Citations

Abstract Glycans decorate most proteins and lipids on the cell surface, and glycoproteins serve a critical role in cell-cell communication. The terminal modification of many glycans is a sialic acid. There are twenty different mammalian sialyltransferases, each with a unique specificity. Recently, we demonstrated that the sialyltransferase ST8Sia6, which adds a terminal alpha-2,8 disialic acid to a preexisting sialic acid on cell surface proteins, generates ligands for the inhibitory receptor Siglec-E. In patients, ST8Sia6 overexpression is associated with worse outcome. We have previously shown that ST8sia6 overexpression in a mouse tumor model accelerates tumor growth, dependent upon Siglec-E expression by immune cells. To understand the basis for ST8Sia6 function in tumors, proteomic analysis of murine tumor lines was performed to identify proteins modified by ST8Sia6. Here, we demonstrate that ST8Sia6 sialylates CD44. Siglecs vary significantly between species, with 5 non-conserved Siglecs in mice compared to 10 non-conserved Siglecs in humans. The closest ortholog to Siglec-E in mice is Siglec-7 in humans. We also demonstrate that ST8Sia6 sialylates CD44, generating ligands for Siglec-7. Thus, ST8sia6 has a conserved role in both mice and humans in modifying glycans on CD44 to generate ligands for Siglecs. As CD44 expression on tumors is associated with increased metastasis, we predict that ST8Sia6 may also have a role in this process which is under investigation. Supported by grants from 2T32AI007425-23 to DJF and NIH Glycobiology Common Fund supplement to R01CA243545-01A1 to VSS.

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 Glycans decorate almost all proteins and lipids on the cell surface, and they play a critical role in cell-cell interactions. Terminal sialic acid addition to glycans helps the immune system distinguish between “self” and “non-self.” The enzyme ST8sia6 adds alpha-2,8 disialic acids to cell surface receptors, and this sialic acid addition promotes binding to the inhibitory receptor Siglec-E. We have previously shown ST8sia6 overexpression in tumors accelerates tumor growth in a Siglec-E dependent manner. Proteomic analysis in murine tumor lines identified CD44 as a target for ST8sia6. CD44 expression on tumors is associated with metastasis and chemoresistance. Since humans and mice have different sialic acid structures, we examined whether ST8sia6 makes CD44 a ligand to the ortholog of Siglec-E in human cells, Siglec-7. When HEK293T cells overexpress ST8sia6 and CD44 together, Siglec-7 binding was significantly increased compared to ST8sia6 or CD44 overexpression alone. Thus, ST8sia6 has a conserved role from mouse to human in decorating CD44 with sialic acid. Our study proposes that ST8sia6 sialylation of CD44 could suppress immune responses and enables cancer progression in human. Supported by grant: 2T32AI007425-23 to DJF and R01 CA243545-01S1 to V.S.S.

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.

After antigenic activation, quiescent naive CD4 + T cells alter their metabolism to proliferate. This metabolic shift increases production of nucleotides, amino acids, fatty acids, and sterols. Here, we show that histone deacetylase 3 (HDAC3) is critical for activation of murine peripheral CD4 + T cells. HDAC3-deficient CD4 + T cells failed to proliferate and blast after in vitro TCR/CD28 stimulation. Upon T-cell activation, genes involved in cholesterol biosynthesis are upregulated while genes that promote cholesterol efflux are repressed. HDAC3-deficient CD4 + T cells had reduced levels of cellular cholesterol both before and after activation. HDAC3-deficient cells upregulate cholesterol synthesis appropriately after activation, but fail to repress cholesterol efflux; notably, they overexpress cholesterol efflux transporters ABCA1 and ABCG1. Repression of these genes is the primary function for HDAC3 in peripheral CD4 + T cells, as addition of exogenous cholesterol restored proliferative capacity. Collectively, these findings demonstrate HDAC3 is essential during CD4 + T-cell activation to repress cholesterol efflux.