Arocha, Sinibaldo Romero

Abstract The transcription factor Runx1 is critical for T cell development and maturation. CD4-Cre Runx1 conditional knockout mice have a block in T cell maturation, leading to complement deposition on CD4+ recent thymic emigrants, leaving very few CD4+ T cells in peripheral lymphoid organs. Consistent with a block in maturation, peripheral Runx1-deficient CD4+ T cells fail to produce normal levels of TNF-a upon stimulation. These CD4+ T cells that persist in CD4-Cre Runx1 cKO mice possess a phenotype not found in other genetically modified mice with a block in T cell maturation. Thymic- and splenic-derived T cells from the CD4-Cre Runx1 cKO exhibit altered expression of markers indicative of anergy including FR4 and CD73. This suggests Runx1 plays a role in regulating the development of T cell tolerance which is disrupted upon deletion of Runx1 in CD4+ T Cells. To test this, we have generated a mixed bone marrow chimera (BMC) system with Estrogen Receptor-Cre Runx1 knockout and B6.SJL bone marrow to knock out Runx1 in mature T cells. Surprisingly, in the mixed BMC both the ER-Cre Runx1 cKO and the B6.SJL peripheral T cells acquire a functionally hyporesponsive phenotype, suggesting that Runx1 regulates an extrinsic signaling mechanism controlling tolerance. Moving forward, this BMC model allows us to investigate the development of this anergic phenotype in mature T cells, and tests the hypothesis that Runx1 plays a critical role in regulating T cell tolerance.

The management of patients with heart failure focuses on halting the progression of disease. To date, no therapeutic approach has been able to restore cardiac function to its original baseline state. Cell therapies, such as cardiopoietic (CP) stem cells, have shown to be promising platforms to restore cardiac function. The CP platform enhances the cardioregenerative potency of mesenchymal stem cells (MSCs). CP cells are characterized by the robust expression and nuclear translocation of cardiac transcription factors such Mesoderm Posterior Basic Helix‐Loop‐Helix Transcription Factor 1 (MESP1), early cardiac transcription factor Nkx2.5, myocyte enhancer factor 2C (Mef2C), and zinc finger‐containing transcription factor GATA4. Clinical trials using CP cells showed signs of safety and efficacy in improving cardiac function in patients with heart failure. However, the costs, time, and complexities associated with the manufacturing process have precluded the adoption of the therapy as a large‐scale clinical practice. In response, we adopted a microencapsulated‐modified‐messenger RNA (M3RNA) platform as a cost‐effective and time‐saving alternative to induce high expression of cardiopoietic markers. We hypothesized that the transfection of human umbilical cord‐derived mesenchymal stem cells (hUC‐MSCs) with M3RNA encoding mesodermal and/or cardiac transcription factor(s) will induce the expression of cardiopoietic markers (MESP1, Nkx2.5, Mef2C, and GATA4). hUC‐MSCs were transfected with M3RNA encoding Brachyury (this protein plays a key role in cardiomyocyte differentiation) and MESP1, Nkx2.5, Mef2C, and GATA4 using the transfection reagent Lipofectamine STEM. Protein expression was measured and quantified using fluorescence microscopy and flow cytometry. hUC‐MSCs transfected with M3RNA encoding mesodermal and/or cardiac transcription factor(s) demonstrated rapid protein expression at 4 hours post‐transfection that reached peak levels between 24 hours and 48 hours post‐transfection. Immunocytochemistry demonstrated similar morphology between transfected and non‐transfected cells. Thus, hUC‐MSCs can be transfected with M3RNA with high efficiency to produce rapid and predictable protein expression in vitro. We here demonstrate a simple, cost‐effective, and time‐saving approach to induce the expression of cardiopoietic markers in UC‐MSCs using M3RNA, which can serve as a highly translatable platform to circumvent many burdens associated with current strategies and development of cell‐based therapies for future clinical trials and practice. Support or Funding Information Mayo Clinic Center for Regenerative Medicine, Van Cleve Cardiac Regenerative Medicine Program, Marriott Foundation, Michael S. and Mary Sue Shannon Family, NIH NHLBI HL134664, Mayo Clinic Regenerative Sciences Post‐Baccalaureate Training Program

