Allogeneic Bone Marrow Transplantation Model Research Articles

The effect of platelet infusion on the repair of bone marrow hematopoietic niche damage in mice induced by (60)Co radiation and hematopoietic reconstruction after bone marrow transplantation

Objective: To observe the effect of platelets on hematopoietic stem cell (HSCs) implantation in mice with radiation-induced bone marrow injury and bone marrow transplantation models. Methods: ①Male C57BL/6 mice were divided into a single irradiation group and a radiation infusion group after receiving (60)Co semimyeloablative irradiation for 18-10 weeks. The irradiation infusion group received 1×10(8) platelets expressing GFP fluorescent protein. ② The allogeneic bone marrow transplantation model was established. The experimental groups included the simple transplantation group (BMT) and the transplantation infusion group (BMT+PLT). The BMT group was infused through the tail vein only 5 × 10(6) bone marrow cells, the BMT+PLT group needs to be infused with bone marrow cells at the same time 1× 10(8) platelets. ③ Test indicators included peripheral blood cell and bone marrow cell counts, flow cytometry to detect the proportion of hematopoietic stem cell (HSC) and hematopoietic progenitor cells, bone marrow cell proliferation and apoptosis, and pathological observation of vascular niche damage and repair. Results: ①On the 3rd, 7th, 14(th), and 21st days after irradiation, the bone marrow cell count of the infusion group was higher than that in the single irradiation group (P<0.05), and the peripheral blood cell count was also higher. A statistically significant difference was found between the white blood cell count on the 21st day and the platelet count on the 7th day (P<0.05). In the observation cycle, the percentage of bone marrow cell proliferation in the infusion group was higher, while the percentage of apoptosis was lower. ② The results of bone tissue immunofluorescence after irradiation showed that the continuity of hematopoietic niche with red fluorescence was better in the irradiation infusion group. ③The chimerism percentage in the BMT+PLT group was always higher than that in the BMT group after transplantation.④ The BMT+PLT group had higher bone marrow cell count and percentage of bone marrow cell proliferation on the 7th and 28th day after transplantation than that in the BMT group, and the percentage of bone marrow cell apoptosis on the 14th day was lower than that in the BMT group (P<0.05). After the 14th day, the percentage of stem progenitor cells in the bone marrow cells of mice was higher than that in the BMT group (P<0.05). ⑤The immunohistochemical results of bone marrow tissue showed that the continuity of vascular endothelium in the BMT+PLT group was better than that in the BMT group. Conclusion: Platelet transfusion can alleviate the injury of vascular niche, promotes HSC homing, and is beneficial to hematopoietic reconstruction.

TNFR2 blockade of regulatory T cells unleashes an antitumor immune response after hematopoietic stem-cell transplantation

BackgroundTargeting immune checkpoints that inhibit antitumor immune responses has emerged as a powerful new approach to treat cancer. We recently showed that blocking the tumor necrosis factor receptor-type 2 (TNFR2)...

Modeling acute graft-versus-host disease (aGVHD) in murine bone marrow transplantation (BMT) models with MHC disparity.

For more than 50years, hematopoietic stem cell transplantation (HSCT) has been the major curative therapy for hematological malignancies and genetic disorders, but its success is limited by the development of graft-versus-host disease (GVHD). GVHD represents a post-transplantation disorder representing the immune-mediated attack of transplant-derived T cells against recipient tissue finally leading to increased morbidity and mortality of the recipient. GVHD develops if donor and recipient are disparate in major or minor histocompatibility antigens (MHC, miHA). Most of the initial knowledge about the biology of GVHD is derived from murine bone marrow transplantation (BMT) models. Of course, GVHD mouse models do not reflect one to one the human situation, but they contribute significantly to our understanding how conditioning and danger signals activate the immune system, enlighten the role of individual molecules, e.g., cytokines, chemokines, death-inducing ligands, define the function of lymphocytes subpopulations for GVHD development and have significant impact on establishing new treatment and prevention strategies used in clinical HSCT. This chapter describes in detail the procedure of allogeneic BMT and the development of GVHD in two commonly used allogeneic murine BMT models (B6→B6.bm1, B6→B6D2F1) with different MHC disparities, which can be used as a basis for advanced studies of GVHD pathology or the development of new treatment strategies.

