NFĸB Targeting in Bone Marrow Mesenchymal Stem Cell-Mediated Support of Age-Linked Hematological Malignancies.

Mesenchymal stem cells (MSCs) can become dysfunctional in patients with hematological disorders. An unanswered question is whether age-linked disruption of the bone marrow (BM) microenvironment is secondary to hematological dysfunction or vice versa. We therefore studied MSC function in patients with different hematological disorders and found decreased MHC-II except from one sample with acute myeloid leukemia (AML). The patients’ MSCs were able to exert veto properties except for AML MSCs. While the expression of MHC-II appeared to be irrelevant to the immune licensing of MSCs, AML MSCs lost their ability to differentiate upon contact and rather, continued to proliferate, forming foci-like structures. We performed a retrospective study that indicated a significant increase in MSCs, based on phenotype, for patients with BM fibrosis. This suggests a role for MSCs in patients transitioning to leukemia. NFĸB was important to MSC function and was shown to be a potential target to sensitize leukemic CD34+/CD38- cells to azacitidine. This correlated with their lack of allogeneic stimulation. This study identified NFĸB as a potential target for combination therapy to treat leukemia stem cells and showed that understanding MSC biology and immune response could be key in determining how the aging BM might support leukemia. More importantly, we show how MSCs might be involved in transitioning the high risk patient with hematological disorder to AML.

Development of LT-HSC-Reconstituted Non-Irradiated NBSGW Mice for the Study of Human Hematopoiesis In Vivo

Humanized immune system (HIS) mouse models are useful tools for the in vivo investigation of human hematopoiesis. However, the majority of HIS models currently in use are biased towards lymphocyte development and fail to support long-term multilineage leucocytes and erythrocytes. Those that achieve successful multilineage reconstitution often require preconditioning steps which are expensive, cause animal morbidity, are technically demanding, and poorly reproducible. In this study, we address this challenge by using HSPC-NBSGW mice, in which NOD,B6.SCID IL-2rγ-/-KitW41/W41 (NBSGW) mice are engrafted with human CD133+ hematopoietic stem and progenitor cells (HSPCs) without the need for preconditioning by sublethal irradiation. These HSPCs are enriched in long-term hematopoietic stem cells (LT-HSCs), while NBSGW mice are permissive to human hematopoietic stem cell (HSC) engraftment, thus reducing the cell number required for successful HIS development. B cells reconstitute with the greatest efficiency, including mature B cells capable of class-switching following allogeneic stimulation and, within lymphoid organs and peripheral blood, T cells at a spectrum of stages of maturation. In the thymus, human thymocytes are identified at all major stages of development. Phenotypically distinct subsets of myeloid cells, including dendritic cells and mature monocytes, engraft to a variable degree in the bone marrow and spleen, and circulate in peripheral blood. Finally, we observe human erythrocytes which persist in the periphery at high levels following macrophage clearance. The HSPC-NBSGW model therefore provides a useful platform for the study of human hematological and immunological processes and pathologies.

Combination Treatment With Metformin and Tacrolimus Improves Systemic Immune Cellular Homeostasis by Modulating Treg and Th17 Imbalance

We examined the effect of combination therapy with metformin and tacrolimus on immune parameters including T regulatory (Treg) and type 17 helper T (Th17) cells in vitro and in vivo in mice and in liver transplantation (LT) patients. T cell proliferation and subtypes after in vitro T cell activation or allogeneic stimulation were evaluated. RNA sequencing and microarray analysis were used to evaluate differences in gene expression. Metformin and tacrolimus were administered to mice with graft-versus-host disease (GVHD) and the effects in vivo were assessed. Five LT patients were treated with metformin and the changes in Treg and Th17 cells examined. Combination therapy decreased Type 1 helper T (Th1) and Th17 cells present after in vitro T cell activation, whereas genes associated with Treg were overexpressed. During in vitro allogeneic stimulation, combination therapy increased Treg cells and decreased T cell proliferation and pro-inflammatory markers. In mice with GVHD, combination treatment decreased the clinical and pathological severity of GVHD. In LT patients, addition of metformin increased the peripheral percentage of CD4+Treg and CD8+Treg cells and decreased CD4+Th17. Our study suggests that the addition of metformin to tacrolimus may improve immunological balance by increasing Treg cells and decreasing Th17 cells.

