T-cell Depleted Bone Marrow Cells Research Articles

Inhibition of Bromodomain and Extraterminal Domain (BET) Proteins Alleviates Chronic Graft- Versus-Host-Disease Via Modulating the CXCR5/CXCL13 Axis

Introduction: Chronic graft-versus-host disease (cGVHD) is a severe complication of allogeneic hematopoietic cell transplant (allo-HCT) often leading to transplant-mediated mortality. Studies have shown that T follicular helper cells (Tfh) upregulate CXCR5 and migrate into the lymphoid organ follicles in response to CXCL13 promoting germinal center (GC) B cell proliferation and differentiation of plasma cells resulting in pathogenic antibody production and profibrotic pathways associated with cGVHD. In contrast, T follicular regulatory cells (Tfr) modulate the magnitude of the GC response; thus, the balance of Tfr/Tfh could be crucial to mitigating cGVHD.Bromodomain and extra-terminal domain (BET) proteins are epigenetic readers influencing transcription of a multitude of inflammatory genes and our lab has previously reported pharmacological inhibition of BET, through OPN-51107, improves survivability and limits disease progression in murine models of acute GVHD that led to opening the first-in-human clinical trial of OPN-51107 in steroid refractory acute GVHD. We demonstrated BET proteins to be important in modulating acute GVHD pathogenesis, however, mechanisms of BET regulation within cGVHD are largely unknown. Here, using OPN-51107 we aim to further investigate mechanisms of cGVHD pathogenesis and identify successful BET inhibition-based therapeutic approaches. Methods: We used a Bronchiolitis Obliterans model (B6->B10.br) of cGVHD. Mice were administered 120mg/kg cyclophosphamide (D-3 and D-2) and 8Gy irradiation (D-1) as a conditioning regimen followed by B6 T-cell depleted bone marrow cells with and without CD45.1 B6 splenocytes. Mice that did not receive allogeneic splenocytes served as controls and did not develop GVHD. Recipients of allogeneic splenocytes were treated with a BET inhibitor - OPN-51107 at 10mg/kg 3x/weekly, or vehicle, from D28-54(+ 4) via oral gavage. Resting lung elastance and resistance was measured using the Flexivent system. At the time of lung function analysis, lung and liver tissue was harvested and stained via Hesson's Trichrome to identify and quantify collagen deposition. We performed immunofluorescence to quantify alveolar alloantibody deposition. Spleen and lung cells were stimulated with PMA/I and analyzed via spectral flow cytometry. An inflammatory murine cytokine array (#ARY006, R&D Systems) was used to analyze circulating plasma cytokine levels. Results: BET inhibition via OPN-51107 treatment decreased lung resistance (vehicle vs OPN-51107 =1.16Rrs vs 0.83Rrs p<0.05) and elastance (vehicle vs. OPN-51107=62Ers vs. 45Ers p<0.05). OPN-51107 treatment also significantly reduced alveolar antibody (Ig) deposition per area compared to vehicle (vehicle vs. OPN-51107= 4.91% vs. 3.3% p=0.01) and collagen deposition (vehicle vs. OPN-51107=19.0% vs. 12.9% p<0.05) and liver (vehicle vs. OPN-51107=19.3% vs. 2.35% p<0.0001). Congruently, BET inhibition decreased B220-CD19- CD138+mature plasma cells (vehicle vs. OPN-51107=15% vs 9.1% p<0.05) in the lung. Serum cytokine array analysis revealed a significant decrease in circulating CXCL13, relevant to Tfh migration as well as a decrease in other proinflammatory cytokines - CXCL9, MCP-1, IL3, RANTES and G-CSF important for macrophage activation and pathogenicity in OPN-51107 compared to vehicle treated mice. BET inhibition reduced splenic Tfh while increasing Tfr, thereby increasing Tfr:Tfh ratio (vehicle vs. OPN-51107= 5% vs. 9% p<0.05). Furthermore, total splenic CD19+ B-cells (vehicle vs. OPN51107= 70% vs. 33% p<0.001) as well as CXCR5 expression on B-cells were decreased in OPN-51107 treated mice (MFI vehicle vs. OPN-51107= 4744 vs. 571.5 p<0.0001). Conclusion: OPN-51107 improved lung function which correlated with significantly decreased lung fibrosis and antibody deposition in a BO model of cGVHD. BET inhibition reduces cGVHD immune pathology through decreased inflammatory cytokine production, promotion of GC Tfr cell population and modulates B-cell proliferation and/or trafficking. Significance: Using OPN-51107 in a BO model of cGVHD we demonstrate that BET inhibition is a feasible therapeutic strategy to reduce disease severity and improve lung function.

Sustained Engraftment in Fanconi Anemia (FA) Mice Using in Utero Hematopoietic Cell Transplantation (IUHCT)

Introduction FA is the most common inherited bone marrow failure syndrome caused by a mutation in one of 23 genes involved in DNA repair; the disease manifests as hematologic abnormalities by age seven with near uniform bone marrow failure in adult patients. The gold standard therapy is bone marrow transplantation (BMT), which portends best outcomes when performed at a young age from Human Leukocyte Antigen (HLA)-matched sibling donors. However, due to the underlying pathophysiology of the disease, FA patients are especially sensitive to the chemoradiation conditioning regimens used with BMT, leading to high short-term morbidity and an increased long-term cancer risk. Due to these challenges of conventional BMT in FA, IUHCT is an attractive non-myeloablative and non-immunosuppressive option capable of inducing donor-specific tolerance when in utero testing has identified pathogenic FA mutations. Here we analyzed whether non-conditioned IUHCT confers fetal engraftment as a potential therapy in the FA mouse disease model. Methods Fanconi anemia type C homozygous mice (Fancc-/-) underwent IUHCT on gestation day 14 (E14) via vitelline vein injections of 1x107 T-cell depleted bone marrow cells from green fluorescent protein (GFP)-expressing congenic donor mice. Littermate wild-type mice served as controls. Donor cell engraftment in peripheral blood and bone marrow was assessed by flow cytometry. To confirm long-term donor HSC engraftment, we performed primary and secondary transplantation of an equivalent number of BM mononuclear cells from either Fancc -/- or wild-type mice that had undergone IUHCT. Statistical analysis was performed via Welch's t-test. P values <0.05 were considered statistically significant; for graphical purposes ***p<0.001; **p<0.002; *p<0.033; nonsignificant, ns. Results Fancc-/- homozygous mice had significantly higher donor GFP+ cell engraftment than wild-type controls at one, three, and six months post-transplantation (Figure 1). This higher engraftment was maintained across cell types in peripheral blood including CD3+ T cells (0.7% vs 3.4%), B220+ B cells (1.2% vs 6.9%), GR1+ granulocytes (1.7% vs 10.6%), and CD11B+ myeloid cells (1.9% vs 11.5%). Analysis of the bone marrow of these mice at 6 months post-transplant similarly showed a trend toward increased donor cell engraftment of progenitor cell populations including LSK, HSPC, LTHSC, STHSC, MPP2, MPP3, MPP4, CLP, CMP, GMP, and MEP cells in Fancc -/- recipients compared to wild-type mice. At all timepoints after primary and secondary transplants, recipient mice engrafted with BM mononuclear cells from prenatally transplanted Fancc -/- mice had higher peripheral blood GFP+ chimerism than those engrafted with BM mononuclear cells from prenatally transplanted wild-type mice (Figure 2). Conclusion Fancc-/- homozygous mice have higher sustained donor cell engraftment compared with wild-type littermates, indicating successful engraftment and possible early selection for wild-type donor cells in this preclinical model. These initial studies support the potential of IUHCT as a therapy for FA and highlight the need for additional studies to identify approaches to enhance donor cell engraftment to more clinically relevant levels. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Indole-3-Carbinol Supplement Reduces GvHD Related Mortality in Allogeneic Hematopoietic Stem Cell Transplant Recipients