Magnetism-Induced Cell-Targeting Transplantation Improves Early Hematopoietic Reconstitution in a Murine Bone Marrow Transplantation Model

BackgroundEarly hematopoietic reconstitution is essential for improving survival and reducing complications after hematopoietic stem/progenitor cell (HSC/HPC) transplantation (HSCT/HPCT). Increasing HSC/HPC homing to the bone marrow is a potential approach for promoting hematopoietic reconstitution.MethodsWe proposed the transplantation of HSCs/HPCs with a magnetism-induced cell-targeting transplantation (MagIC-TT) strategy. HSCs/HPCs were magnetized with CD45 microbeads. The biological characteristics (morphology, proliferation, viability, and ferroptosis) and target migration ability of these cells were studied in vitro. The hematopoietic reconstitution experiments were constructed in vivo in autologous and allogeneic bone marrow transplantation models with grouping showed as Table 1. The therapeutic effects were assessed by survival, donor chimerism, routine blood examination and histological analysis. We also performed transcriptomic sequencing for further mechanistic studies.ResultsThe biological characteristics was found no significant difference between the MagIC-TT and non-MagIC-TT groups, while migration ability was greatly improved with MagIC-TT (Data not showed). The survival rate was higher in the MagIC-TT group and significantly different in the allogeneic model (P<0.05). Hematopoietic reconstitution of donor chimerism, WBCs, HGB, and PLTs was faster in the MagIC-TT groups (within 22 days) (Figure 1). Confocal observation showed that more donor cells (eGFP +) were retained in the injected femur of the MagIC-TT group than in the femur of the non-MagIC-TT group 7 days after transplantation (P<0.05) (Figure 2). The severity of aGVHD (Assessed by survival, body weight, back arching, fur losting, diarrhea etc.) was reduced in the MagIC-TT groups in the allogeneic model (P<0.05) (Data not showed). Transcriptome sequencing revealed differentially expressed genes (DEGs) involved in localization/locomotion and pathways, cytokine-cytokine receptor interactions and chemokine signaling pathways between the two groups (Figure 3).ConclusionThe MagIC-TT strategy improves HSC/HPC homing, resulting in faster hematopoietic reconstitution in a murine bone marrow transplantation model.Key wordsMagnetism; Hematopoietic Stem Cell Transplantation (HSCT); Cell Homing; Hematopoietic Reconstitution; Ferroptosis [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Graft-Versus-Host Disease Prevention by In Vitro-Generated Myeloid-Derived Suppressor Cells Is Exclusively Mediated by the CD11b+CD11c+ MDSC Subpopulation.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of myeloid progenitor cells that dampen overwhelming adaptive immune responses through multiple mechanisms and are recognized as an attractive novel immune intervention therapy for counteracting the destructive effects of graft- versus -host disease (GVHD) developing after allogeneic bone marrow transplantation (BMT). MDSCs can be produced in great numbers for cellular therapy, but they present a mixture of subsets whose functions in GVHD prevention are undefined. Here, we generated MDSCs in vitro from murine BM cells in the presence of GM-CSF and defined the integrin CD11c as a marker to subdivide MDSCs into two functional subgroups: CD11b+CD11c+ and CD11b+CD11c− MDSCs. Isolated CD11b+CD11c+ and CD11b+CD11c− MDSCs both inhibited alloantigen-stimulated T-cell proliferation in vitro, although CD11b+CD11c+ MDSCs were more efficient and expressed higher levels of different immunosuppressive molecules. Likewise, expression of surface markers such as MHC class II, CD80, CD86, or PD-L1 further delineated both subsets. Most importantly, only the adoptive transfer of CD11b+CD11c+ MDSCs into a single MHC class I-disparate allogeneic BMT model prevented GVHD development and strongly decreased disease-induced mortality, while CD11b+CD11c− MDSCs were totally ineffective. Surprisingly, allogeneic T-cell homing and expansion in lymphatic and GVHD target organs were not affected by cotransplanted CD11b+CD11c+ MDSCs indicating a clear contradiction between in vitro and in vivo functions of MDSCs. However, CD11b+CD11c+ MDSCs shifted immune responses towards type 2 immunity reflected by increased Th2-specific cytokine expression of allogeneic T cells. Induction of type 2 immunity was mandatory for GVHD prevention, since CD11b+CD11c+ MDSCs were ineffective if recipients were reconstituted with STAT6-deficient T cells unable to differentiate into Th2 cells. Most importantly, the beneficial graft- versus -tumor (GVT) effect was maintained in the presence of CD11b+CD11c+ MDSCs since syngeneic tumor cells were efficiently eradicated. Strong differences in the transcriptomic landscape of both subpopulations underlined their functional differences. Defining CD11b+CD11c+ MDSCs as the subset of in vitro-generated MDSCs able to inhibit GVHD development might help to increase efficiency of MDSC therapy and to further delineate relevant target molecules and signaling pathways responsible for GVHD prevention.

