Chromatin-Modifying Enzymes in T Cell Development.

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.

Transcription factor Tox2 is required for metabolic adaptation and tissue residency of ILC3 in the gut.

Group 3 innate lymphoid cells (ILC3) are the major subset of gut-resident ILC with essential roles in infections and tissue repair, but how they adapt to the gut environment to maintain tissue residency is unclear. We report that Tox2 is critical for gut ILC3 maintenance and function. Gut ILC3 highly expressed Tox2, and depletion of Tox2 markedly decreased ILC3 in gut but not at central sites, resulting in defective control of Citrobacter rodentium infection. Single-cell transcriptional profiling revealed decreased expression of Hexokinase-2 in Tox2-deficient gut ILC3. Consistent with the requirement for hexokinases in glycolysis, Tox2-/- ILC3 displayed decreased ability to utilize glycolysis for protein translation. Ectopic expression of Hexokinase-2 rescued Tox2-/- gut ILC3 defects. Hypoxia and interleukin (IL)-17A each induced Tox2 expression in ILC3, suggesting a mechanism by which ILC3 adjusts to fluctuating environments by programming glycolytic metabolism. Our results reveal the requirement for Tox2 to support the metabolic adaptation of ILC3 within the gastrointestinal tract.

Cutting Edge: Enhanced Antitumor Immunity in ST8Sia6 Knockout Mice.

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.

Identification and characterization of the antigen recognized by the germ cell mAb TRA98 using a human comprehensive wet protein array.

TRA98 is a rat monoclonal antibody (mAb) which recognizes a specific antigen in the nuclei of germ cells. mAb TRA98 has been used to understand the mechanism of germ cell development and differentiation in many studies. In mice, the antigen recognized by mAb TRA98 or GCNA1 has been reported to be a GCNA gene product, but despite the demonstration of the immunoreactivity of this mAb in human testis and sperm in 1997, the antigen in humans remains unknown, as of date. To identify the human antigen recognized by mAb TRA98, a human comprehensive wet protein array was developed containing 19,446 proteins derived from human cDNAs. Using this array, it was found that the antigen of mAb TRA98 is not a GCNA gene product, but nuclear factor-κB activating protein (NKAP). In mice, mAb TRA98 recognized both the GCNA gene product and NKAP. Furthermore, conditional knockout of Nkap in mice revealed a phenotype of Sertoli cell-only syndrome. Although NKAP is a ubiquitously expressed protein, NKAP recognized by mAb TRA98 in mouse testis was SUMOylated. These results suggest that NKAP undergoes modifications, such as SUMOylation in the testis, and plays an important role in spermatogenesis.

Cutting Edge: ST8Sia6-Generated α-2,8-Disialic Acids Mitigate Hyperglycemia in Multiple Low-Dose Streptozotocin-Induced Diabetes.

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 ß 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.

NKAP Must Associate with HDAC3 to Regulate Hematopoietic Stem Cell Maintenance and Survival.

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.

Murine T Cell Maturation Entails Protection from MBL2, but Complement Proteins Do Not Drive Clearance of Cells That Fail Maturation in the Absence of NKAP.

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.

Treg-specific deletion of NKAP results in severe, systemic autoimmunity due to peripheral loss of Tregs.

Regulatory T cells are critical for the generation and maintenance of peripheral tolerance. Conditional deletion of the transcriptional repressor NKAP in Tregs using Foxp3-YFP-cre NKAP conditional knockout mice causes aggressive autoimmunity characterized by thymic atrophy, lymphadenopathy, peripheral T cell activation, generation of autoantibodies, immune infiltration into several organs, and crusty skin at 3 weeks of age, similar to that of "scurfy" Foxp3-mutant mice. While Treg development in the thymus proceeds normally in the absence of NKAP, there is a severe loss of thymically-derived Tregs in the periphery. NKAP-deficient Tregs have a recent thymic emigrant phenotype, and are attacked by complement in a cell-intrinsic manner in the periphery. Previously, we demonstrated that NKAP is required for conventional T cell maturation as it prevents complement-mediated attack in the periphery. We now show that Tregs undergo a similar maturation process as conventional T cells, requiring NKAP to acquire complement resistance after thymic egress.

NKAP is a transcriptional repressor of notch signaling and is required for T cell development.

