Shapiro, Michael J.

Abstract Many tumors exhibit increased incorporation of sialic acids into cell-surface glycans, which impact the tumor microenvironment. Sialic acid immunoglobulin-like lectins (Siglec) are receptors that recognize sialic acids and modulate immune responses, including responses to tumors. However, the roles of individual sialyltransferases in tumorigenesis and tumor growth are not well understood. Here, we examined the sialyltransferase ST8Sia6, which generated α2,8-linked disialic acids that bind to murine Siglec-E and human Siglec-7 and -9. Increased ST8Sia6 expression was found on many human tumors and associated with decreased survival in several cancers, including colon cancer. Because of this, we engineered MC38 and B16-F10 tumor lines to express ST8Sia6. ST8Sia6-expressing MC38 and B16-F10 tumors exhibited faster growth and led to decreased survival, which required host Siglec-E. ST8Sia6 expression on tumors also altered macrophage polarization toward M2, including upregulation of the immune modulator arginase, which also required Siglec-E. ST8Sia6 also accelerated tumorigenesis in a genetically engineered, spontaneous murine model of colon cancer, decreasing survival from approximately 6 months to 67 days. Thus, ST8Sia6 expression on tumors inhibits antitumor immune responses to accelerate tumor growth.

Abstract The immune system contains a series of checks and balances that maintain tolerance and prevent autoimmunity. Sialic acid-binding Ig-type lectins (Siglecs) are cell surface receptors found on immune cells and inhibit inflammation by recruiting protein tyrosine phosphatases to ITIMs. Islet-resident macrophages express Siglec-E, and Siglec-E expression decreases on islet-resident macrophages as insulitis progresses in the NOD mouse. The sialyltransferase ST8Sia6 generates α-2,8-disialic acids that are ligands for Siglec-E in vivo. We hypothesized that engaging Siglec-E through ST8Sia6-generated ligands may inhibit the development of immune-mediated diabetes. Constitutive overexpression of ST8Sia6 in pancreatic β cells mitigated hyperglycemia in the multiple low-dose streptozotocin model of diabetes, demonstrating that engagement of this immune receptor facilitates tolerance in the setting of inflammation and autoimmune disease.

T cell development involves stepwise progression through defined stages that give rise to multiple T cell subtypes, and this is accompanied by the establishment of stage-specific gene expression. Changes in chromatin accessibility and chromatin modifications accompany changes in gene expression during T cell development. Chromatin-modifying enzymes that add or reverse covalent modifications to DNA and histones have a critical role in the dynamic regulation of gene expression throughout T cell development. As each chromatin-modifying enzyme has multiple family members that are typically all coexpressed during T cell development, their function is sometimes revealed only when two related enzymes are concurrently deleted. This work has also revealed that the biological effects of these enzymes often involve regulation of a limited set of targets. The growing diversity in the types and sites of modification, as well as the potential for a single enzyme to catalyze multiple modifications, is also highlighted.

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 Recent thymic emigrants that fail postpositive selection maturation are targeted by complement proteins. T cells likely acquire complement resistance during maturation in the thymus, a complement-privileged organ. To test this, thymocytes and fresh serum were separately obtained and incubated together in vitro to assess complement deposition. Complement binding decreased with development and maturation. Complement binding decreased from the double-positive thymocyte to the single-positive stage, and within single-positive thymocytes, complement binding gradually decreased with increasing intrathymic maturation. Binding of the central complement protein C3 to wild-type immature thymocytes required the lectin but not the classical pathway. Specifically, MBL2 but not MBL1 was required, demonstrating a unique function for MBL2. Previous studies demonstrated that the loss of NKAP, a transcriptional regulator of T cell maturation, caused peripheral T cell lymphopenia and enhanced complement susceptibility. To determine whether complement causes NKAP-deficient T cell disappearance, both the lectin and classical pathways were genetically ablated. This blocked C3 deposition on NKAP-deficient T cells but failed to restore normal cellularity, indicating that complement contributes to clearance but is not the primary cause of peripheral T cell lymphopenia. Rather, the accumulation of lipid peroxides in NKAP-deficient T cells was observed. Lipid peroxidation is a salient feature of ferroptosis, an iron-dependent nonapoptotic cell death. Thus, wild-type thymocytes naturally acquire the ability to protect themselves from complement targeting by MBL2 with maturation. However, NKAP-deficient immature peripheral T cells remain scarce in complement-deficient mice likely due to ferroptosis.

