients. At 8 weeks, NOX2(-/-) and rIPC recipients of 60-minute CI grafts exhibited smaller neointimal lesions than B6 recipients (NOX2(-/-): p = 0.0009; rIPC: p = 0.0005). Impaired medial SMC recovery in murine aortic allografts at 2 weeks occurs in the absence of adaptive immunity. Enhanced medial SMC recovery and reduced neointimal lesion formation in NOX2(-/-) and rIPC recipients of 60-minute CI grafts suggest a causal role for NØs in impaired medial SMC repopulation and the development of AV.

TLR3 deficiency induces chronic inflammatory cardiomyopathy in resistant mice following coxsackievirus B3 infection: role for IL-4

Recent findings indicate that TLR3 polymorphisms increase susceptibility to enteroviral myocarditis and inflammatory dilated cardiomyopathy (iDCM) in patients. TLR3 signaling has been found to inhibit coxsackievirus B3 (CVB3) replication and acute myocarditis in mouse models, but its role in the progression from myocarditis to iDCM has not been previously investigated. In this study we found that TLR3 deficiency increased acute (P = 5.9 × 10(-9)) and chronic (P = 6.0 × 10(-7)) myocarditis compared with WT B6.129, a mouse strain that is resistant to chronic myocarditis and iDCM. Using left ventricular in vivo hemodynamic assessment, we found that TLR3-deficient mice developed progressively worse chronic cardiomyopathy. TLR3 deficiency significantly increased viral replication in the heart during acute myocarditis from day 3 through day 12 after infection, but infectious virus was not detected in the heart during chronic disease. TLR3 deficiency increased cytokines associated with a T helper (Th)2 response, including IL-4 (P = 0.03), IL-10 (P = 0.008), IL-13 (P = 0.002), and TGF-β(1) (P = 0.005), and induced a shift to an immunoregulatory phenotype in the heart. However, IL-4-deficient mice had improved heart function during acute CVB3 myocarditis by echocardiography and in vivo hemodynamic assessment compared with wild-type mice, indicating that IL-4 impairs cardiac function during myocarditis. IL-4 deficiency increased regulatory T-cell and macrophage populations, including FoxP3(+) T cells (P = 0.005) and Tim-3(+) macrophages (P = 0.004). Thus, TLR3 prevents the progression from myocarditis to iDCM following CVB3 infection by reducing acute viral replication and IL-4 levels in the heart.

Assessing the Role of Tissue Infiltrating APCs in Graft-Versus-Host Disease Through Two Photon Intravital Microscopy

AbstractAbstract 342Graft-versus-host disease (GVHD) limits the broader application of allogeneic bone marrow transplantation. While initial T cell activation in GVHD occurs predominantly in secondary lymphoid organs, we have consistently observed MHCII+ donor-derived APCs in histopathologic GVHD lesions in tissues such as skin and intestine, frequently adjacent to infiltrating T cells. We hypothesized that productive interactions occur between donor APCs and T cells in situ in GVHD target tissues, which propagate disease. To address this hypothesis we utilized two photon intravital microscopy to analyze interactions between fluorescently labeled donor CD8+ T cells and tissue infiltrating donor dendritic cells (DCs), within skin lesions of living mice in a murine GVHD model. Lethally irradiated 129 mice received T cell-depleted (TCD) marrow from C57BL6 (B6) mice expressing yellow fluorescent protein (YFP) driven by the CD11c promoter (B6-CD11cYFP), B6 red fluorescent protein (RFP)+ CD8+ T cells and WT (unlabeled) B6 CD4+ T cells. The skin of the ear was imaged in anesthetized, living mice, between days 21 and 28 post-transplant, using a LaVision two photon laser scanning microscope, scanning 60uM thick z-stacks every 30 seconds for 1 hour. Individual RFP+ CD8+ T cells were tracked over time throughout the 3 dimensional image. Detailed surface maps of the YFP+ dendritic cell (DC) network were generated. A distance transformation to calculate the distance of each RFP+ CD8+ T cell from the surface of the YFP+ DC network at all times was performed. Through these analyses, we observed both highly motile donor CD8+ T cells making contact with donor DCs (defined as <=2μM between RFP+ CD8+ T cell and YFP+ DC surface), and a proportion which make long term, stable contact (continuous contact between RFP+ CD8+ T cell and YFP+ DC for at least 30 minutes, during which the RFP+ CD8+ T cell speed is below 5.5μM/min). To test whether CD8+ T cell:DC interactions required cognate TCR:MHCI interactions, lethally irradiated 129 mice received a 1:1 mixture of WT B6 and MHCI-deficient B6 BM. In group 1, labeled marrow was from B6-CD11cYFP, and unlabeled marrow from B6-β2-microglobulin (β2m)−/− donors. In group 2, labeled marrow was from B6-CD11cYFP/β2m−/− mice, and unlabeled marrow from B6. In addition to these bone marrow mixtures, all mice received B6 RFP+ CD8+ T cells and unlabeled B6 CD4+ T cells. Mice were imaged as above. Long lasting contacts between donor RFP+ CD8+ T cells and YFP+ donor DCs in skin lesions were less frequent when YFP+ DCs were β2m−/−, and therefore MHCI-deficient. We have also examined whether MHCII-dependent interactions occur between CD4+ T cells and DCs in situ in skin lesions. In preliminary experiments 129 mice received B6-CD11cYFP bone marrow, B6 RFP+ CD4+ T cells, and B6 unlabeled CD8+ T cells. After 1 hour of imaging, mice received anti-MHCII antibody or isotype control and imaging was continued for 2 hours thereafter. RFP+ CD4+ T cell motility increased after anti-MHCII but not after isotype control antibody treatment. Because GVL occurs primarily in BM and spleen, targeting of tissue-infiltrating APCs could represent a unique strategy to ameliorate GVHD while preserving GVL. 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