T cell development depends on the coordinated interplay between receptor signaling and transcriptional regulation. Through a genetic complementation screen a transcriptional repressor, NKAP, was identified. NKAP associated with the histone deacetylase HDAC3 and was shown to be part of a DNA-binding complex, as demonstrated by chromatin immunoprecipitation. NKAP also associated with the Notch corepressor complex. The expression of NKAP during T cell development inversely correlated 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, we ablated NKAP by Lck(cre). Loss of NKAP blocked development of alphabeta but not gammadelta T cells, and Nkap(fl/o)Lck(cre) DP T cells expressed 8- to 20-fold higher amounts of Hes1, Deltex1, and CD25 mRNA. Thus, NKAP functions as a transcriptional repressor, acting on Notch target genes, and is required for alphabeta T cell development.

NKAP Regulates Invariant NKT Cell Proliferation and Differentiation into ROR-γt-Expressing NKT17 Cells.

Invariant NKT (iNKT) cells are a unique lineage with characteristics of both adaptive and innate lymphocytes, and they recognize glycolipids presented by an MHC class I-like CD1d molecule. During thymic development, iNKT cells also differentiate into NKT1, NKT2, and NKT17 functional subsets that preferentially produce cytokines IFN-γ, IL-4, and IL-17, respectively, upon activation. Newly selected iNKT cells undergo a burst of proliferation, which is defective in mice with a specific deletion of NKAP in the iNKT cell lineage, leading to severe reductions in thymic and peripheral iNKT cell numbers. The decreased cell number is not due to defective homeostasis or increased apoptosis, and it is not rescued by Bcl-xL overexpression. NKAP is also required for differentiation into NKT17 cells, but NKT1 and NKT2 cell development and function are unaffected. This failure in NKT17 development is rescued by transgenic expression of promyelocytic leukemia zinc finger; however, the promyelocytic leukemia zinc finger transgene does not restore iNKT cell numbers or the block in positive selection into the iNKT cell lineage in CD4-cre NKAP conditional knockout mice. Therefore, NKAP regulates multiple steps in iNKT cell development and differentiation.

HDAC3 Is Required for the Downregulation of RORγt during Thymocyte Positive Selection.

To generate functional peripheral T cells, proper gene regulation during T cell development is critical. In this study, we found that histone deacetylase (HDAC) 3 is required for T cell development. T cell development in CD2-icre HDAC3 conditional knockout (cKO) mice (HDAC3-cKO) was blocked at positive selection, resulting in few CD4 and CD8 T cells, and it could not be rescued by a TCR transgene. These single-positive thymocytes failed to upregulate Bcl-2, leading to increased apoptosis. HDAC3-cKO mice failed to downregulate retinoic acid-related orphan receptor (ROR) γt during positive selection, similar to the block in positive selection in RORγt transgenic mice. In the absence of HDAC3, the RORC promoter was hyperacetylated. In the periphery, the few CD4 T cells present were skewed toward RORγt(+) IL-17-producing Th17 cells, leading to inflammatory bowel disease. Positive selection of CD8 single-positive thymocytes was restored in RORγt-KO Bcl-xL transgenic HDAC3-cKO mice, demonstrating that HDAC3 is required at positive selection to downregulate RORγt.

Update on Gender Equity in Immunology, 2001 to 2016.

In 2001, The American Association of Immunologists Committee on the Status of Women conducted a survey examining the percentage of women faculty members within immunology departments or women in immunology graduate programs across 27 institutions in the United States, comparing it to the percentage of women receiving a Ph.D. Here, we examine the representation of women across these same 27 immunology departments and programs to examine changes in gender equity over the last 15 years.

T Cell Adolescence: Maturation Events Beyond Positive Selection.

Single-positive thymocytes that successfully complete positive and negative selection must still undergo one final step, generally termed T cell maturation, before they gain functional competency and enter the long-lived T cell pool. Maturation initiates after positive selection in single-positive thymocytes and continues in the periphery in recent thymic emigrants, before these newly produced T cells gain functional competency and are ready to participate in the immune response as peripheral naive T cells. Recent work using genetically altered mice demonstrates that T cell maturation is not a single process, but a series of steps that occur independently and sequentially after positive selection. This review focuses on the changes that occur during T cell maturation, as well as the molecules and pathways that are critical at each step.