Introduction: Following allogeneic hematopoietic stem cell transplant (allo-HCT), donor T cells react against host antigens and cause severe graft versus host disease (GvHD) (Majhail N.S. et al., 2015. Biol Blood Marrow Transplant). Disruption of gut integrity is one of the major contributors to GvHD morbidity and mortality. Dysbiosis due to GvHD decreases host immunity and increases transplant-related mortality (Taur Y. et al., 2014. Blood). Indole, a bacterial metabolite, and indole derivatives are known to ameliorate host immunity and gut epithelial barrier function through modulating goblet cells, innate lymphoid cells (ILCs), dendritic cells (DCs) and T cell subsets (Gutiérrez-Vázquez C. & Quintana F.J., 2018. Immunity; Hubbard T.D. et al., 2015.Sci Rep). Indoles act via the aryl hydrocarbon receptor (AHR) expressed in innate lymphoid cells to generate regulatory T cells (Tregs) while inhibiting Th1 polarization (Gutiérrez-Vázquez C. & Quintana F.J., 2018. Immunity; Hubbard T.D. et al., 2015. Sci Rep). Previously, we showed that enteral administration of indole 3-carboxaldehyde (ICA) to murine allo-transplant recipients reduces GvHD severity and mortality (Swimm A. et al., 2018. Blood). However, since ICA is not available as a GMP-compliant FDA- approved pharmaceutical, our goal is to identify indoles for clinical research that have protective effects against GvHD. Here, we report that indole-3-carbinol (I3C), an indole currently marketed as a nutritional supplement, induces Treg generation and higher survival in allo-HCT. Methods: To study modulation of T cell subsets by indoles, mice were gavaged daily for 14 days with corn-oil suspensions of 50, 100, 200 or 400mg/kg of I3C, 150mg/kg of ICA, or corn oil alone. On day 15, mice were euthanized and splenocytes were isolated to study T cell subsets. To investigate the effect of I3C on GvHD, lethally irradiated (9Gy) B10.BR (H-2Kk) mice received 5 x 106 T cell-depleted bone marrow (TCDBM) cells with or without 2 x 106 T cells from MHC mismatched C57BL/6 (H-2Kb) donors. Allo-transplanted mice received either vehicle or 200mg/kg of I3C daily from day -2 to day 40. Mice were weighed and assessed twice a week for first 30 days and once a week afterwards. Lethally irradiated (9Gy) C57BL/6 mice were received 5 x 106 TCDBM cells with or without 5 x 105 T cells from FVB (H2Kq) donors and 14 days of daily gavage with vehicle or 200 mg/kg of I3C or 150mg/kg of ICA to study gut bacterial translocation into mesenteric lymph nodes (MLN). On the following day, MLNs were collected, immediately homogenized in 0.25ml sterile PBS and cultured (100ul) on Blood Agar plate to determine CFU/g tissue. Results: Percentages of regulatory T cells (Tregs) in the spleen were significantly increased at 14 days in non-transplanted mice treated with 200mg/kg of I3C (11.38%, n=5), 400mg/kg of I3C (11.48%%, n=5) and ICA (11%%, n=5) compared to mice treated with vehicle (6.74%%, n=5) (Figure 1), whereas frequencies of CD8+ T cells were unchanged. Allo-transplanted mice treated with I3C had higher survival (57.14%, n=7) compared to the vehicle-treated control group (33.33%, n=9) (Figure 2). However, weight gain was slower in the I3C-treated mice compared to surviving vehicle-treated mice. Allo-transplanted recipient mice showed trend of reduced bacterial translocation into MLNs of mice that received 200mg/kg of I3C and ICA. Our results indicate I3C supplement enhances rate of survival and decreases gut bacterial translocation into MLNs in allo-HCT recipients. Continuing experiments will study bacterial composition, histological changes, and determine mechanisms for I3C restoration of gut integrity in all-HCT recipients. Conclusion: The results above suggest indoles increase regulatory T cells. Allo-bone marrow transplant recipients treated with indoles had improved survival. We propose I3C as a prospective supplement to treat GvHD in all-HCT patients. Keywords: Allo-HCT, GvHD, Indoles, I3C, Tregs, Survival Acknowledgement: Research reported in this publication was supported in part by the Morningside Center for Innovative and Affordable Medicine through the generous support of donors and Emory University. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Donor peritoneal-derived cells can attenuate graft-versus-host disease after MHC-incompatible bone marrow transplantation in mice

BackgroundGraft-versus-host disease (GVHD) remains one of the most important barriers to bone marrow transplantation (BMT) and quality of life. Peritoneal-derived cells (PCs) can regulate immune function. MethodsThe GVHD mouse model, which was established by injection with T-cell depleted bone marrow cells and spleen cells, was treated with donor PCs. The survival time, weight, GVHD score, and pathological organ damage were used to assess the efficacy of PCs. Various cytokines (e.g., TNF-α, IFN-γ, IL-2, IL-17, IL-15, IL-4, IL-10, and TGF-β) and immunocytes (e.g., CD4+, CD8+, Treg, and NK cells) were employed to investigate the preliminary mechanism. The cytokine levels were tested via Luminex liquid chips, real-time PCR, and Western blotting, and the proportion of each T-cell subpopulation was detected via flow cytometry. One-way Analysis of Variance (ANOVA) followed by Tukey's post-test was employed to perform multiple comparisons, and Kaplan–Meier method was used to perform survival analysis. ResultsCompared to the saline group, the PC group harbored a significant reduction in pathological damage to the colon and small intestine, a longer survival time, and a lower GVHD score. The mice in the PC group had significantly higher levels of TGF-β, IL-10, and IL-4 and lower levels of IFN-γ, TNF-α, and IL-15 than those in the saline group. In the PC group, CD8+ and Th1 ratios decreased, while CD4+, Th2, NK, and Treg ratios increased. ConclusionsDonor PCs can attenuate GVHD caused by MHC-incompatible BMT by regulating cytokine levels and immunocyte ratios.