AbstractAbstract 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.

Hematopoietic Stem Cells Are Dependent On Homologous Recombination Only During Active Cell Cycle in Nuclease-Mutant Exonuclease1mut mice

Abstract 1225Hematopoietic stem cell (HSC) maintenance and self-renewal is crucial for long term tissue repopulation and immune function. HSC populations require functional DNA repair pathways in order to maintain their reconstitution capabilities but the pathways involved and the mechanisms of regulation are still being elucidated. It has been proposed that quiescent HSCs rely on the error prone non homologous end joining pathway for DNA double strand break (DSB) repair while HSCs in cycle use both NHEJ and the high fidelity homologous recombination (HR), but functional in vivo studies have not yet been completed. Exonuclease 1 participates in homologous recombination. We used Exo1mut fibroblasts to demonstrate that loss of Exo1 function results in a defective HR response, increased sensitivity to DSB inducing agents, and aberrant DNA damage signaling. However, Exo1mut mice did not appear to require HR to maintain quiescent HSCs at steady state or to respond to DNA damage. Exo1mutmice were able to sustain long term serial repopulation, displayed no defect in competitive repopulation or quiescence maintenance, and did not display increased sensitivity to whole body ionizing radiation (IR). In contrast, when Exo1mut HSCs were pushed into cell cycle with 5-Fluorouracil, the hematopoietic population and HSCs became hypersensitive to IR stress relative to WT B6 mice, as shown by decreased bone marrow cellularity, colony forming unit defects, loss of the HSC population, and finally animal death. Thus, loss of Exo1, and in turn fully functional HR, in quiescent HSC is not critical to stem cell function, survival, or recovery after DNA damage, whereas HR mediated repair of DNA damage is essential for HSC maintenance after cell cycle entry. In HSCs, DNA damage repair response, and sensitivity is dependent on cell cycle. Disclosures:No relevant conflicts of interest to declare.

Hepatic Stellate Cell Conditioned Myeloid Cells Provide a Novel Therapy for Prevention of Factor VIII Antibody Formation in the Hemophilia A Mouse Model