Abstract Background Clinical trials utilizing mesenchymal stem cells (MCSs) for the treatment of perianal Crohn disease are expanding. Most enrolled Crohn patients are being actively treated with corticosteroids, immunomodulators, and biologic therapy for their luminal and perianal disease at the time of enrollment and treatment. Aim We sought to broaden the understanding of the effect of corticosteroids, immunomodulators, and biologic therapy on the viability and function of MCSs. This information is important for tailoring inclusion and exclusion criteria of clinical trials. Methods Human adipose–derived mesenchymal stem cells (hAMCSs) were harvested and isolated from healthy patient donors. At Passage 3, hAMCSs were treated with 7 commonly used immunosuppressive therapies used to treat Crohn disease at increasing concentrations: dexamethasone, methotrexate, azathioprine, 6-mercaptopurine, infliximab, vedolizumab, and ustekinumab. Cell proliferation, migration, and cytokine secretion were analyzed at Day 4. Results Dexamethasone and azathioprine and 6-mercaptopurien affected cell proliferation and migration. Dexamethasone even resulted in cell death at high physiologic concentrations. The same drugs also had the most profound impacts on IL-6, IL-8, and monocyte chemoattractant protein-1 secretion profiles. Biologic therapies, including anti-tumor necrosis factor, anti-interleukin, and anti-integrins, had the smallest impact on hAMSC proliferation, migration, and cytokine secretion profile. Conclusions In clinical trials with MCSs, a washout period may be recommended for corticosteroids and immunomodulators to minimize any effect of systemic immunosuppression on MSC function and efficacy.

Abstract NKAP and HDAC3 are critical for T cell maturation. NKAP and HDAC3 physically associate, and a point mutation in NKAP, NKAP(Y352A), abrogates this interaction. To evaluate the significance of NKAP and HDAC3 association in T cell maturation, transgenic mice were engineered for cre-mediated endogenous NKAP gene deletion coupled to induction of NKAP(Y352A) or a wild type (WT) control transgene, NKAP(WT), in double positive thymocytes or regulatory T cells (Tregs). T cell maturation was normal in mice with endogenous NKAP deletion coupled to NKAP(WT) induction. However, severe defects occurred in T cell and Treg maturation and in iNKT cell development when NKAP(Y352A) was induced, recapitulating NKAP deficiency. Conventional T cells expressing NKAP(Y352A) failed to enter the long-term T cell pool, did not produce cytokines, and remained complement susceptible, whereas Tregs expressing NKAP(Y352A) were eliminated as recent thymic emigrants leading to lethal autoimmunity. Overall, these results demonstrate the significance of NKAP–HDAC3 association in T cells.

Abstract NKAP is a multifunctional nuclear protein that associates with the histone deacetylase HDAC3. Although both NKAP and HDAC3 are critical for hematopoietic stem cell (HSC) maintenance and survival, it was not known whether these two proteins work together. To assess the importance of their association in vivo, serial truncation and alanine scanning was performed on NKAP to identify the minimal binding site for HDAC3. Mutation of either Y352 or F347 to alanine abrogated the association of NKAP with HDAC3, but did not alter NKAP localization or expression. Using a linked conditional deletion/re-expression system in vivo, we demonstrated that re-expression of the Y352A NKAP mutant failed to restore HSC maintenance and survival in mice when endogenous NKAP expression was eliminated using Mx1-cre and poly-IC, whereas re-expression of wild type NKAP maintained the HSC pool. However, Y352A NKAP did restore proliferation in murine embryonic fibroblasts when endogenous NKAP expression was eliminated using ER-cre and tamoxifen. Therefore, Y352 in NKAP is critical for association with HDAC3 and for HSC maintenance and survival but is not important for proliferation of murine embryonic fibroblasts, demonstrating that NKAP functions in different complexes in different cell types.

CD4 and CD8 T cells are vital components of the immune system. We found that histone deacetylase 3 (HDAC3) is critical for the development of CD4 T cells, as HDAC3-deficient DP thymocytes generate only CD8SP thymocytes in mice. In the absence of HDAC3, MHC Class II-restricted OT-II thymocytes are redirected to the CD8 cytotoxic lineage, which occurs with accelerated kinetics. Analysis of histone acetylation and RNA-seq reveals that HDAC3-deficient DP thymocytes are biased towards the CD8 lineage prior to positive selection. Commitment to the CD4 or CD8 lineage is determined by whether persistent TCR signaling or cytokine signaling predominates, respectively. Despite elevated IL-21R/γc/STAT5 signaling in HDAC3-deficient DP thymocytes, blocking IL-21R does not restore CD4 lineage commitment. Instead, HDAC3 binds directly to CD8-lineage promoting genes. Thus, HDAC3 is required to restrain CD8-lineage genes in DP thymocytes for the generation of CD4 T cells.