Regulatory T Cells Prevent Donor Cell Rejection in a Mouse Model of Delayed Gestational Age in Utero Hematopoietic Cell Transplantation

Background: In utero hematopoietic cell transplantation (IUHCT) results in long-term, multilineage chimerism without myeloablation/immunosuppression. It has the potential to treat a number of congenital hematologic disorders, including as Sickle cell disease. Central tolerance, through which donor-reactive host T cells and host-reactive donor T cells are deleted, is instrumental to the induction of donor-specific tolerance (DST) and the prevention of graft-versus-host-disease (GVHD). The window of central tolerance induction in humans occurs at a gestational age that will pose significant technical challenges and exclude from treatment all fetuses evaluated after 14 weeks gestation. We have previously demonstrated that IUHCT results in the peripheral induction of traditional and non-traditional regulatory T cells that contribute to the suppression of GVHD (Riley et al. ASH 2016 Abstract #4540). We hypothesized that regulatory T cells also contribute to DST and, furthermore, may be used to expand the window of central tolerance induction to a later point in gestation.Methods: A mouse model of delayed gestational age IUHCT was developed in which 9.7x10^6 C57/Bl6 (B6) GFP T cell-depleted (TCD) bone marrow (BM) cells were injected intravenously into day of life 0 (P0) naive Balb/c pups via the facial vein. The ability of IUHCT-induced regulatory T cells to prevent donor cell rejection was assessed by injecting 9.7x10^6 B6 GFP TCD BM cells plus 5x10^6 CD4+ splenocytes harvested from 2-4 week old chimeric mice. Fetuses injected with 9.7x10^6 B6 GFP TCD BM cells at gestational day 14 (E14) served as positive controls for tolerance and engraftment. Peripheral blood (PB) was analyzed at 4, 8, 12, and 24 weeks of age for donor cell chimerism (%CD45+H2kb+GFP+/CD45+H2kd+) and for engraftment (defined as chimerism >1% at 4 weeks). Weight gain and histology of the skin, liver, lungs, and small bowel were analyzed to confirm the absence of GVHD. PB was analyzed at 6 months of age to confirm multilineage engraftment. An in vivo mixed lymphocyte reaction (MLR) was performed at 6 months of age to confirm long-term DST. B6-sensitized Balb/c mice served as positive controls for alloreactivity. Comparison of continuous data was performed using Student's t test. Binary data were compared using Fischer's exact test. All tests were two-sided with an alpha level set at 0.05 for significance.Results: Our model of delayed gestational age IUHCT was associated with donor cell rejection in 52 of 56 (92.9%) pups injected with TCD BM only. Co-injection of CD4+ chimeric splenocytes significantly improved engraftment (100% vs. 7.1%, p<0.001) and chimerism (7.56% vs. 0.46% at 4 weeks, p=0.003) compared to TCD BM alone. No difference in engraftment (100% vs. 90%, p=0.501) or chimerism (7.56% vs. 7.66% at 4 weeks, p=0.961) was observed compared to fetuses injected with TCD BM at E14 (Figures 1A and 1B). These trends persisted at subsequent time points. Pups injected with TCD BM plus CD4+ chimeric splenocytes displayed normal weight gain and no histologic evidence of GVHD. Survival to adulthood was 100%. Donor cells of all hematopoietic lineages were present in the PB at 6 months of age. On in vivo MLR, pups injected with TCD BM plus CD4+ chimeric splenocytes displayed significantly reduced T cell proliferation compared to B6-sensitized Balb/c mice and P0 pups injected with TCD BM only. T cell proliferation did not differ significantly from fetuses injected with TCD BM at E14 (Figure 1C).Conclusion: The induction of tolerance following IUHCT is important for maintaining successful long-term engraftment and requires injection at an early gestational age to avoid donor cell rejection by the maturing fetal immune system. This study demonstrates an important role for regulatory T cell populations in the induction of DST following IUHCT. The adoptive transfer of regulatory T cells from a chimeric mouse to a naïve P0 pup inhibits donor cell rejection in our model of delayed gestational age IUHCT, allowing central tolerance induction and long-term engraftment to occur despite the increased maturity of the recipient immune system. These findings represent proof-of-principle that augmentation of regulatory T cell-dependent peripheral tolerance is one approach by which IUHCT could be delayed to a later point in gestation, decreasing the technical difficulty of the procedure and allowing fetuses evaluated after 14 weeks to be eligible for treatment. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Role of Glucose Transporter 1 in Graft-Versus-Host Disease and Graft-Versus-Tumor Effect

Graft-versus-host disease (GVHD) is a major obstacle to allogeneic hematopoietic cell transplantation. GVHD is induced when donor mature T cells encounter host antigens and get activated. Activated T cells require high metabolic flux through growth-promoting pathways to support clonal expansion. It is generally thought that glycolysis is employed in this process. However, conflicting data have been reported by different groups regarding the role of glycolysis in GVHD. Since glucose transporter 1 (Glut1) is essential for T cell glycolysis, we attempted to define the role of glycolysis in GVHD using donor T cells that are deficient of Glut1.One million T cells from Glut1-knockout (CD4CreGlut1fl/fl) or wild-type (control) C57BL/6 donors were transplanted into lethally irradiated (9.5 Gy) C3H/HeJ mice along with 1×107 donor T-cell-depleted bone marrow (TCDBM) cells. While all recipients of control T cells developed lethal GVHD and died within 20 days after transplantation, eight out of ten CD4CreGlut1fl/fl T cell recipients survived more than 60 days after transplantation (Figure 1, P<0.01). Body weights were slightly lower and clinical scores were slightly higher (P=not significant) in the surviving CD4CreGlut1fl/fl T cell recipients at day +56 when compared to those in the TCDBM alone group, suggesting that CD4CreGlut1fl/fl T cell recipients developed very mild GVHD if any. We further studied the role of Glut1 in CD4 and CD8 T cell mediated GVHD separately using C57BL/6 into BALB/c model. Similar to the results from the total T cell experiment, all CD4CreGlut1fl/fl CD4 and CD8 T cell recipients survived more than 60 days after transplantation. By contrast, 100% control CD4 and 50% CD8 T cell recipients died of lethal GVHD within 60 days after transplantation. These data demonstrate that Glut1 is required for alloresponses and glycolysis is indispensasble in the development of lethal GVHD.To understand the mechanism by which CD4CreGlut1fl/fl T cells mediate less GVHD, we first studied their ability to respond to alloantigens in mixed lymphocyte reaction. The results indicated that CD4CreGlut1fl/fl T cells have dramatically decreased ability to respond to BALB/c antigens when compared with the control T cells. Consistent with the in vitro data, T cell expansion was 59.4% less in CD4CreGlut1fl/fl T cell recipients than that in control T cell recipients when measured at day +7 after GVHD induction. Apoptosis was increased in alloantigen-activated (CD69+) CD4 but not in CD8 T cells from CD4CreGlut1fl/fl mice when compared with that from control mice when measured 16 hours after the initiation of culture. However, more cell death was observed in both activated CD4 and CD8 T cells from CD4CreGlut1fl/fl mice than that from control mice. In addition, alloreactive CD4CreGlut1fl/fl T cells failed to sustain activated mTORC1 signaling, as assessed by phosphorylation of the downstream p70 S6 kinase target, small ribosomal subunit S6. These results indicate that, upon response to alloantigens, T cells are prone to apoptosis and cell death and lead to decreased clonal expansion in the absence of Glut1, suggesting a mechanism by which CD4CreGlut1fl/fl T cells have decreased ability to induce GVHD.To determine whether absence of Glut1 in T cells affects graft-versus-tumor (GVT) effects, we transplanted 1×106 T cells from Glut1-knockout (CD4CreGlutfl/fl) C57BL/6 donors into lethally irradiated (8.5 Gy) BALB/c mice along with 1×107 donor TCDBM cells. At the time of transplantation, the recipients were challenged with 1×105 BCL1 cells, a BALB/c-derived leukemia/lymphoma cell line. While all TCDBM alone recipients died of leukemia/lymphoma and all control T cell recipients died of lethal GVHD, all CD4CreGlut1fl/fl T cell recipients survived more than 100 days after transplantation without leukemia/lymphoma (Figure 2), indicating that T cells deficient of Glut1 preserve GVT effect.The results from this study definitely demonstrate that glycolysis is essential for the development of GVHD. Inhibition of glycolysis in T cells increases apoptosis and cell death and suppresses clonal expansion of allospecific T cells. Interestingly, GVT effect is preserved despite the inhibition of glycolysis. These data suggest that glycolysis could potentially be targeted for prevention and treatment of GVHD. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Endothelial Protective Effects of Defibrotide Reduce Acute Graft Versus Host Disease after Experimental Allogeneic Hematopoietic Stem Cell Transplantation