AbstractAbstract 4117 Background:Hemophilia is an X-linked bleeding disorder resulting from a mutation in coagulation factor VIII (F.VIII). A major drawback of current plasma-derived or recombinant F.VIII therapy is the formation of F.VIII antibodies (inhibitors). Inhibitor formation is a T cell-dependent, B cell-mediated immune response to foreign infused F.VIII. Myeloid derived suppressor cells (MDSCs) are potent suppressors of T cell and B cell responses and are currently under study for therapeutic applications in transplantation and autoimmune diseases. However, the mechanisms of MDSC development and function remain unknown, and in vitro propagation of MDSCs has been a challenge. We hypothesized that MDSCs might be effective in inhibiting F.VIII inhibitor formation in the hemophilia A model. Methods:We developed a novel method for generating MDSCs in vitro by culturing bone marrow cells from hemophilia A mice with hepatic stellate cells (HSCs), hereafter referred to as HSC-conditioned myeloid cells (H-MCs). DCs were propagated from the bone marrow with GM-CSF and IL-4, whereas H-MCs were propagated from the bone marrow with GM-CSF and HSCs. Granulocyte contaminants were removed on day 2 and the remaining monocytic populations were harvested on day 5. Expression of cell surface antigens was analyzed by flow cytometry. Arginase1 and iNOS levels were compared by qPCR, with or without LPS stimulation. The in vitro suppressive capacity of the H-MCs was determined by a mixed leukocyte reaction culture. Splenic T cells from hemophilia A mice were stimulated by irradiated DCs (at a 1–20 ratio, APC to T cell) and recombinant F.VIII. Additional irradiated DCs or H-MCs were added in graded numbers as regulators. The proliferative response was determined by 3H-thymidine incorporation. The phenotype of cultured CD4+ T cells was characterized by intracellular staining for Foxp3 and IFN-gamma and analyzed by flow cytometry. Inhibition of B cells by H-MCs was determined by a CFSE dilution assay. Purified splenic B cells were labeled with CFSE and stimulated by Ig-M and IL-4. APCs (spleen cells) or H-MCs were added at a ratio of 1:10 (APC to B cell). The proportion of proliferating B cells was determined by CFSE dilution of B220 stained cells. In the COX-2 suppression assay, CFSE labeled B cells were treated with varying concentrations of the selective inhibitor of COX-2, NS398. The suppressive effect of H-MCs on B cells in vivo was determined by simultaneously administering H-MCs (I.V) and F.VIII (I.V.) to hemophila A mice on day 0 and rechallenging with recombinant F.VIII on days 2 and 4. WT B6 mice and hemophilia A mice without H-MC transfer served as controls. Plasma anti-F.VIII antibody titers were measured on day 12 by a modified ELISA assay. Results:H-MCs expressed low levels of costimulatory molecules but high levels of the inhibitory molecule B7-H1 and immunoregulatory enzyme arginase-1. In contrast, DCs expressed high levels of costimulatory molecules and MHC class II. In vitro studies demonstrated that the H-MCs markedly inhibited antigen specific T cell proliferation induced by dendritic cells in response to recombinant F.VIII (Fig. 1). H-MCs altered the T cell response in hemophilia A mice by promoting the expansion of regulatory T cells and inhibiting IFN-γ producing CD4+ T cells. When the H-MCs were cocultured with B cells isolated from hemophilia A mice, in the presence of Ig-M and IL-4, the H-MCs abrogated B cell activation and proliferation directly (Fig. 2). H-MCs may be modulating the B cell response through the Cox-2 pathway, as inhibition of Cox-2 through NS398 led to the restoration of B cell proliferation. More importantly, adoptive transfer of H-MCs into hemophilia Amice, at the time of F.VIII infusion, markedly suppressed anti-F.VIII antibody formation (Fig. 3). Conclusion:These results suggest that HSC conditioned myeloid cells may represent a potential therapeutic approach to induction of immune tolerance in patients with hemophilia A andother immune disorders. [Display omitted] [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Cognate Antigen Is Necessary and Sufficient for the Migration of Effector and Memory T Cells to Vascularized Allografts