Combined CCR5 and CXCR3 Blockade Attenuates Murine Agvhd through Alternating Donor-Derived T Cell Distribution and Function

Acute graft-versus-host disease (aGVHD) is one of the major complications and a leading cause of death after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Chemokines and their cognate receptors play an important role in directing the migration of donor derived T cells to target organs. Chief among these chemokine receptors are CXCR3 and CCR5. Our previous study found that CCR5 blockade combined with cyclosporine A in a murine model could attenuate the severity of aGVHD in a less extent. This might be due to multiple expression of chemokine receptors on immune cell, only one kind receptor blockade might not completely block the migration of T cells to aGVHD target organs. In this study, we investigated whether combined CCR5 and CXCR3 blockade could further attenuate murine aGVHD, and also explored its immunologic mechanism.BALB/c(H-2Kd)recipients received 8.0 Gy of total body irradiation (60Coγsource) prior to transplantation followed by the infusion of bone marrow cells (BMCs) and splenic cells (SCs) from C57BL/6J(H-2Kb)or CCR5 knockout mice(H-2Kb)to establish an allogeneic bone marrow transplantation model. Body weight change, clinical scores of aGVHD, degree of histopathologic damage and survival rate were dynamically recorded to assess the efficacy of CCR5 and CXCR3 blockade in this transplantation model. Mechanically, donor derived lymphocytes were labeled with fluorescence dye to detect their distribution in vivo. Immunohistochemical analysis for CD4+ and CD8+ were performed on target organs of recipient mice at 7 days post-transplantation. Flow cytometry was used to calculate the absolute number of T cells in target organs, and to detect its proliferation, activation and differentiation in the spleen. ELISA was used to determine the concentration of T cells related cytokines.The results showed that both CCR5 and CXCR3 are enhanced during the aGVHD process. Although transplantation of allogeneic BMCs and SCs induced severe clinical aGVHD with high aGVHD scores (4.67±0.33) and a lethality of 90%, combined CCR5 and CXCR3 blockade attenuated clinical signs of aGVHD, reduced aGVHD scores (1.25±0.25, P< 0.001), reduced aGVHD-induced death (a lethality of 40%, P< 0.05). Compared with aGVHD group, combined CCR5 and CXCR3 blockade reduced liver histologic score (3.3±0.3 versus 1.3±0.3, P< 0.05), lung histologic score (3.6±0.3 versus 1.3±0.3, P< 0.05) and intestine histologic score (3.7±0.3 versus 1.3±0.3, P< 0.05). On days 7 after transplantation, more fluorescent signal emanated from the spleen and from lymph nodes after the use of CCR5 and CXCR3 blockade. Also combined CCR5 and CXCR3 blockade reduced the infiltration of CD4+ and CD8+ T cells into the liver and intestine, and retained part of CD4+ and CD8+ T cells within SLOs. As for the effector function of donor T cells, the results showed that combined CCR5 and CXCR3 blockade dampened T-cell activation, suppressed the percentage of Th1 and Tc1, and induced Treg induction. Additionally, combined CCR5 and CXCR3 blockade altered the concentrations of IFN-γ and IL10. Furthermore, the transplantation of CCR5-/- splenic cells with CXCR3 blockade showed a stronger protection effect on aGVHD.Combined CCR5 and CXCR3 blockade further attenuated aGVHD. aGVHD inhibition was associated with alternated donor T-cell distribution, limited donor T-cell activation and negatively impacted T-cell effector function in vivo. These data suggest that the combined use of CCR5 and CXCR3 blockade might be applicable for aGVHD prophylaxis in clinical settings. DisclosuresNo relevant conflicts of interest to declare.

Bone marrow-derived macrophages converted into cancer-associated fibroblast-like cells promote pancreatic cancer progression.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by a desmoplastic reaction caused by cancer-associated fibroblasts (CAFs), which provokes treatment resistance. CAFs are newly proposed to be heterogeneous populations with different functions within the PDAC microenvironment. The most direct sources of CAFs are resident tissue fibroblasts and mesenchymal stem cells, however, the origins and functions of CAF subtypes remain unclear. Here, we established allogeneic bone marrow (BM) transplantation models using spontaneous PDAC mice, and then investigated what subtype cells derived from BM modulate the tumor microenvironment and affect the behavior of pancreatic cancer cells (PCCs). BM-derived multilineage hematopoietic cells were engrafted in recipient pancreas, and accumulated at the invasive front and central lesion of PDAC. We identified BM macrophages-derived CAFs in tumors. BM-derived macrophages treated with PCC-conditioned media expressed CAF markers. BM-derived macrophages led the local invasion of PCCs in vitro and enhanced the tumor invasive growth in vivo. Our data suggest that BM-derived cells are recruited to the pancreas during carcinogenesis and that the specific subpopulation of BM-derived macrophages partially converted into CAF-like cells, acted as leading cells, and facilitated pancreatic cancer progression. The control of the conversion of BM-derived macrophages into CAF-like cells may be a novel therapeutic strategy to suppress tumor growth.