Introduction Graft versus host disease (GVHD) is a major complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT). The pathophysiology of acute GVHD involves donor T cell- and inflammatory cytokine-mediated injury to the patient's tissues and causes significant morbidity and mortality. Defibrotide is a mixture of single stranded nucleotides that has a number of pharmacological effects that contribute to its endothelial protective properties. We hypothesized that preventive treatment with defibrotide will reduce the incidence and severity of GVHD due to its endothelial protective and anti-inflammatory properties and potentially does not alter graft versus leukemia (GVL) responses. Methods Recipient B10.BR mice received Cyclophosphamide 120 mg/kg per day on day-3 and -2, and received lethal total body irradiation (750cGy) on day 0, followed by the infusion of donor C57BL/6 T-cell depleted bone marrow cells. To induce GVHD, respective groups were co-infused with splenocytes, while non-GVHD controls received BM cells only. Mice were treated with defibrotide at the dose of 800mg/kg per body weight or vehicle control for the first week daily and then three times/week thereafter. For GVL experiments, C57BL/6 recipients were lethally irradiated (12Gy) with TBI and transplanted with C3H.SW donor TCD bone marrow alone or in combination with 3 million T-cells along with C1498-luc and treated with defibrotide or vehicle controls. Results Allogeneic defibrotide treated recipients demonstrated significantly better survival when compared to control group (83.34% vs 54.55% on day +28). Clinical GVHD scores were significantly worse after day +14 in control group when compared to defibrotide treated recipients. Both at day +7 and on day +28 allogeneic defibrotide treated recipients showed significantly less organ GVHD in gut, lung and liver (p Conclusion Defibrotide treatment through its anti-inflammatory and endothelium-protective effects reduces the incidence and severity of acute GVHD, while not impairing GVL activity.

Microrna-191 Regulates T-Cell Clonal Expansion during Graft-Versus-Host Disease

Allogeneic hematopoietic cell transplantation is a potentially curative treatment choice for a wide variety of hematological malignancies. However, graft-versus-host disease (GVHD), which is mediated by donor alloreactive T cells, limits the success of this procedure. Previous studies have demonstrated that several microRNAs (miRs) modulate graft-versus-host disease. miR-191 was previously reported to be able to support T cell survival after TCR stimulation. We hypothesize that miR191 regulates T cell response during GVHD. To test this hypothesis, we first studied miR-191 expression in alloreactive T cells. The result demonstrated that miR-191 was up-regulated in donor T cells isolated from murine GVHD recipients, suggesting that miR-191 may play a role in GVHD induction. We further studied the role of miR-191in GVHD using miR-191 deficient T cells (KO). Lethally irradiated (8.5 Gy) BALB/c mice were injected intravenously with 1×107 T cell-depleted bone marrow (TCDBM) cells along with 1×106 purified T cells from wild-type (WT) or KO mice, which are in C57BL/6 background. Interestingly, all recipients in the WT group died within 35 days after transplantation, while only one out of ten animals died in the KO group during an observation period of 56 days. Body weights and clinical scores were also improved in KO T cell recipients when compared with the WT controls. Similar results were also observed in a second GVHD model (C57BL/6→C3H/HeJ). To understand the mechanism by which miR-191 KO T cells have decreased ability to mediate GVHD, we first measured the ability of KO T cells to respond to alloantigens in vitro in a mixed lymphocytes reaction assay. Dramatically decreased alloresponse was observed with KO T cells as compared with WT T cells. Similarly, decreased clonal expansion was observed in KO T cells in vivo upon challenge with alloantigens as measured by bioluminescent imaging (Figure 1A). These results were further supported by data from a co-transfer experiment, in which equal numbers of WT and KO T cells were transplanted into the same GVHD recipient. At day7 after transplantation, KO T cells showed significantly reduced expansion in the spleen and liver compared with WT T cells. Reduced alloresponses mediated by KO T cells may not due to decreased proliferative capability directly as an in vivo carboxyfluorescein succinimidyl ester (CFSE) assay showed a comparable cell division between WT and KO T cells upon challenge with alloantigens. Rather, increased cell death is responsible for decreased alloresponse observed in KO T cells because dramatically increased number of dead cells was observed in KO group compared with WT group upon response to alloantigens in vitro and vivo. To determine the genes that are regulated by miR-191, we did a screening based on the prediction. Humans and mice share more than 100 predicted targets for miR-191. We chose top 20 of these targets for RT-qPCR screening. The result demonstrated that Taf5 was a target gene of miR-191. Expression of TAF5 protein was down-regulated in activated KO T cells when compared with the WT T cells. Finally, we investigated whether miR-191 KO T cells preserve graft-versus-leukemia effects. 1×106 T cells from WT or KO mice were transplanted into lethally irradiated BALB/c mice along with 1×107 TCDBM cells and 1×105 host-type BCL-1 cells. While all recipients that received only TCDBM and tumor cells developed lethal leukemia/lymphoma, none of WT and KO T cells recipients developed tumor. In conclusion, our findings reveal a critical role of miR-191 during GVHD process and demonstrate that miR-191 is a novel therapeutic target for GVHD. Figure 1 Disclosures No relevant conflicts of interest to declare.