Background: A key step in the rejection process is the transendothelial migration of Ag-specific effector and memory T cells into the graft. It is generally assumed that this step is mediated by Gai-coupled chemokine receptor signaling. Here, we revisited this paradigm by investigating the migration of polyclonal and monoclonal effector and memory T cells to vascularized transplants in the presence or absence of Gai signaling. Methods: B6 CD4 and CD8 polyclonal effector and memory T cells primed against BALB/c were treated with pertussis toxin (PTx) to block Gai signaling or not and co-transferred to B6 wt or alymphoplastic (aly/aly) mice 2 days after transplanting BALB/c hearts. Ag(OVA)-specific (OT-I) and non-specific (P14) CD8 effector/memory T cells were transferred to B6 recipients of B6 Act-OVA or wt heart or kidney grafts to investigate the role of cognate Ag in T cell migration. Migration of transferred T cells to the grafts was quantified by flow cytometry and 2-photon intravital microscopy using congenic markers, MHC-tetramers and cell tracker reagents. Results: PTx-treated and untreated polyclonal effector and memory T cells migrated to and rejected heart allografts in equal numbers and with the same tempo. Ag(OVA)-specific (OT-I) effector/memory T cells migrated only to heart grafts that expressed the cognate Ag (OVA) and their migration was not inhibited by PTx. Non-specific (P14) T cells, on the other hand, did not migrate to Act-OVA grafts unless Ag-specific (OT-I) T cells were present, and their migration was abrogated by PTx. Intravital imaging of transplanted Act-OVA kidneys established that PTx did not inhibit the firm adhesion or transendothelial migration of Ag-specific (OT-I) effector/memory T cells. In contrast, adhesion and transmigration of non-specific (P14) T cells occurred only if their Gai signaling was intact. Conclusion: In contrast to the chemokine-dependent paradigm, the migration of Ag-specific effector/memory T cells to vascularized allografts requires interaction with cognate Ag and is independent of chemokine signaling. Entry of non-specific effector/memory T cells, on the other hand, requires chemokines but occurs only if Ag-specific T cells are present. These findings underscore the critical role of cognate Ag in directing the entry of primed T cells to non-lymphoid tissues and suggest novel mechanisms for their transmigration across the endothelium.

IL-1 signal affects both protection and pathogenesis of virus-induced chronic CNS demyelinating disease

BackgroundTheiler’s virus infection induces chronic demyelinating disease in mice and has been investigated as an infectious model for multiple sclerosis (MS). IL-1 plays an important role in the pathogenesis of both the autoimmune disease model (EAE) and this viral model for MS. However, IL-1 is known to play an important protective role against certain viral infections. Therefore, it is unclear whether IL-1-mediated signaling plays a protective or pathogenic role in the development of TMEV-induced demyelinating disease.MethodsFemale C57BL/6 mice and B6.129S7-Il1r1tm1Imx/J mice (IL-1R KO) were infected with Theiler’s murine encephalomyelitis virus (1 x 106 PFU). Differences in the development of demyelinating disease and changes in the histopathology were compared. Viral persistence, cytokine production, and immune responses in the CNS of infected mice were analyzed using quantitative PCR, ELISA, and flow cytometry.ResultsAdministration of IL-1β, thereby rending resistant B6 mice susceptible to TMEV-induced demyelinating disease, induced a high level of Th17 response. Interestingly, infection of TMEV into IL-1R-deficient resistant C57BL/6 (B6) mice also induced TMEV-induced demyelinating disease. High viral persistence was found in the late stage of viral infection in IL-1R-deficient mice, although there were few differences in the initial anti-viral immune responses and viral persistent levels between the WT B6 and IL-1R-deficiecent mice. The initial type I IFN responses and the expression of PDL-1 and Tim-3 were higher in the CNS of TMEV-infected IL-1R-deficient mice, leading to deficiencies in T cell function that permit viral persistence.ConclusionsThese results suggest that the presence of high IL-1 level exerts the pathogenic role by elevating pathogenic Th17 responses, whereas the lack of IL-1 signals promotes viral persistence in the spinal cord due to insufficient T cell activation by elevating the production of inhibitory cytokines and regulatory molecules. Therefore, the balance of IL-1 signaling appears to be extremely important for the protection from TMEV-induced demyelinating disease, and either too much or too little signaling promotes the development of disease.

Intrinsic unresponsiveness of Mertk−/− B cells to chronic graft-versus-host disease is associated with unmodulated CD1d expression

