Shapiro, Virginia Smith

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.

Abstract The production of naïve B cells in bone marrow (BM) is critical for maintaining a diverse peripheral B cell pool to fight infection and establish lifelong immunity. The generation of naïve B cells is impaired in Flt3-ligand (FL-/-) mice contributing to reductions in splenic B cells. The goal of this study was to determine the consequence of FL-deficiency on splenic B cell maturation. We found significant reductions in numbers of transitional (TS) and follicular (FO), but not marginal zone (MZ) B cells in FL-/- mice. Mixed BM chimeras revealed that the reduction in TS B cells was cell extrinsic, linking microenvironmental signals to TS B cell maturation. FL administration into FL-/- mice restored the deficiency in TS B cells that do not express Flt3, suggesting that a FL responsive component of the microenvironment is critical for TS maturation. Intracellular Ki67 revealed a significant decrease in the proliferative capacity of TS cells in FL-/- mice. Expression of a Bcl2 transgene did not rescue TS cells, uncoupling FL-deficiency to Bcl2-regulated survival pathways. BAFF-R expression and serum levels of BAFF were comparable in WT and FL-/- mice, supporting BAFF-independent regulation of TS cells. These observations support an integral indirect role for Flt3 signaling in regulation of TS B cell maturation in the spleen.

Abstract Invariant Natural Killer T (iNKT) cells are an innate lineage of lymphocytes that can produce copious amount of cytokines within hours of stimulation. In the thymus, positive selection into the iNKT cell lineage occurs at the double positive (DP) stage, before maturation through various stages (stage 0-3) to complete development. NKAP, a transcriptional repressor, is required for conventional T lymphocyte development and maturation. NKAP interacts with Hdac3 for its repressor function. Previously, using CD4-cre NKAP cKO mice, we have shown that NKAP is required for positive selection into the iNKT cell lineage. To study the role of NKAP in later developmental stages, we generated PLZF-cre NKAP cKO mice with NKAP deletion occurring after entry into the iNKT lineage at stage 0. In the PLZF-cre NKAP cKO mice we observed a significant decrease in the absolute number of iNKTs with a 20-fold reduction at stage 2 and 200-fold reduction at stage 3. This defect is cell intrinsic and cannot be rescued by over-expression of Bcl-XL. Our preliminary results indicate that there may be a block in iNKT cell maturation, as expression of transcription factors Tbet, Gata3, and ROR-γt that define NKT1, NKT2 and NKT17 lineages respectively, are decreased in PLZF-cre NKAP cKO. PLZF-cre Hdac3 cKO mice have a similar defect in iNKT development. Thus, NKAP together with Hdac3 may regulate iNKT cell development and maturation.

Abstract T cell development depends on the coordinated interplay between receptor signaling and transcriptional activation/repression. We performed a genetic complementation screen, and identified a novel transcriptional repressor, NKAP. NKAP associates with HDAC3 and is part of a DNA-binding complex, as shown by chromatin immunoprecipitation. NKAP also associates with CIR, a part of the Notch corepressor complex. The expression of NKAP during T cell development inversely correlates with the expression of Notch target genes, implying that NKAP may modulate Notch-mediated transcription. To examine the function of NKAP in T cell development, Lck-cre NKAP conditional knockout mice were generated. Interestingly, loss of NKAP blocks development of alpha/beta but not gamma/delta T cells. In addition, lck-cre NKAP cKO DP T cells express 8- to 20-fold higher levels of Hes1, Deltex1 and CD25, providing in vivo evidence that NKAP functions as a transcriptional repressor, acting at least in part, by repression of the Notch pathway.

Abstract During hematopoiesis, proper differentiation of the long-term, multi-potent, self-renewing hematopoietic stem cell (HSC) results in the cellular components of blood. The lineage fate decisions that occur during hematopoiesis result from the coordinated regulation of transcriptional activators, repressors, and chromatin modifiers. We have identified NKAP as a novel regulator of hematopoiesis, that functions at least in part as a transcriptional repressor. In this study we employ a combination of conditional and inducible mouse models of NKAP deletion to explore the role of NKAP in hematopoietic development starting at the HSC stage. Our models allow for both the pan-deletion of NKAP starting in the HSC stage by using a vav-cre transgenic, as well as temporally regulated deletion of NKAP in the hematopietic compartment through the use of the interferon-inducible mx1-cre model system. These complimentary approaches allow us to conclude that NKAP functions in a cell intrinsic manner, as a member of a unique group of transcriptional regulators, and is necessary in the earliest stage of definitive hematopoiesis.

Abstract Inhibitory receptors have a critical role in the regulation of immunity. Siglecs are a family of primarily inhibitory receptors expressed by immune cells that recognize specific sialic acid modifications on cell surface glycans. Many tumors have increased sialic acid incorporation. Overexpression of the sialyltransferase ST8Sia6 on tumors led to altered immune responses and increased tumor growth. In this study, we examined the role of ST8Sia6 on immune cells in regulating antitumor immunity. ST8Sia6 knockout mice had an enhanced immune response to tumors. The loss of ST8Sia6 promoted an enhanced intratumoral activation of macrophages and dendritic cells, including upregulation of CD40. Intratumoral regulatory T cells exhibited a more inflammatory phenotype in ST8Sia6 knockout mice. Using adoptive transfer studies, the change in regulatory T cell phenotype was not cell intrinsic and depended on the loss of ST8Sia6 expression in APCs. Thus, ST8Sia6 generates ligands for Siglecs that dampen antitumor immunity.

Iron-sulfur (Fe-S) clusters are cofactors essential for the activity of numerous enzymes including DNA polymerases, helicases, and glycosylases. They are synthesized in the mitochondria as Fe-S intermediates and are exported to the cytoplasm for maturation by the mitochondrial transporter ABCB7. Here, we demonstrate that ABCB7 is required for bone marrow B cell development, proliferation, and class switch recombination, but is dispensable for peripheral B cell homeostasis in mice. Conditional deletion of ABCB7 using Mb1-cre resulted in a severe block in bone marrow B cell development at the pro-B cell stage. The loss of ABCB7 did not alter expression of transcription factors required for B cell specification or commitment. While increased intracellular iron was observed in ABCB7-deficient pro-B cells, this did not lead to increased cellular or mitochondrial reactive oxygen species, ferroptosis, or apoptosis. Interestingly, loss of ABCB7 led to replication-induced DNA damage in pro-B cells, independent of VDJ recombination, and these cells had evidence of slowed DNA replication. Stimulated ABCB7-deficient splenic B cells from CD23-cre mice also had a striking loss of proliferation and a defect in class switching. Thus, ABCB7 is essential for early B cell development, proliferation, and class switch recombination.

The clock is ticking for senescent cells Senescent cells promote their own recognition and removal through the immune system by generating a bioactive secretome called the senescence-associated secretory phenotype (SASP). Sturmlechner et al . report that the cell cycle regulator p21 directs an early form of the SASP, which they call the p21-activated secretory phenotype (PASP) (see the Perspective by Reen and Gil). As part of the PASP, the chemokine CXCL14 attracts macrophages, which monitor stressed cells expressing elevated p21. If stressed cells recuperate and p21 levels return to normal within 4 days, then macrophages disengage from their targets. Otherwise, macrophages recruit cytotoxic T cells that facilitate target cell removal. Other cell cycle regulators such as p16 can induce many factors overlapping with the PASP, but p21 uniquely drives this CXCL14-mediated “timer” mechanism of senescent cell immunosurveillance. —STS