Recipient-Derived BAFF and Alloantigen Synergistically Activate B Cells in Murine Chronic Gvhd

Increased B cell-activating factor (BAFF) and aberrant B cell survival and activation are associated with chronic graft versus host disease (cGVHD) in patients. Whether excessive BAFF production has a pathologic role in the development of cGVHD after allogeneic hematopoietic stem cell transplantation (allo-HCT) remains unknown. Herein, we address this hypothesis by employing BAFF knockout (KO) and BAFF overexpressing (Tg) donor mice in the major MHC mismatched C57BL/6 (B6)-to-BALB/c cGVHD model (Wu et al J Immunol 2013;191:488), except we increased the T cell depleted bone marrow (TCDBM) dose to 1x107 per recipient to improve engraftment. BALB/c recipients receiving TCDBM plus 1x106 splenocytes (Sp) are the disease group, and those receiving TCDBM only are controls. After verifying cGVHD phenotypic manifestations (including longevity, weight loss, eye and lung findings), we examined whether cGVHD mice had the increased BAFF and B cell findings as occur in human cGVHD (Sarantopoulos et al, Blood 2009; 113: 3865). We observed that cGVHD mice had higher BAFF/B cell ratios compared to controls (Fig. 1A, n=10 each). We fully characterized the peripheral B cell compartment in these mice, and found that the relative composition of B cell subsets was significantly altered in diseased mice. CD93 cell surface expression has been used in mice to define B cells that emigrate from BM and circulate through secondary lymphoid organs. In cGVHD mice, we found a significant increase in the relative proportion of CD93+ B cells and a significant decrease in the proportion of CD93- B cells (Fig1B). Also, a significant increase in the frequency of cells positive for the germinal center and activation marker GL7 was found only within CD93- B cell subset of cGVHD mice (Fig 1C). When stimulated via BCR ex vivo, this GL7+ mature B cell subset had significantly increased Syk and BLNK activation, measured by phosphoflow (n=5, p=0.008). We next aimed to determine whether high BAFF alone or high BAFF and alloantigen together lead to altered B cell homeostasis and to the promotion of GL7+ BCR-activated B cells. C57BL/6 (B6) BAFF Tg TCDBM cells were used as donor to afford excessive and persistent BAFF levels after HCT in either syngeneic or BALB/c recipients. After B6 BAFF Tg TCDBM to BALB/c HCT, body weight decreased significantly. By contrast, B6 BAFF Tg TCDBM to B6 syngeneic HCT recipients remained healthy (Fig.1D, left panel,*p=0.004). Notably, the frequency of the CD93-GL7+ B cell subset was increased in BALB/c mice that received B6 BAFF Tg TCDBM only, compared to recipients of syngeneic BAFF Tg or WT TCDBM only (Fig.1D, right panel, p=0.008). Thus, both BAFF and alloantigen are required for B cell activation after HCT. Together, our data also suggest that GL7+ B cells identify aberrantly BCR-activated B cells in murine cGVHD, arising in the setting of BAFF-driven altered B cell homeostasis.While polymorphisms in either donor or recipient BAFF genes are associated with human GVHD (Clark et al Blood 2011; 118: 1140), the source of excess BAFF after allo-HCT remains unknown. We addressed whether pathologic BAFF was derived from donor or recipient hematopoietic cells using TCDBM from B6 BAFF KO mice vs WT as donor cells for HCT to BALB/c recipients. Consistent with the known requirement of recipient BAFF for recovery of the B cell compartment after syngeneic transplant (Gorelik et al JEM 2003; 198:937), recovery of a donor peripheral B cell pool was not significantly different between groups. Remarkably, plasma BAFF levels were also not different between BALB/c recipients of B6 BAFF KO TCDBM +Sp vs recipients of WT TCDBM +Sp, suggesting that radioresistant recipient cells produced BAFF in cGVHD. Chronic GVHD-associated weight loss was also not different between groups (Fig.1E, representative of 3 separate experiments). We found that BAFF production in the engrafted KO recipient BM was uniformly low. Thus, we have now determined that non-BM, radioresistant recipient cells are the principal source of soluble pathologic BAFF in murine cGVHD. Further characterization of BAFF-producing cells is underway in order to identify therapeutic targets.In summary, we have now demonstrated that recipient-derived BAFF has a mechanistic role in aberrant activation of B cells in cGVHD. Our findings will lead to the development of anti-BAFF and BCR targeting agents for patients.This work was supported the National Institutes of Health (NHLBI R01 HL 129061-01). [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Impaired expansion of CD11b+Gr-1+ cells from MyD88-deficient donor bone marrow cells aggravates graft-versus-host disease (TRAN2P.966)

Abstract Myeloid differentiation 88 (MyD88), an adaptor molecule in TLR-associated signaling, has recently been known to play a role in expansion/activation of CD11b+Gr-1+ myeloid-derived suppressor cells. To understand role of MyD88 expressed by donor-originating myeloid cells in GVHD development, we investigated dynamics of T cell-depleted bone marrow (TCD-BM) cells and gene-expressions of CD11b+Gr-1+ cells originated from Myd88-/- donor mice after transplantation into allogeneic BALB.B hosts with WT T cells. The allogeneic recipients of Myd88-/- TCD-BM and WT T cells showed aggravated GVHD, with significant loss of CD11b+Gr-1+ population, compared with BALB.B recipients of WT TCD-BM and T cells. Impaired expansion of Myd88-/- TCD-BM cells in BALB.B GVHD hosts was confirmed via in vivo tracking analysis. However, their expansion was intact in syngeneic C57BL/6 recipients, indicating that impaired CD11b+Gr-1+ cell expansion from Myd88-/- TCD-BM cells was specific to GVHD circumstance. In transcriptome analysis, CD11b+Gr-1+ cells of MyD88-/- TCD-BM origin exhibited higher expression of genes related to apoptosis and antigen-presentation than did those of WT TCD-BM origin. These results suggest that impaired expansion of the CD11b+Gr-1+ cells from Myd88-/- TCD-BM cells in allogeneic hosts is related to enhanced apoptosis in combination with enhanced differentiation into DC-like cells in the presence of activated allo-reactive T cells, and provide insight into the control of GVHD.

MyD88 in Donor Bone Marrow Cells Is Critical for Protection from Acute Intestinal Graft-Versus-Host Disease