Activation and migration of marginal zone B (MZB) cells into follicular (FO) regions of the spleen has been proposed as one of the mechanisms that regulate the development of autoreactive B cells. The mer receptor tyrosine kinase (Mertk) mediates apoptotic cell clearance and regulates activation and cytokine secretion. In the well-studied class II chronic GVH model of bm12 cells into B6 hosts, we observed that Mertk deficient B6 mice did not generate autoantibodies in response to this allogeneic stimulus. We posited that Mertk is important in MHC-II-mediated B cell signaling. In the present study, we show that B cells from Mertk−/− mice but not WT B6 mice exhibited decreased calcium mobilization and tyrosine phosphorylation when stimulated by MHC-II cross-linking. The finding that Mertk was important for class II signaling in B cells was further supported by the preponderance of a-allotype autoantibodies in cGVH in RAG-KO mice reconstituted with a mixture of bone marrow from Mertk−/− mice (b-allotype) and C20 mice (a-allotype). MZB cells from Mertk−/− mice were unable to down regulate surface CD1d expression and subsequent inclusion in the MZ, associated with significantly lower germinal center responses compared to MZB cells from WT. Moreover, Mertk−/− mice treated with an anti-CD1d down regulating antibody responded significantly to bm12 cells, while no response was observed in Mertk−/− mice treated with control antibodies. Taken together, these findings extend the role of Mertk to include CD1d down regulation on MZB cells, a potential mechanism limiting B cell activation in cGVH.

Cognate antigen is necessary and sufficient for the migration of effector and memory T cells to vascularized allografts (173.19)

Abstract A key step in rejection is the transendothelial migration (TEM) of Ag-specific effector and memory (E&amp;M) T cells into the graft. It is assumed that this step is mediated by Gαi-coupled chemokine receptor signaling. This migratory paradigm was revisited by treating Ag(OVA)-specific(OT-I) and non-specific(P14) CD8 E&amp;M T cells with pertussis toxin (PTx) to block Gαi signaling or not and co-transferring to B6 recipients of B6 Act-OVA or wt vascularized allografts to investigate the roles of cognate Ag and Gαi signaling in T cell TEM. Flow cytometry quantification demonstrated that OVA-specific(OT-I) E&amp;M T cells migrated only to heart grafts that expressed cognate Ag(OVA) and their migration was not inhibited by PTx. Non-specific(P14) T cells, on the other hand, did not migrate to OVA grafts unless OT-I T cells were present and their migration was abrogated by PTx. Two-photon intravital imaging of transplanted OVA kidneys established that PTx only inhibited the firm adhesion and TEM of non-specific(P14) E&amp;M T cells and not the Ag(OVA)-specific(OT-I) E&amp;M T cells. The migration of Ag-specific E&amp;M T cells to vascularized allografts requires cognate Ag and is independent of chemokine signaling. Entry of non-specific E&amp;M T cells, on the other hand, requires chemokines but occurs only if Ag-specific T cells are present. These findings underscore a critical role for cognate Ag in directing the entry of primed T cells to non-lymphoid tissues and suggest novel mechanisms for T cell TEM.

B7x deficiency exacerbates renal injury in antibody mediated nephritis (60.7)

Abstract B7x (B7-H4, B7S1) is an important member of the B7/CD28 superfamily of T cell costimulatory proteins. B7x is a negative regulator of CD4+ and CD8+ T cell activation and proliferation, and is expressed in both hematopoietic and non-hematopoietic tissues. We hypothesized that B7x is instrumental in the pathogenesis of antibody mediated nephritis. To examine this question we induced nephritis in B7x-deficient (B7x-/-) (n=10) and WT B6 mice (n=10) by passive transfer (i.v.) of nephritogenic antibodies. Following induction of nephritis, we found a marked increase in the levels of serum IgG (p=0.023), IgG2b (p=0.055), and IgG1 (p=0.064) in B7x-/- mice compared to WT mice. Moreover, renal damage was more severe in B7x deficient mice. Serum creatinine and BUN were elevated in the B7x-/- group compared to the WT mice. Furthermore, B7x-/- mice demonstrated worse kidney histopathology, with an increase in infiltrating PMN cells in glomeruli (p&amp;lt;0.0001) and interstitium (p=0.004), crescent formation, and tubular atrophy/dilatation. Immunohistochemical studies revealed increased glomerular infiltration of CD3+ T cells (p=0.034) and CD68+ macrophages (p=0.003) in B7x-/- mice kidneys. At the mRNA level, IL-23 was significantly upregulated in kidneys of B7x-/- mice. Thus, B7x plays a protective role in the pathogenesis of antibody mediated nephritis. The relative contributions of systemic versus local immune effects of B7x in nephrotoxic nephritis are currently being investigated.

