Wilfahrt, Drew Nicholas

Abstract After activation, CD4+ T cells undergo metabolic modifications in order to orchestrate successful immune responses. In particular, naïve T cells shift to a more glycolysis-heavy metabolic program that produces the necessary metabolites for growth, division, and exertion of effector function. Histone Deacetylase 3 (HDAC3) has been shown to epigenetically regulate many T cell fate decisions and is also capable of post-translationally modifying non-histone proteins. Deletion of HDAC3 in mature CD4+ T cells using a distal Lck-Cre HDAC3 cKO mouse revealed a reduction in differentiated T-helper cell populations in vivo. In contrast, in vitro differentiation of HDAC3-deficient CD4+ T cells occurred normally, however, the numbers of T cells present within each culture was greatly diminished. CD3/CD28-stimulated HDAC3-deficient cells exhibited poor proliferation with decreased blastogenesis, decreased CD25 expression, and decreased IL-2 production. Signaling downstream of TCR is disrupted in the HDAC3-deficient T cells. HDAC3-deficient CD4+ T cells have an overabundance of Foxo1 protein, and diminished signaling downstream of mTOR, including reduced phosphorylation of Ribosomal Protein S6, and decreased expression of Egr2. Key transporters involved in cholesterol efflux, ABCA1 and ABCG1, were upregulated in HDAC3-deficient CD4+ T cells. Strikingly, the proliferation and blasting defects of HDAC3-deficient CD4+ T cells are restored with the addition of exogenous cholesterol, but the signaling defects remain. Thus, the failure of HDAC3-deficient CD4+ T cells to proliferate is due to alterations in cholesterol homeostasis.

Abstract T cell anergy is a state of cellular hyporesponsiveness that prevents the propagation of an immune response to self. Consequently, the development and maintenance of the anergy program in T cells is tightly controlled. We found in wild type (WT) mice that anergic T cells, defined by co-expression of the cell surface markers CD73 and FR4, express lower expression of the transcription factor Runx1. To test if Runx1 acts as a negative regulator of the anergy program, we utilized a 1:1 mixed bone marrow chimera (BMC) system with ER-Cre Runx1 cKO and WT B6.SJL bone marrow to delete Runx1 in mature CD4+ T cells. Upon tamoxifen treatment and deletion of Runx1, ER-Cre Runx1 cKO CD4+ T cells develop a CD73+FR4+ anergic phenotype. This effect is cell-intrinsic since WT T cells from the same bone marrow chimera do not exhibit the anergic phenotype. Functionally, ER-Cre Runx1 cKO T cells fail to proliferate and produce less IL-2 after TCR/CD28 stimulation, consistent with anergy. Interestingly, WT B6.SJL T cells from the ER-Cre Runx1 cKO:B6.SJL BMC mice fail to proliferate in response to TCR/CD28 stimulation, suggesting Runx1 additionally regulates a cell-extrinsic suppressive mechanism. Using depletion experiments, we found this extrinsic suppression requires FR4+ anergic T cells, but not CD25+ Tregs. Interestingly, the loss of Runx1 leads to the generation of CD73+FR4+ anergy in OT-II T cells, which do not engage their TCR. Thus, the loss of Runx1 drives anergy in the absence of TCR signaling. We conducted RNA-sequencing on ER-Cre Runx1 cKO and WT anergic T cells, and have identified several genes regulated by Runx1 that could play a role in anergy. Further analysis will serve to define the molecular mechanisms Runx1 employs to regulate T cell anergy.

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 recent thymic emigrants, and leaving very few CD4+T cells in peripheral lymphoid organs. Interestingly, thymic- and splenic-derived T cells from CD4-Cre Runx1 cKO mice exhibit increased expression of markers indicative of anergy including FR4 and CD73, which is not seen in other genetically modified mice with a block in T cell maturation. These data suggest Runx1 plays a role in regulating the development of T cell anergy in CD4+T cells. We have generated a 1:1 mixed bone marrow chimera (BMC) system with Estrogen Receptor-Cre (ER-Cre) Runx1 conditional knockout and wild type B6.SJL bone marrow to delete Runx1 in mature CD4+ T cells. Upon tamoxifen treatment and deletion of Runx1, the ER-Cre Runx1 cKO CD4+ T cells spontaneously develop an anergic phenotype as they fail to proliferate upon TCR/CD28 stimulation, and co-express CD73 and FR4. This cell surface phenotype is cell intrinsic since wild type B6.SJL CD4+ T cells in the mixed bone marrow chimera do not upregulate CD73 and FR4 upon tamoxifen treatment. Surprisingly, the mixed BMC system reveals both the ER-Cre Runx1 cKO and the wild type B6.SJL peripheral T cells fail to proliferate in response to TCR/CD28 stimulation, suggesting Runx1 also regulates a cell-extrinsic suppressive mechanism controlling T cell tolerance. This cell-extrinsic induction of anergy requires Runx1-deficient CD4+ T cells, but it is not dependent on Runx1-deficient Tregs. Thus, Runx1 is a key regulator of T cell anergy, and further analysis of this BMC model will serve to uncover the mechanism.

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.