Allogeneic hematopoietic stem cell transplantation is an important therapeutic modality used to treat malignancies of hematopoietic origin, such as leukemia and lymphoma. However, development of graft-versus-host disease (GVHD) causes non-relapse mortality and substantial morbidity of recipients. Myeloid differentiation factor 88 (MyD88), a major adaptor mediating TLR signaling, is also known to deliver pro-inflammatory signals. Activation of inflammatory signaling through MyD88 plays a key role in the expansion of myeloid-derived suppressor cells (MDSC) which are a heterogeneous population of immature myeloid ells with anti-inflammatory activity. To explore the contribution of MyD88 expressed by donor bone marrow (BM) cells to development of GVHD, we induced GVHD using T-cell-depleted BM (TCD-BM) isolated from MyD88-deficient (MyD88KO) mice and T cells isolated from wild-type (WT) mice. We employed C57BL/6 (H-2b) → B6D2F1 (H-2b/d) mouse model of GVHD, which differ at major and minor histocompatibility loci. Lethally irradiated B6D2F1 recipient mice were transplanted with either T cell-depleted bone marrow (TCD BM, 5 x 106) from either WT or MyD88KO mice together with WT spleen T cells (1 x 106). Transplantation with MyD88KO TCD BM aggravated GVHD; serious gut damage was evident, with infiltration of T cells specifically into the intestines of recipients. GVHD hosts transplanted with MyD88KO TCD BM exhibited markedly reduced expansion of MDSC. GVHD aggravation after transplantation with MyD88KO TCD-BM, associated with high-level T cell infiltration into the intestine and insignificant expansion of MDSC, was reproduced in another minor histocompatibility mismatch model (C57BL/6 → BALB.B). We next examined allogeneic T cells in the spleens of GVHD hosts in terms of the expression levels of CCR9, which are known to be associated with T cell migration to the intestinal mucosa and the proportion of CCR9 positive cells in CFSE low CD8+ T cells was higher in recipients of MyD88KO TCD BM than WT controls. In parallel, the levels of CCL25 were more highly expressed in the gut of MyD88KO recipients than WT controls. Mixed leukocyte cultures of CFSE- labeled C57BL/6 T cells and irradiated B6DF1 feeder cells were prepared in the presence of MDSC isolated from MyD88KO or WT mice. T cells, co-incubated with MDSC isolated from MyD88KO BM, exhibited a greater extent of CFSE dilution and less Annexin V staining, compared to T cells co-incubated with cells from WT BM. Moreover, MDSC from recipients of MyD88KO TCD BM exhibited a reduced suppressive function, compared to their WT counterparts. Next, we determined whether insufficient expansion of and ineffective suppression by MDSC caused severe GVHD in recipients of MyD88KO TCD BM. Supplementation of transplanted mice with MDSC from WT mice, not from MyD88KO mice, attenuated the severity of GVHD and reduced intestinal T cell infiltration in recipients of MyD88KO TCD BM. To verify the importance of MyD88-mediated signaling by MDSC in protection against severe GVHD, we determined if transplantation with TCD-BM cells containing high levels of MDSC attenuated the severity of GVHD. Pre-treatment of BM donors with lipopolysaccharide increased the frequencies of MDSC and the amounts of MyD88 transcripts in TCD-BM transplant, and alleviated the severity of GVHD and intestinal T-cell infiltration. To explore whether MDSC expansion levels could be used to predict the severity of intestinal GVHD, the T/MDSC ratios were calculated in blood of patients at the time of engraftment and were significantly higher in patients with intestinal GVHD ≥ grade 2. In conclusion, we have shown that MyD88-dependent MDSC expansion from donor BM is critical for protection against fatal acute intestinal GVHD. DisclosuresNo relevant conflicts of interest to declare.

Central Memory T Cells That Express High Level Of CD44 Mediate Strong Anti-Tumor Response Without Gvhd

Several groups have demonstrated directly and indirectly that central memory T cells (TCM) have decreased ability to induce graft-versus-host disease (GVHD) compared with naïve T cells (TN). We have previously demonstrated that TCM expressing high level of CD44 (CD44high TCM) do not cause GVHD. In this study, we further defined the role of CD4+ and CD8+ subsets of this population in GVHD using the same GVHD model as we used previously (C57BL/6→BALB/c). While all CD4+ TN recipients developed lethal GVHD and died within 28 days after transplantation, none of the recipients of CD4+CD44high TCM developed GVHD and all survived more than 100 days after transplantation (P<0.01). Similarly, 36% of CD8+ TN recipients developed lethal GVHD but none of CD8+CD44high TCM recipients developed GVHD (P<0.05). One of the unique features of effector memory T cells (TEM) is that these cells are able to facilitate stem cell engraftment and mediate some degree of anti-tumor response without causing GVHD. To test the ability of TCM to deplete host radioresistant T cells, BALB/c recipients were lethally irradiated (8.5 Gy) and transplanted with 1x107 T-cell-depleted (TCD) bone marrow cells together with graded numbers of T cell subsets from C57BL/6. Host T cells persist in all mice receiving only TCD bone marrow cells. Addition of 1x105 of either TN or CD44high TCM to the graft led to 100% depletion of all host radioresistant T cells. In contrast, none of 1x105 TEM recipients and only 50% of 5x105 TEM recipients depleted all host radioresistant T cells. These data indicate that TN and CD44high TCM have equivalent abilities to deplete host radioresistant T cells and both of them are at least 5 times more potent than TEM. To test the ability of CD44high TCM to mediate anti-tumor effects, host-type leukemia/lymphoma cells (BCL1) cells were added to the stem cell graft. All recipients transplanted with TCD bone marrow together with BCL1 cells developed tumor and died within 28 days after transplantation. Addition of 1x104 TN and CD44high TCM protected 42% and 36% of the recipients from developing tumor respectively. In contrast, only 7% of TEM recipients were protected (P<0.05) at the same dose level. Similarly, addition of 1x105 TN and CD44high TCM protected 79% and 80% of the recipients from developing tumor respectively. However, only 25% of 1x105 TEM recipients were protected (P<0.01). These data indicate that TN and CD44high TCM have equal anti-tumor activity and both of them are at least 10 times more potent than TEM. Similar results were obtained in CD8 subset of CD44high TCM. A critical question is why CD44high TCM are able to kill host radioresistant T cells and tumor cells so efficiently without inducing GVHD. Even though CD44high TCM were able to respond to alloantigens, bioluminescence imaging analyses suggest that CD44high TCM also mediate an abortive alloresponses upon transfer into allogeneic recipients. Using an in vivo functional assay, we further demonstrated that alloreactive CD44high TCM lost the reactivity over time, suggesting a mechanism by which CD44high TCM were able to eliminate host resistance without causing GVHD. In conclusion, these data demonstrate that selective transplantation of CD44high TCM is able to efficiently separate anti-tumor effects from GVHD. Disclosures:No relevant conflicts of interest to declare.

FTY720 Markedly Increases Alloengraftment but Does Not Eliminate Host Anti-Donor T Cells that Cause Graft Rejection on Its Withdrawal

The immunomodulator FTY720 (FTY) is beneficial in models of graft-versus-host disease, solid organ transplantation, and autoimmunity and has been approved for use in patients with multiple sclerosis. FTY modifies the homing and migration of many cell types. We report that FTY has profound positive and negative effects on allogeneic bone marrow (BM) engraftment in sublethally irradiated recipients. FTY increased donor hematopoietic progenitors in the BM, resulting in high donor engraftment in the B cell, myeloid cell, and natural killer cell, but not T cell, lineages. Donor T cell progenitors within the thymus of FTY-treated recipients were dramatically reduced, resulting in a lack of donor T cell reconstitution. In addition to preventing the ingress of donor (and host) T cell progenitors, FTY prevented the egress of fully functional host CD4+CD8- and CD4-CD8+ thymocytes that on cessation of FTY administration were able to exit from the thymus and contribute to a rapid and complete rejection of a well-established donor BM graft. When used in combination with anti-CD40L mAbs to block the CD40L:CD40 costimulatory pathway, FTY markedly enhanced anti-CD40L mAb-mediated alloengraftment promotion. In contrast to FTY alone, the combination of anti-CD40L mAb and FTY resulted in a surprisingly stable, multilineage, long-term donor chimerism. These data illustrate FTY's profound migration modulating effects and suggest a use in combinatorial therapy in achieving stable alloengraftment under nonmyeloablative conditions.