Antigen-specific B cells are not required for soluble OVA protein induced T-cell tolerance through i.v. route (65.8)

Abstract Intravenous exposure to soluble proteins can lead to antigen-specific immune tolerance. Moreover, resting B cells have been shown to be tolerogenic antigen-presenting cells (APC) in vivo. However, it is still not clear whether this APC function is fulfilled by the rare antigen-specific B cells or the abundant antigen non-specific B cells. To address this, we used soluble chicken ovalbumin (OVA) as a model antigen and employed B-cell receptor (BCR) transgenic mice (8.18C5 Tg) of C57BL6 (B6) background. In the homozygous 8.18C5 Tg mice, ~97% of peripheral B cells contain recombined immunoglobulin heavy chain specific for non-related myelin oligodendrocyte glycoprotein. As expected, a single i.v. dose of soluble OVA in wt B6 mice induced significant B+T cell tolerance. The 8.18C5 Tg mice immunized with OVA+CFA failed to produce any detectible anti-OVA antibody response, confirming the lack of OVA-specific endogenous B cells in these mice. Importantly, pretreatment of the 8.18C5 Tg mice with 25µg soluble OVA i.v. also resulted in profound T-cell tolerance upon OVA+CFA challenge a week later. Since antigen non-specific B cells can present processed antigen after soluble protein exposure in vivo, our results support the notion that antigen non-specific resting B cells in the steady state may participate in the peripheral tolerance to self proteins.

Abstract 4842: p300 targeting impairs treg function and promotes host anti-tumor immunity

Abstract The long-held view that tumors were not sufficiently immunogenic to promote host immune responses has now been revised to the assessment that at least for some tumors, Foxp3+ T-regulatory (Treg) cells actively suppress anti-tumor immunity. Seeking to build on our ongoing assessment of the regulation of Foxp3+ Treg biology by HDAC and HAT enzymes, we have begun to use combined genetic and pharmacologic approaches to identify and modulate key mechanism within Tregs that limit host immunity. Microarray studies of gene expression in WT, p300-/- and CBP-/- Tregs showed that p300 or CBP deletion led to down-regulation of Foxp3 expression, as well as that of a number of signature Treg genes. Cells co-transfected with Foxp3 and increasing amounts of p300 expression vectors showed that Foxp3 acetylation was enhanced by increasing levels of p300 levels, whereas a p300 small molecule inhibitor, C646 (p300i), impaired Foxp3 acetylation and inhibited Treg function using in vitro suppression assays. In vivo use of C646 showed negligible effects on CD4, CD8 or Treg cell numbers, but qPCR studies showed C646 decreased Treg expression of CTLA-4, GITR, IL-10 and TGF-b, in vitro assays again showed impaired Treg suppression. Additional studies showed, critically, that use of the p300i impaired Treg but not T effector cells in allograft recipients. Thus, parent-to-F1 assays involving alloactivation and proliferation of CD4 an dCD8 T cells were unaffected by C646, and this p300i restored allograft rejection in Treg-dependent models of allograft survival. Turning to tumor models, TC1 lung cancer cells were injected in the flanks of WT B6 mice, B6 mice in which p300 was deleted in CD4+ T cells (Treg plus Teff using CD4-Cre) or just in Tregs (Foxp3-Cre) (n=10/grp), p300 was inhibited using C646 (Alzet pumps, 14 d, 0.9 mg/kg/d) from day 6 post-tumor injection; in both genetic and pharmacologic approaches, p300 targeting suppressed tumor cell growth. qPCR analysis of tumors in C646-treated mice showed increased CD4 and granzyme B mRNA but decreased Foxp3; these data were confirmed by immunohistology, along with increased IFN-g production by purified CD8 cells (ELISPOT), along with reductions in tumor volumes and weights in these C646-treated mice. C646 use did not impair tumor growth in immunodeficient (RAG-/-) mice, in which tumors were exposed to drug in RAG-/- mice as seen by decreased acetylation of histone 3 in tumor extracts (Western blot); no effects on acetylation of certain p300-independent targets within tumor extracts was seen (e.g. acetylation of alpha-tubulin was unimpaired). The p300i also had no direct effect on tumor cell proliferation in vitro. We conclude that our comprehensive genetic and pharmacologic studies show that p300 is an important target for modulation of host Foxp3+ Treg functions, such that Treg suppression can be incrementally reduced and host anti-tumor responses promoted without inducing concomitant autoimmunity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4842. doi:1538-7445.AM2012-4842