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.

Abstract Recent thymic emigrants (RTEs) must undergo phenotypic and functional maturation to become long-lived mature naive T cells. In CD4-cre NKAP conditional knockout mice, NKAP-deficient RTEs fail to complete T cell maturation. In this study, we demonstrate that NKAP-deficient immature RTEs do not undergo apoptosis, but are eliminated by complement. C3, C4, and C1q are bound to NKAP-deficient peripheral T cells, demonstrating activation of the classical arm of the complement pathway. As thymocytes mature and exit to the periphery, they increase sialic acid incorporation into cell surface glycans. This is essential to peripheral lymphocyte survival, as stripping sialic acid with neuraminidase leads to the binding of natural IgM and complement fixation. NKAP-deficient T cells have a defect in sialylation on cell surface glycans, leading to IgM recruitment. We demonstrate that the defect in sialylation is due to aberrant α2,8-linked sialylation, and the expression of three genes (ST8sia1, ST8sia4, and ST8sia6) that mediate α2,8 sialylation are downregulated in NKAP-defcient RTEs. The maturation of peripheral NKAP-deficient T cells is partially rescued in a C3-deficient environment. Thus, sialylation during T cell maturation is critical to protect immature RTEs from complement in the periphery.

Abstract The activation of T cells and the initiation of an immune response is tightly controlled through the crosstalk of both positive and negative regulators. Two adaptors that function as negative regulators of T cell activation are adaptor in lymphocytes of unknown function X (ALX) and linker for activation of X cell (LAX). Previously, we showed that T cells from mice deficient in ALX and LAX display similar hyperresponsiveness, with increased IL-2 production and proliferation upon TCR/CD28 stimulation, and that these adaptors physically associate. In this study, we analyze the nature of the association between ALX and LAX. We demonstrate that this association occurs in the absence of TCR/CD28 signaling via a mechanism independent of both tyrosine phosphorylation of LAX and the SH2 domain of ALX. Cotransfection of ALX with LAX resulted in LAX tyrosine phosphorylation in the absence of TCR/CD28 stimulation. ALX-mediated LAX phosphorylation depends upon the ALX SH2 domain, which functions to recruit Lck to LAX. We also show that LAX, like ALX, can inhibit RE/AP reporter activation. However, in contrast to its inhibition of NFAT, the inhibition of RE/AP by LAX is independent of its tyrosine phosphorylation. Therefore, it can be concluded that inhibition of signaling events involved in T cell activation by LAX occurs through mechanisms both dependent on and independent of its tyrosine phosphorylation.

Abstract Steady-state hematopoiesis is sustained through differentiation balanced with proliferation and self-renewal of hematopoietic stem cells (HSCs). Disruption of this balance can lead to hematopoietic failure, as hematopoietic differentiation without self-renewal leads to loss of the HSC pool. We find that conditional knockout mice that delete the transcriptional repressor NKAP in HSCs and all hematopoietic lineages during embryonic development exhibit perinatal lethality and abrogation of hematopoiesis as demonstrated by multilineage defects in lymphocyte, granulocyte, erythrocyte and megakaryocyte development. Inducible deletion of NKAP in adult mice leads to lethality within 2 weeks, at which point hematopoiesis in the bone marrow has halted and HSCs have disappeared. This hematopoietic failure and lethality is cell intrinsic, as radiation chimeras reconstituted with inducible Mx1-cre NKAP conditional knockout bone marrow also succumb with a similar time course. Even in the context of a completely normal bone marrow environment using mixed radiation chimeras, NKAP deletion results in HSC failure. NKAP deletion leads to decreased proliferation and increased apoptosis of HSCs, which is likely due to increased expression of the cyclin-dependent kinase inhibitors p21Cip1/Waf1 and p19Ink4d. These data establish NKAP as one of a very small number of transcriptional regulators that is absolutely required for adult HSC maintenance and survival.