RNA-seq of Human T-Cells After Hematopoietic Stem Cell Transplantation Identifies Linc00402 as a Novel Regulator of T-Cell Alloimmunity

ABSTRACTMechanisms governing allogeneic T-cell responses after allogeneic hematopoietic stem cell (HSC) and solid organ transplantation are incompletely understood. Long non-coding RNAs (lncRNA) do not code for, but control gene expression with tissue specificity. However, their role in T-cell alloimmunity is unknown. We performed RNA-seq on donor T-cells from HSCT patients and found that increasing strength of allogeneic stimulation caused greater differential expression of lncRNAs. The differential expression was validated in an independent patient cohort, and also following ex vivo allogeneic stimulation of healthy human T-cells. Linc00402, a novel, conserved lncRNA, was identified as the most differentially expressed and was enriched 88 fold in human T-cells. Mechanistically, it was mainly located in the cytoplasm, and its expression was rapidly reduced following T-cell activation. Consistent with this, tacrolimus preserved the expression of Linc00402 following T-cell activation, and lower levels of Linc00402 were found in patients who subsequently went on to develop acute graft versus host disease (GVHD). The dysregulated expression of Linc00402 was also validated in murine T-cells, both in vitro and in vivo. Functional studies using multiple modalities to deplete Linc00402 in both mouse and human T-cells, demonstrated a critical role for Linc00402 in the T-cell proliferative response to an allogeneic stimulus but not a non-specific anti-CD3/CD28 stimulus. Thus, our studies identified Linc00402 as a novel, conserved regulator of allogeneic T-cell function. Because of its T-cell specific expression and its impact on allogeneic T-cell responses, targeting Linc00402 may improve outcomes after allogeneic HSC and solid organ transplantation.One sentence summaryLncRNAs are differentially expressed by allogeneic antigen-stimulated T-cells, and the novel lncRNA, Linc00402, is a specific regulator of mouse and human allogeneic T-cells.

Expression of CD4+CD25high+ Regulatory T Cells in Miniature Swine

In mice, there has been increasing evidence that FOXP3-expressing CD4+CD25+ regulatory T cells (Tregs) inhibit potentially reactive T cells to allo-antigens and play an important role in transplant (Tx) tolerance. Human CD4+CD25 high+ T cells represent Tregs, characterized by anergic and antigen-specific suppressive properties. Despite the significance of miniature swine as a preclinical model, only limited data are available regarding the existence of a porcine counterpart to the murine or human Tregs. Methods: From peripheral blood of five adult miniature swine, CD4+ cells (CD4+), CD4+CD25 high+ cells (CD25high+), CD4+CD25low+ cells (CD25low+), and CD4+CD25- cells (CD25-) were isolated with a cell sorter. Expression of the homologous gene to murine FOXP3 was quantified by RT-PCR in isolated fractions, and normalized to GAPDH. Proliferation of CD25high+ and suppressive property of CD25high+ to CD4+ responder cells upon allogeneic stimulation was evaluated by MLR and dilution assay, respectively, after isolation and 9-day culture with allogeneic cells in the presence of IL-2. Results: FOXP3 was expressed ten times higher in CD25 high+, compared to that in CD25low+ (p<0.05). CD25- expressed almost no FOXP3. CD25high+ was anergic and could suppress the proliferation of responder CD4+ upon allogeneic stimulation after isolation. After culture, CD25high+ remained anergic and showed a more suppressive effect on cells used for culture than on third party cells. Conclusions: 1) CD25high+ in miniature swine peripheral blood expressed an extremely high level of FOXP3. 2) This fraction was anergic and has suppressive properties, showing antigen-specificity after exposure to alloantigens. 3) We found for the first time that miniature swine CD25high+ represents the homologous population to the murine and human Tregs. Miniature swine Tx models, therefore, may provide opportunities for pre-clinical evaluation of innovative Tregs-based tolerance strategy.