Microrna-31 Regulates T-Cell Metabolism Via HIF1α and Promotes Effector Function

MicroRNAs (miRs) repress gene expression at the post-transcriptional level via binding target mRNAs subsequently promoting mRNA degradation or impeding translation. Certain miRs influence the survival, differentiation, and function of T cells in cancer, infections, and autoimmunity. Uncontrolled T-cell allogeneic responses contribute to chronic graft-versus-host disease (cGVHD), a major cause of non-relapse mortality following allogeneic hematopoietic stem-cell transplantation (allo-HCT). Dysregulation of miR-31 is implicated in cancer, but how miR-31 impacts allogeneic T-cell response is unclear. Using oligonucleotide array, we found miR-31 was dramatically up-regulated in T cells under alloantigen-driven vs. homeostatic proliferation in recipient mice following bone marrow transplantation (BMT) (Figure 1A-B). To understand how miR-31 regulates T-cell responses to alloantigens, we utilized murine models of allogeneic BMT and donor mice with miR-31 conditional knock-out on their T cells. In a sclerodermatous cGVHD model (B6→BDF1), we observed that the recipients transferred with donor T cells deficient for miR-31 exhibited significantly alleviated disease (Figure 1C), reflected by attenuated fibrosis and pathologic damage in skin. In a bronchiolitis obliterans cGVHD model (B6→B10.BR), we found that the recipients of miR-31-deficient T cells had reduced airway resistance, elastance (Figure 1D) and pathological damage in the lungs as compared with those of WT T cells. The role of miR-31 in promoting T-cell pathogenicity was further confirmed when miR-31 was inhibited by administration of specific antagomir (locked nuclear acid anti-miR-31). We used mass cytometry to analyze donor immune cell reconstitution in recipient spleens after BMT. Consistent with attenuated disease manifestation, the recipients of miR-31-deficient T cells displayed improved reconstitution of donor T- and B- lymphocytes. The follicular T helper cells (TFH) instruct germinal center (GC) B-cell expansion, affinity maturation, and plasma cell differentiation, whereas follicular regulatory T cells (TFR) inhibit TFH -mediated B-cell activation and antibody production. We found that miR-31 deficient T cells differentiated into more TFR, but fewer TFH during alloresponses. As a result, the differentiation and activation of GC B cells and the generation of plasma cells were attenuated in the recipients of miR-31-deficient T cells. Furthermore, miR-31-deficient T cells exhibited defects in proliferation and survival in allogeneic recipients, resulting in fewer donor T cells in recipient thymus, skin and lungs. In the absence of miR-31, T cells differentiated less into Th17 cells but more towards Tregs (Figure 1E) in vivo. Those miR-31-deficient Tregs expressed higher levels of Neuropilin 1 and PD-L1, the markers associated with superior stability and suppressive function of Tregs. While miR-31 played little role on Th1 cell differentiation, it facilitated alloantigen-reactive iTregs losing Foxp3 and producing IFNγ after being transferred into allogeneic recipients. Hypoxia, a hallmark of inflamed and damaged tissue, can drive fibrosis and disease development through immune cell dysregulation. Upon activation, T cells rapidly increase their metabolic rate and switch from oxidative phosphorylation to aerobic glycolysis. We found that miR-31-deficient T cells exhibited reduced hypoxia-inducible factor 1α (HIF1α) signaling (glycolysis promotor), surface GLUT1 expression, and glucose uptake, but increased lipid droplet accumulation in allogeneic recipients, suggesting that miR-31 promotes metabolic switch from fatty acid β-oxidation to aerobic glycolysis in allo-reactive T cells (Figure 1F). Furthermore, under hypoxia (3% oxygen) but not normoxia (21% oxygen) conditions, miR-31 increased Th17 but decreased iTreg differentiation from naïve CD4 T cells in the presence of IL-6 and TGFβ in vitro. Taken together, miR-31 regulates T-cell expansion, differentiation, and metabolism via promoting HIF1α expression and hypoxia adaptation in allo-reactive T cells, which enhances T-cell effector function and pathogenicity after allogeneic BMT. Disclosures No relevant conflicts of interest to declare.