Histone Deacetylase 3 Is Required for T Cell Maturation.

Recent thymic emigrants are newly generated T cells that need to undergo postthymic maturation to gain functional competency and enter the long-lived naive T cell pool. The mechanism of T cell maturation remains incompletely understood. Previously, we demonstrated that the transcriptional repressor NKAP is required for T cell maturation. Because NKAP associates with histone deacetylase 3 (HDAC3), we examined whether HDAC3 is also required for T cell maturation. Although thymic populations are similar in CD4-cre HDAC3 conditional knockout mice compared with wild-type mice, the peripheral numbers of CD4(+) and CD8(+) T cells are dramatically decreased. In the periphery, the majority of HDAC3-deficient naive T cells are recent thymic emigrants, indicating a block in T cell maturation. CD55 upregulation during T cell maturation is substantially decreased in HDAC3-deficient T cells. Consistent with a block in functional maturation, HDAC3-deficient peripheral T cells have a defect in TNF licensing after TCR/CD28 stimulation. CD4-cre HDAC3 conditional knockout mice do not have a defect in intrathymic migration, thymic egress, T cell survival, or homeostasis. In the periphery, similar to immature NKAP-deficient peripheral T cells, HDAC3-deficient peripheral T cells were bound by IgM and complement proteins, leading to the elimination of these cells. In addition, HDAC3-deficient T cells display decreases in the sialic acid modifications on the cell surface that recruit natural IgM to initiate the classical complement pathway. Therefore, HDAC3 is required for T cell maturation.

Immature recent thymic emigrants are eliminated by complement.

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.

Lineage(-)Sca1+c-Kit(-)CD25+ cells are IL-33-responsive type 2 innate cells in the mouse bone marrow.

IL-33 promotes type 2 immune responses, both protective and pathogenic. Recently, targets of IL-33, including several newly discovered type 2 innate cells, have been characterized in the periphery. In this study, we report that bone marrow cells from wild-type C57BL/6 mice responded with IL-5 and IL-13 production when cultured with IL-33. IL-33 cultures of bone marrow cells from Rag1 KO and Kit(W-sh/W-sh) mice also responded similarly; hence, eliminating the possible contributions of T, B, and mast cells. Rather, intracellular staining revealed that the IL-5- and IL-13-positive cells display a marker profile consistent with the Lineage(-)Sca-1(+)c-Kit(-)CD25(+) (LSK(-)CD25(+)) cells, a bone marrow cell population of previously unknown function. Freshly isolated LSK(-)CD25(+) cells uniformly express ST2, the IL-33 receptor. In addition, culture of sorted LSK(-)CD25(+) cells showed that they indeed produce IL-5 and IL-13 when cultured with IL-33 plus IL-2 and IL-33 plus IL-7. Furthermore, i.p. injections of IL-33 or IL-25 into mice induced LSK(-)CD25(+) cells to expand, in both size and frequency, and to upregulate ST2 and α(4)ß(7) integrin, a mucosal homing marker. Thus, we identify the enigmatic bone marrow LSK(-)CD25(+) cells as IL-33 responsive, both in vitro and in vivo, with attributes similar to other type 2 innate cells described in peripheral tissues.

Adult hematopoietic stem cells require NKAP for maintenance and survival.

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.

Transcriptional repressors, corepressors and chromatin modifying enzymes in T cell development.

Gene expression is regulated by the combined action of transcriptional activators and transcriptional repressors. Transcriptional repressors function by recruiting corepressor complexes containing histone-modifying enzymes to specific sites within DNA. Chromatin modifying complexes are subsequently recruited, either directly by transcriptional repressors, or indirectly via corepressor complexes and/or histone modifications, to remodel chromatin into either a transcription-friendly 'open' form or an inhibitory 'closed' form. Transcriptional repressors, corepressors and chromatin modifying complexes play critical roles throughout T cell development. Here, we highlight those genes that function to repress transcription and that have been shown to be required for T cell development.

The mitochondrial iron transporter ABCB7 is required for B cell development, proliferation, and class switch recombination in mice.

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.

ST8Sia6 Promotes Tumor Growth in Mice by Inhibiting Immune Responses.

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.