Recipient Type-Sepcific Engineered Regulatory T Cells Prevent Graft-vs-Host Disease After Allogeneic Bone Marrow Transplantation in Mice

Adoptive transfer of regulatory T cells (Tregs), in particular, recipient-type specific Tregs (sTregs), represents a promising approach to prevent graft-vs-host disease (GvHD) after allogeneic bone marrow transplantation. The objective of this study was to investigate whether engineered sTregs could prevent GvHD after bone marrow transplantation. Lentiviral vector forkhead box P3 (Foxp3)/pXZ9 containing Foxp3-IRES-GFP fragment and its mock control pXZ9 was constructed. Lentiviruses were produced via transient 3-plasmid transfection. BALB/c CD4+CD25−T cells were infected with lentiviruses and further stimulated using anti-CD3ε and anti-CD28 antibody (engineered irrelevant-Tregs [irTregs]) or C57BL/6 splenocytes (engineered sTregs). The expression of Tregs marks, production of cytokines, cell proliferation rate, and suppression function of Foxp3/pXZ9 infected cells were similar to natural Tregs. Irradiation BABL/c recipient were injected with C57BL/6 donor T cell–depleted bone marrow (TCD-BM) cells (1 × 10 7) and C57BL/6 splenocytes (1 × 10 7) together with engineered sTregs, irTregs, or natural Tregs (5 × 10 6). Irradiated BABL/c mice received TCD-BM cells only, TCD-BM cells plus splenocytes, or splenocytes and pXZ9-transduced cells (control). Recipient survival, short-term GvHD scores, and the Th1 subpopulation were monitored. Recipients of a combination of TCD-BM cells and splenocytes developed lethal GVHD. When engineered sTregs were added, 80% of recipients survived at least 60 days after transplantation; this survival rate was much higher than in any other group. The GvHD scores between the 3 Tregs groups did not demonstrate significance. Compared with other sources of Tregs in vivo, engineered sTregs strongly suppressed Th1 cell expansion. Therefore, a an in vitro strategy was developed to generate engineered sTregs. These cells demonstrated similar phenotypes and stable suppressive capacity as natural Tregs. Like natural Tregs, co-injection of engineered Tregs protected recipients from lethal GvHD in a murine model of GvHD. The engineered sTregs were superior to irTregs in minimizing murine GvHD.

Increased numbers of circulating ECs are associated with systemic GVHD

Circulating endothelial cells (ECs) are known to reflect endothelial injury, and endothelial injury is associated with graft-versus-host disease (GVHD). We hypothesised that circulating ECs might be associated with systemic acute graft-versus-host disease (aGVHD). BALB/c (H-2k(d) ) mice were treated with total body irradiation and then infused with C57B/6-derived T-cell-depleted bone marrow (TCD-BM) cells or TCD-BM cells and splenocytes. Cyclosporine was used to prevent aGVHD. Circulating ECs and allogeneic lymphocytes were analysed by flow cytometry at multiple time points. The morphology and ultrastructure of the endothelium were examined by light microscopy or transmission electron microscopy. The results indicated that the number of circulating ECs peaked at day 5 after lethal irradiation in all mice; allogenic transplanted mice (TCD-BM cells and splenocytes) developed typical aGVHD beginning at day 7, exhibiting both histological and clinical symptoms of disease. Circulating ECs peaked a second time at day 9 with aGVHD progression. However, following the administration of CSA, an absence of or a reduction in the amount of subsequent endothelial injury was observed. Circulating ECs might be associated with systemic aGVHD.

Mast Cells Reduce Gvhd Severity In Allogeneic Transplantation by Reducing the Proliferation of Conventional T Cells

AbstractAbstract 243Mast cells are important effector cells in many innate and adaptive immune responses, and can influence the function of several immune cells including granulocytes, monocytes/macrophages, dendritic cells, T cells, B cells, NK cells, and NKT cells. In contrast to their well-know pro-inflammatory roles, in certain models, mast cells have been shown to decrease inflammation and tissue injury. We explored the biological activity of mast cells in a well-established model of mouse graft-versus-host disease (GVHD) by comparing C57BL/6-KitW-sh/W-sh mice (which lack mast cells) to wild-type (WT) C57BL/6 (H-2b) recipients in a major-MHC mismatched model of allogeneic transplantation. After myeloablative irradiation, recipients received 5×106 T-cell depleted bone marrow cells from FVB/N donors (H-2q) on day 0 followed by transfer of 2×106 FVB/N CD4 and CD8 conventional T cells (Tcon) at a 2:1 ratio on day 4. A dramatic decrease in survival was observed in the animals lacking mast cells where 100% of KitW-sh/W-sh recipients died by day 15 following transplantation while over 50% of WT recipients were alive at day 60 (p < 0.0001). The exacerbated GVHD in KitW-sh/W-sh mice correlated with an increase in Tcon proliferation as indicated by bioluminescence imaging (BLI), particularly in lymph node, liver, and gastrointestinal tract tissue sites (p < 0.001). The percentage of Foxp3 positive Treg cells was similar before and after transplantation in KitW-sh/W-sh and WT recipients, and CD4+CD25hi Treg from KitW-sh/W-sh mice were capable of suppressing T cell proliferation in a mixed leukocyte reaction. When KitW-sh/W-sh mice were given 5×106 bone marrow-derived cultured mast cells (BMCMCs) i.p., mast cell re-population of the gut and peritoneal cavity was indicated by both BLI and traditional staining methods. Preliminary experiments with KitW-sh/W-sh recipients engrafted with BMCMCs i.p. 6 weeks before allogenic transplantation demonstrated improved survival in the above GVHD model, but not if the BMCMCs were derived from IL-10-/- C57BL/6 mice. Together, these results support the hypothesis that mast cells can decrease Tcon proliferation and reduce GVHD severity by a mechanism that involves production of IL-10 by mast cells but which is independent of CD4+CD25+ Treg. Disclosures:No relevant conflicts of interest to declare.

A Second Prophylactic MHC-Mismatched Bone Marrow Transplantation Protects Against Rat Acute Myeloid Leukemia (BNML) Without Lethal Graft-Versus-Host Disease

We have employed a rat model for human acute myeloid leukemia, a promyelocytic leukemia in the BN rat strain (BNML), to develop new protocols for immunotherapy in combination with allogeneic bone marrow transplantation (alloBMT). The status of mixed chimerism in allotransplanted rats provided an opportunity for immunotherapy using alloreactive donor cells. In addition to T or natural killer (NK) cells, we introduced a second infusion of bone marrow cells as prophylactic donor lymphocyte infusions (DLI) to test whether an effective graft-versus-leukemia (GVL) response could be obtained without clinical graft-versus-host disease (GVHD). BN rats were sublethally irradiated and transplanted with T-cell depleted bone marrow cells from either fully major histocompatibility complex (MHC)-mismatched (PVG) donor rats or MHC-matched (PVG.1N) as controls. Seven days after transplantation, rats were given 500 leukemic cells to mimic minimal residual disease. Additional cellular therapy was given at day +7. The efficiency of DLI was monitored by chimerism analysis in peripheral blood. Rats receiving infusions of NK cells succumbed to leukemia. T-DLI induced complete donor T-cell chimerism and lethal GVHD. A second alloBMT protected against leukemia. This effect was dependent on an MHC incompatibility between the donor and host and also on the presence of alloreactive T cells in the second bone marrow inoculum, resulting in an increased, mixed donor T-cell chimerism. A second prophylactic transplantation influenced the degree of T-cell chimerism to balance favorably between GVL and GVHD. If applicable to humans, repeated alloBMT may provide a novel approach to leukemia therapy.