Focused Regulatory T Cell Repertoires Are Indicative of Successful GvHD Control in Experimental and Clinical Stem Cell Transplantation

Acute Graft versus Host Disease (aGvHD) remains a major complication and leading cause of mortality after allogeneic stem cell or bone marrow transplantation (BMT). Current strategies for treatment are still based on unspecific immunosuppressive therapy. Over the last decade, there have been major advances in the field of adoptive immunotherapy using regulatory CD4+CD25+Foxp3+ T cells (Treg cells). Nonetheless, not much is known about the exact mechanisms of Treg-mediated suppression, and even less about the importance of T cell receptor (TCR) specificity and its diversity on the functionality of Tregs.We hypothesized that an optimal Treg TCR repertoire is necessary for successful prevention of aGvHD. To test this hypothesis, we sequenced the TCR repertoire of 8 patients who were diagnosed with aGvHD on day 30 post transplantation and compared it with the TCR repertoire of nine GvHD-free patients. Analysis of GvHD-free patients on day 30 (and 100 days-follow up) revealed a lower TCR diversity when compared to the patients suffering from GvHD. A more detailed analysis of the TCR repertoire showed that in patients without GvHD, fewer clonotypes were needed to comprise 50% of the whole repertoire as compared with samples from patients with GvHD (Figure 1A). Thus, expansion of protective clones indicates their potent immunosuppressive capabilities.Next, we employed a well-described murine model of allogeneic BMT (BL/6–>Balb/c) with co-injection of Tregs. Recipient Balb/c mice transplanted in this fashion were previously shown to be protected from aGvHD. However, the mechanisms involved in this Treg-mediated protection are not fully understood. Therefore, Tregs were FACS sorted from B6.Cg-Foxp3tm1Mal/J mice based on their Foxp3 expression. Recipient mice were transplanted with T-cell depleted bone marrow and a mixture of conventional T cells (Tconv) and Tregs in 1:1 ratio. Transferred Tregs were re-sorted on day 7 and day 14 from secondary lymphoid organs based on the congenic marker Thy 1.1 and Foxp3 expression. Using this model, we investigated the kinetics of the Treg TCR repertoire early after BMT in 5 independent experiments. We found a consistently similar narrowing of the repertoire and clonal expansion in mice protected from GvHD (Figure 1B). Diversity analysis using inverse Simpson Index also confirmed our findings. These data further support the notion that a clonal expansion of Tregs is necessary for an optimal immunosuppression of an allogeneic response, both in human and in mice. To test the functionality and phenotype of such expanded Tregs, they were re-sorted from BMT-recipient mice 14 days after transplantation. These Tregs were expanded using α-CD3 and α-CD28 antibodies and were functionality tested in an in vitro Treg suppression assay. Re-sorted Tregs after expansion showed expression of established Treg surface and intracellular markers such as Helios, CD25, GITR and CTLA-4. For the suppression assay, responder CD4 Tconv were stained with a proliferation tracking dye eFluor670 and stimulated in vitro with CD3 and CD28 beads in the presence of different ratios of re-sorted and expanded, or polyclonaly activated Tregs as the control. Allo-specific ex vivo Tregs exhibited a superior suppressive potential when compared with polyclonaly activated Tregs in vitro.Taken together, our current study highlights the importance of specific Treg driven allo-response in GvHD prevention. Further studies are needed, particularly in larger patient cohorts to confirm these findings. However, we propose that this approach might lead to identification and subsequent use of specific Treg clones with high immunosuppressive capacity for the prevention of aGvHD. [Display omitted] DisclosuresGanser:Novartis: Membership on an entity's Board of Directors or advisory committees. Koenecke:abbvie: Consultancy; BMS: Consultancy; Roche: Consultancy; Amgen: Consultancy.

Flt-3L Expansion of Recipient CD8α+ Dendritic Cells Deletes Alloreactive Donor T Cells and Represents an Alternative to Posttransplant Cyclophosphamide for the Prevention of GVHD.

Purpose: Allogeneic bone marrow transplantation (BMT) provides curative therapy for leukemia via immunologic graft-versus-leukemia (GVL) effects. In practice, this must be balanced against life threatening pathology induced by graft-versus-host disease (GVHD). Recipient dendritic cells (DC) are thought to be important in the induction of GVL and GVHD.Experimental Design: We have utilized preclinical models of allogeneic BMT to dissect the role and modulation of recipient DCs in controlling donor T-cell-mediated GVHD and GVL.Results: We demonstrate that recipient CD8α+ DCs promote activation-induced clonal deletion of allospecific donor T cells after BMT. We compared pretransplant fms-like tyrosine kinase-3 ligand (Flt-3L) treatment to the current clinical strategy of posttransplant cyclophosphamide (PT-Cy) therapy. Our results demonstrate superior protection from GVHD with the immunomodulatory Flt-3L approach, and similar attenuation of GVL responses with both strategies. Strikingly, Flt-3L treatment permitted maintenance of the donor polyclonal T-cell pool, where PT-Cy did not.Conclusions: These data highlight pre-transplant Flt-3L therapy as a potent new therapeutic strategy to delete alloreactive T cells and prevent GVHD, which appears particularly well suited to haploidentical BMT where the control of infection and the prevention of GVHD are paramount. Clin Cancer Res; 24(7); 1604-16. ©2018 AACR.

Interleukin-21 promotes thymopoiesis recovery following hematopoietic stem cell transplantation

BackgroundImpaired T cell reconstitution remains a major deterrent in the field of bone marrow (BM) transplantation (BMT) due to pre-conditioning-induced damages inflicted to the thymi of recipient hosts. Given the previously reported thymo-stimulatory property of interleukin (IL)-21, we reasoned that its use post-BMT could have a profound effect on de novo T cell development.MethodsTo evaluate the effect of IL-21 on de novo T cell development in vivo, BM derived from RAG2p-GFP mice was transplanted into LP/J mice. Lymphocyte reconstitution was first assessed using a hematological analyzer and a flow cytometer on collected blood samples. Detailed flow cytometry analysis was then performed on the BM, thymus, and spleen of transplanted animals. Finally, the effect of human IL-21 on thymopoiesis was validated in humanized mice.ResultsUsing a major histocompatibility complex (MHC)-matched allogeneic BMT model, we found that IL-21 administration improves immune reconstitution by triggering the proliferation of BM Lin−Sca1+c-kit+ (LSK) subsets. The pharmacological effect of IL-21 also culminates in the recovery of both hematopoietic (thymocytes) and non-hematopoietic (stromal) cells within the thymi of IL-21-treated recipient animals. Although T cells derived from all transplanted groups proliferate, secrete various cytokines, and express granzyme B similarly in response to T cell receptor (TCR) stimulation, full regeneration of peripheral naïve CD4+ and CD8+ T cells and normal TCRvβ distribution could only be detected in IL-21-treated recipient mice. Astonishingly, none of the recipient mice who underwent IL-21 treatment developed graft-versus-host disease (GVHD) in the MHC-matched allogeneic setting while the graft-versus-tumor (GVT) effect was strongly retained. Inhibition of GVHD onset could also be attributed to the enhanced generation of regulatory B cells (B10) observed in the IL-21, but not PBS, recipient mice. We also tested the thymopoiesis-stimulating property of human IL-21 in NSG mice transplanted with cord blood (CB) and found significant improvement in de novo human CD3+ T cell development.ConclusionsIn sum, our study indicates that IL-21 represents a new class of unforeseen thymopoietin capable of restoring thymic function following BMT.

Enhance T Cell Immunotherapy By Targeting PIM-2 Kinase

PIM kinases, a family of serine/threonine kinases, play an important role in regulating cell survival, cell proliferation, transcription activation and protein translation. Of the three isoforms, PIM-1, PIM-2 and PIM-3, some of them are overly expressed in several hematological malignancies as well as solid tumors. Thus, these kinases have been targeted clinically in several cancer studies. However, the roles of PIM kinases in T cells from previous studies are inconclusive and the functions of each isoform in these cells are yet to be investigated.In our study, we focused on the role of PIM-2 kinase in T cell responses to allo-antigen and tumor using pre-clinical models. Using allogeneic bone marrow transplantation (allo-BMT) models, we transferred allogeneic T cells isolated from mice deficient for Pim-2 (Pim-2-/-, FVB background) in comparison to WT and PIM1/3double deficient mice (PIM1/3-/-) into lethally irradiated Balb/C recipients. Our data revealed that upon allogeneic stimulation, PIM-2-/- T cells induced accelerated graft-versus-host-disease (GVHD) compared to WT or PIM1/3-/- T cells (Fig.1A). In addition, PIM-2-/- donor T cells were highly activated with significantly increased proliferation and IFN-γ production in recipient spleen and GVHD target organs. Donor PIM-2-/- T cells also expressed high levels of chemokine receptor, CXCR3 and a4b7 integrin (gut homing receptor) that correlated with increased capacity of donor T-cell migration to GVHD target organs such as liver and gut. In graft-versus-leukemia (GVL) study, PIM-2-/- donor T cells had roughly 4-fold higher ability to mediate GVL effect to WT and PIM1/3-/- T cells (Fig.1B). Negative regulation of T-cell responses to allo-antigens by PIM-2 was independently verified by restoring PIM-2 expression on PIM-2-/- T cells or inhibiting PIM kinases in PIM1/3-/- T cells. Altogether, the data from allo-BMT studies suggested that PIM-2 isoform functions as a negative regulator in T cell allo-response in contrast to PIM-1 and PIM-3 kinases.We then focused on evaluating T-cell immunity against syngeneic tumor. By administering tumor (TS-1 breast tumor in FVB background) into WT and PIM-2-/- mice either subcutaneously or intravenously, we observed that PIM-2-deficient mice efficiently control tumor growth in sharp contrast to WT mice (Fig. 1C). Mechanistic study revealed that tumor rejection in PIM-2-/- recipient relies on CD8+ tumor infiltrating lymphocytes (TILs) with a significant up-regulation of IFN-g, TNF-a and Fas-L expression. Lower infiltration of regulatory T cells and down regulation of PD-1 expression on T cells recovered from tumor site were also observed in PIM-2 deficient mice. Moreover, the tumor growth rate in PIM-2-/- mice after T-cell depletion with antibody was similar to that of WT mice. To further exclude the effect of other immune cells involved in tumor regression, we adoptively transferred WT and PIM-2-/- CD8+ T cells into WT mice with pre-established TS-1 tumor. CD8+ T cells deficient for PIM-2 was more efficient in controlling of tumor growth than WT or PIM1/3-/- CD8+ T cells. These data indicate that inhibition of PIM-2 in T cells display superior anti-tumor activity. In conclusion, PIM-2 kinase possesses a distinct role in anti-tumor immunity compared to the other two isoforms and it represents a potential target to enhance anti-tumor activity of adoptive T-cell immunotherapy. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