Short-Term Ionomycin Exposure Activates Naive Murine T-Cells and Induces a Rapid Phenotypic Shift to Memory T-Cell Status: Potential for Use as a Method To Reduce GvHD Activity of Allogeneic T-Cells.

We are investigating methods to reduce the graft-versus-host disease (GVHD) potential of donor T-cells while retaining graft-versus-leukemia (GVL) activity in allogeneic HSCT. Previous investigations by our group and others in have shown that naive CD4 T-cells induce severe acute GVHD, while memory CD4 T-cells do not induce GVHD but retain GVL activity in murine transplant models. These findings have led to studies for the development of methods to increase the number of memory T-cells available for transplant. The calcium ionophore, ionomycin, is a T-cell activating agent and mitogen. By increasing intracellular Ca2+ levels, ionomycin is induces T-cell activation through signaling mechanisms including phospholipase C activation, hydrolysis of phosphoinositides, and activation of protein kinase C. Differences in memory and naive T-cell responses to ionomycin have been attributed to resistance of memory T-cells to increases in Ca2+. Memory T-cells lack intracellular Ca2+ stores, and are also resistant to influx of Ca2+. Brief low dose ionomycin exposure (20min, 2μM) of T-cells, leading to increased density of naive T-cells, has previously been exploited as a method for separating memory and naive T-cells by Percoll gradient separation. Since ionomycin exposure induces T-cell activation through native Ca2+ dependent signaling mechanisms, we hypothesized that ionomycin-treated T-cells would shift to an activated/memory T-cell phenotype. Murine splenic T-cells were treated with 1.3μM ionomycin for 4hr. Memory and naive T-cell subsets and activation markers were analyzed by flow cytometry. 75% and 85% of untreated CD4 and CD8 T-cells, respectively, had the CD62L+ naive phenotype. These numbers were dramatically reduced to 7% and 17% after ionomycin exposure, representing a shift to the memory T-cell phenotype. Viability of T-cells was not significantly affected. The majority of remaining CD62L+ naive T-cells expressed activation markers CD25 and CD69. The fraction of CD4+CD25+Foxp3+ regulatory T-cells was also determined by intracellular staining of the transcription factor and co-expression of surface markers. CD4+CD25+Foxp3+ regulatory T-cells represented 4% of untreated CD4 T-cells and 3% of ionomycin-treated CD4 T-cells. While ionomycin has been used for many years in studies of T-cell activation, to our knowledge this is the first demonstration of a rapidly-induced shift of naive T-cells to a memory phenotype. A pilot experiment was conducted testing the GVHD activity of ionomycin-treated splenocytes (SP) in B6→ (B6 × Balb/C)CB6F1 recipients. 5 × 106 T-cell depleted bone marrow cells (TCD-BM) were transplanted along with 10 × 106 treated or untreated SP. Mice that received untreated SP all died from acute GvHD by 34 days after transplant, while all recipients of ionomycin-treated SP survived until the experiement was terminated at day 49 (average weight loss was 25%, data not shown). Continuing experients will refine the dose to further reduce GVHD symptoms and also test GVL activity of the treated cells. Treatment of donor T-cells with ionomycin may represent a clinically applicaple method to engineer donor lymphocyte infusions that are safer for HSCT patients. [Display omitted]

Donor-derived thymic-dependent T cells cause chronic graft-versus-host disease

AbstractChronic graft-versus-host disease (GVHD) is the most common cause of poor long-term outcomes after allogeneic bone marrow transplantation (BMT), but the pathophysiology of chronic GVHD still remains poorly understood. We tested the hypothesis that the impaired thymic negative selection of the recipients will permit the emergence of pathogenic T cells that cause chronic GVHD. Lethally irradiated C3H/HeN (H-2k) recipients were reconstituted with T-cell–depleted bone marrow cells from major histocompatibility complex [MHC] class II–deficient (H2-Ab1−/−) B6 (H-2b) mice. These mice developed diseases that showed all of the clinical and histopathological features of human chronic GVHD. Thymectomy prevented chronic GVHD, thus confirming the causal association of the thymus. CD4+ T cells isolated from chronic GVHD mice were primarily donor reactive, and adoptive transfer of CD4+ T cells generated in these mice caused chronic GVHD in C3H/HeN mice in the presence of B6-derived antigen-presenting cells. Our results demonstrate for the first time that T cells that escape from negative thymic selection could cause chronic GVHD after allogeneic BMT. These results also suggest that self-reactivity of donor T cells plays a role in this chronic GVHD, and improvement in the thymic function may have a potential to decrease chronic GVHD.

Critical Roles of Memory T Cells and Antidonor Immunoglobulin in Rejection of Allogeneic Bone Marrow Cells in Sensitized Recipient Mice

Allosensitization is a major risk factor for graft failure in clinical bone marrow transplantation, even with an human leukocyte antigen (HLA)-matched combination under radiation-based conditioning regimens. The critical components of immunological memory in donor bone marrow graft rejection in allosensitized hosts remain unclear at present. C57BL/6-recipient mice, which had been intraperitoneally injected with splenocytes from donor C3H mice on day -35 (sensitized recipients), had been lethally irradiated with 10-Gy whole-body irradiation and were intravenously injected with T-cell-depleted bone marrow cells (TCD-BMC) from C3H mice or third-party SJL mice. Lethally irradiated recipient mice, which had been sensitized by donor splenocytes 5 weeks before the transplantation of TCD-BMC, completely rejected the donor-BMC in a donor-specific manner, whereas none of the nonsensitized recipient mice, all of which showed full allogeneic chimerism, rejected the donor TCD-BMC. Antibody-mediated T cell and/or Natural Killer (NK) cell depletion did not improve the ability of the sensitized recipients to overcome the rejection even when a megadose of TCD-BMC was administered to the sensitized recipients. Furthermore, BMC rejection occurred in sensitized B cell-deficient mice. In adoptive transfer experiments, naive mice, which received a transfer of purified T cells from sensitized mice, rejected the donor BMC, but not those from nonsensitized mice. Moreover, naive mice, which received a transfer of serum containing antidonor immunoglobulin, rejected the donor BMC. These findings suggest that alloreactive memory T cells and antidonor immunoglobulin independently function to reject donor BMC in sensitized recipients.