PreImplantation factor (PIF) therapy provides comprehensive protection against radiation induced pathologies.

Acute Radiation Syndrome (ARS) may lead to cancer and death and has few effective countermeasures. Efficacy of synthetic PIF treatment was demonstrated in preclinical autoimmune and transplantation models. PIF protected against inflammation and mortality following lethal irradiation in allogeneic bone marrow transplant (BMT) model. Herein, we demonstrate that PIF imparts comprehensive local and systemic protection against lethal and sub-lethal ARS in murine models. PIF treatment 2 h after lethal irradiation led to 100% survival and global hematopoietic recovery at 2 weeks after therapy. At 24 h after irradiation PIF restored hematopoiesis in a semi-allogeneic BMT model. PIF-preconditioning provided improved long-term engraftment. The direct effect of PIF on bone marrow cells was also demonstrated in vitro: PIF promoted pre-B cell differentiation and increased immunoregulatory properties of BM-derived mesenchymal stromal cells. PIF treatment also improved hematopoietic recovery and reduced systemic inflammatory cytokine production after sub-lethal radiation exposure. Here, PIF also prevented colonic crypt and basal membrane damage coupled with reduced nitric oxide synthetase (iNOS) and increased (B7h1) expression. Global upper GI gene pathway analysis revealed PIF's involvement in protein-RNA interactions, mitochondrial oxidative pathways, and responses to cellular stress. Some effects may be attributed to PIF's influence on macrophage differentiation and function. PIF demonstrated a regulatory effect on irradiated macrophages and on classically activated M1 macrophages, reducing inflammatory gene expression (iNOS, Cox2), promoting protective (Arg1) gene expression and inducing pro-tolerance cytokine secretion. Notably, synthetic PIF is stable for long-term field use. Overall, clinical investigation of PIF for comprehensive ARS protection is warranted.

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

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

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

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

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

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

Interleukin-22 directly regulates intestinal stem cell regeneration after tissue damage (MUC8P.722)

Abstract Little is known about regulation of the intestinal stem cell (ISC) compartment after injury. We hypothesized that IL-22 could promote intestinal regeneration by either acting on ISCs or by acting on Paneth cells (PCs) making up the ISC niche. Utilizing an in vitro regeneration model, we found that organoids derived from murine small intestine (SI) and large intestine (LI) crypts demonstrated substantially increased size after culture with IL-22. Innate lymphoid cell co-culture also increased organoid size in an IL-22-dependent fashion. IL-22 expanded the Lgr5+ ISC pool within SI organoids, activated STAT3 phosphorylation in Lgr5+ cells, and increased size of organoids cultured from purified SI ISCs. Using a murine allogeneic bone marrow transplant (BMT) model, we found that IL-22 administration led to reduced intestinal graft vs. host disease pathology, increased maintenance of Lgr5+ ISCs, and significantly greater ISC proliferation. ISC protection post-BMT was not due to niche augmentation, as PC numbers, PC-derived growth factors (EGF, Wnt3), and stroma-derived growth factors (R-spondin3) were all unchanged after IL-22 administration. IL-22 receptor (IL-22R) staining further indicated a direct effect of IL-22 on ISCs rather than the niche, as PCs were found to express very little IL-22R and demonstrated no evidence of STAT3 phosphorylation upon in vitro culture. In summary, we found that IL-22 regulates epithelial regeneration after tissue damage by acting directly on ISCs.

BET bromodomain inhibition suppresses graft-versus-host disease after allogeneic bone marrow transplantation in mice

AbstractAcute graft-versus-host disease (GVHD) is the major obstacle of allogeneic bone marrow transplantation (BMT). Bromodomain and extra-terminal (BET) protein inhibitors selectively block acetyl-binding pockets of the bromodomains and modulate histone acetylation. Here, we report that inhibition of BET bromodomain (BRD) proteins with I-BET151 alters cytokine expression in dendritic cells (DCs) and T cells, including surface costimulatory molecules, in vitro and in vivo cytokine secretion, and expansion. Mechanistic studies with I-BET151 and JQ1, another inhibitor, demonstrate that these effects could be from disruption of association between BRD4 and acetyl-310 RelA of nuclear factor kappa B. Short-term administration early during BMT reduced GVHD severity and improved mortality in two different allogeneic BMT models but retained sufficient graft-versus-tumor effect. Thus inhibiting BRD proteins may serve as a novel approach for preventing GVHD.

Mouse bone marrow-derived mesenchymal stem cells inhibit leukemia/lymphoma cell proliferation in vitro and in a mouse model of allogeneic bone marrow transplant.

The allogeneic hematopoietic stem cell (HSC) transplantation of mesenchymal stem cells (MSCs) contributes to the reconstitution of hematopoiesis by ameliorating acute graft-versus-host disease (aGVHD). However, the role of MSCs in graft-versus-leukemia remains to be determined. In the present study, we co-cultured C57BL/6 mouse bone marrow (BM)-derived MSCs with A20 murine B lymphoma, FBL3 murine erythroleukemia and P388 murine acute lymphocytic leukemia cells. Cell proliferation, apoptosis, cell cycle progression and the amount of cytokine secretion were then measured using a Cell Counting kit-8, Annexin V/propidium iodide staining, flow cytometry and ELISA, respectively. We also established a model of allogeneic bone marrow transplantation (BMT) using BALB/c mice. Following the administration of A20 cells and MSCs, we recorded the symptoms and the survival of the mice for 4 weeks, assessed the T cell subsets present in peripheral blood, and, after the mice were sacrifice, we determined the infiltration of MSCs into the organs by histological staining. Our results revealed that the MSCs inhibited the proliferation of the mouse lymphoma and leukemia cells in vitro, leading to cell cycle arrest and reducing the secretion of interleukin (IL)-10. In our model of allogeneic BMT, the intravenous injection of MSCs into the mice injected wth A20 cells decreased the incidence of lymphoma, improved survival, increased the fraction of CD3+CD8+ T cells, decreased the fraction of CD3+CD4+ T cells and CD4+CD25+ T cells in peripheral blood, and ameliorated the manifestation of aGVHD. The results from the present study indicate that MSCs may be safe and effective when used in allogeneic BMT for the treatment of hemotological malignancies.

MSC therapy attenuates obliterative bronchiolitis after murine bone marrow transplant.

RationaleObliterative bronchiolitis (OB) is a significant cause of morbidity and mortality after lung transplant and hematopoietic cell transplant. Mesenchymal stromal cells (MSCs) have been shown to possess immunomodulatory properties in chronic inflammatory disease.ObjectiveAdministration of MSCs was evaluated for the ability to ameliorate OB in mice using our established allogeneic bone marrow transplant (BMT) model.MethodsMice were lethally conditioned and received allogeneic bone marrow without (BM) or with spleen cells (BMS), as a source of OB-causing T-cells. Cell therapy was started at 2 weeks post-transplant, or delayed to 4 weeks when mice developed airway injury, defined as increased airway resistance measured by pulmonary function test (PFT). BM-derived MSC or control cells [mouse pulmonary vein endothelial cells (PVECs) or lung fibroblasts (LFs)] were administered. Route of administration [intratracheally (IT) and IV] and frequency (every 1, 2 or 3 weeks) were compared. Mice were evaluated at 3 months post-BMT.Measurements and Main ResultsNo ectopic tissue formation was identified in any mice. When compared to BMS mice receiving control cells or no cells, those receiving MSCs showed improved resistance, compliance and inspiratory capacity. Interim PFT analysis showed no difference in route of administration. Improvements in PFTs were found regardless of dose frequency; but once per week worked best even when administration began late. Mice given MSC also had decreased peribronchiolar inflammation, lower levels of hydroxyproline (collagen) and higher frequencies of macrophages staining for the alternatively activated macrophage (AAM) marker CD206.ConclusionsThese results warrant study of MSCs as a potential management option for OB in lung transplant and BMT recipients.