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Binding of a T cell to an appropriate antigen-presenting cell (APC) induces the rapid reorientation of the T cell cytoskeleton and secretory apparatus towards the cell-cell contact site in a T cell antigen receptor (TCR) and peptide/major histocompatibility complex-dependent process. Such T cell polarization directs the delivery of cytokines and cytotoxic mediators towards the APC and contributes to the highly selective and specific action of effector T cells. To study the signaling pathways that regulate cytoskeletal rearrangements in T lymphocytes, we set up a conjugate formation assay using Jurkat T cells as effectors and cell-sized latex beads coated with various antibodies as artificial APCs. Here, we report that beads coated with antibodies specific for the TCR-CD3 complex were sufficient to induce T cell polarization towards the bead attachment site, as judged by reorientation of the microtubule-organizing center (MTOC) and localized actin polymerization. Thus, these cytoskeletal changes did not depend on activation of additional coreceptors. Moreover, single subunits of the TCR complex, namely TCR-zeta and CD3epsilon, were equally effective in inducing cytoskeletal polarization. However, mutagenesis of the immunoreceptor tyrosine-based activation motifs (ITAMs), present three times in TCR-zeta and once in CD3epsilon, revealed that the induction of cytoskeletal rearrangements required the presence of at least one intact ITAM. In agreement with this result, lack of functional Lck, the protein tyrosine kinase responsible for ITAM phosphorylation, abolished both MTOC reorientation and polarized actin polymerization. Both inhibitor and transient overexpression studies demonstrated that MTOC reorientation could occur in the absence of Ras activation. Our results suggest that APC-induced T cell polarization is a TCR-mediated event that is coupled to the TCR by the same signaling motif as TCR-induced gene activation, but diverges in its distal signaling requirements.

T cell activation by antigen/MHC induces the expression of several genes critical to the immune response, including interleukin-2. T cells from mice deficient for the NF-kappa B family member c-rel cannot activate IL-2 gene expression. However, mutating the NF-kappa B site in the IL-2 promoter has only moderate effects. To investigate additional ways c-Rel could regulate IL-2 gene expression, we determined whether c-rel overexpression could increase the activity of other transcription factors involved in IL-2 promoter regulation: NF-AT, Oct/OAP (ARRE-1), and AP-1. In Jurkat TAg cells, overexpression of c-Rel increased AP-1 activation approximately 17-fold. Moreover, AP-1 activity stimulated by anti-TCR Abs or PMA/ionomycin was further increased by c-Rel overexpression. c-Rel overexpression did not affect NF-AT or ARRE-1 activity. Additionally, overexpression of c-Rel activated the nonconsensus AP-1 site from the IL-2 promoter (NF-IL-2B), although to a lesser extent, approximately sixfold. AP-1 activation required both the DNA binding and transactivation domains of c-Rel. Our results may provide an explanation for the effect on IL-2 gene activation in c-rel-deficient mice.

Germinal center (GC) formation, which is an integrant part of humoral immunity, involves energy-consuming metabolic reprogramming. Rag-GTPases are known to signal amino acid availability to cellular pathways that regulate nutrient distribution such as the mechanistic target of rapamycin complex 1 (mTORC1) pathway and the transcription factors TFEB and TFE3. However, the contribution of these factors to humoral immunity remains undefined. Here, we show that B cell-intrinsic Rag-GTPases are critical for the development and activation of B cells. RagA/RagB deficient B cells fail to form GCs, produce antibodies, and to generate plasmablasts during both T-dependent (TD) and T-independent (TI) humoral immune responses. Deletion of RagA/RagB in GC B cells leads to abnormal dark zone (DZ) to light zone (LZ) ratio and reduced affinity maturation. Mechanistically, the Rag-GTPase complex constrains TFEB/TFE3 activity to prevent mitophagy dysregulation and maintain mitochondrial fitness in B cells, which are independent of canonical mTORC1 activation. TFEB/TFE3 deletion restores B cell development, GC formation in Peyer's patches and TI humoral immunity, but not TD humoral immunity in the absence of Rag-GTPases. Collectively, our data establish the Rag GTPase-TFEB/TFE3 pathway as a likely mTORC1 independent mechanism to coordinating nutrient sensing and mitochondrial metabolism in B cells., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

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., Competing Interests: Declaration of interests The authors declare no competing interests., (Published by Elsevier Inc.)

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., (Copyright © 2022 by The American Association of Immunologists, Inc.)

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., Competing Interests: DW, RP, JL, MK, MS, SM, MK, MR, HZ, VS No competing interests declared, (© 2021, Wilfahrt et al.)

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., Competing Interests: ML, MS, MR, MK, SM, KM, VS No competing interests declared, (© 2021, Lehrke et al.)

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., (©2021 American Association for Cancer Research.)

The meninges contain adaptive immune cells that provide immunosurveillance of the central nervous system (CNS). These cells are thought to derive from the systemic circulation. Through single-cell analyses, confocal imaging, bone marrow chimeras, and parabiosis experiments, we show that meningeal B cells derive locally from the calvaria, which harbors a bone marrow niche for hematopoiesis. B cells reach the meninges from the calvaria through specialized vascular connections. This calvarial-meningeal path of B cell development may provide the CNS with a constant supply of B cells educated by CNS antigens. Conversely, we show that a subset of antigen-experienced B cells that populate the meninges in aging mice are blood-borne. These results identify a private source for meningeal B cells, which may help maintain immune privilege within the CNS., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Tissue macrophages self-renew during homeostasis and produce inflammatory mediators upon microbial infection. We examined the relationship between proliferative and inflammatory properties of tissue macrophages by defining the impact of the Wnt/β-catenin pathway, a central regulator of self-renewal, in alveolar macrophages (AMs). Activation of β-catenin by Wnt ligand inhibited AM proliferation and stemness, but promoted inflammatory activity. In a murine influenza viral pneumonia model, β-catenin-mediated AM inflammatory activity promoted acute host morbidity; in contrast, AM proliferation enabled repopulation of reparative AMs and tissue recovery following viral clearance. Mechanistically, Wnt treatment promoted β-catenin-HIF-1α interaction and glycolysis-dependent inflammation while suppressing mitochondrial metabolism and thereby, AM proliferation. Differential HIF-1α activities distinguished proliferative and inflammatory AMs in vivo. This β-catenin-HIF-1α axis was conserved in human AMs and enhanced HIF-1α expression associated with macrophage inflammation in COVID-19 patients. Thus, inflammatory and reparative activities of lung macrophages are regulated by β-catenin-HIF-1α signaling, with implications for the treatment of severe respiratory diseases., Competing Interests: Declaration of interests A.W.G. is a member of the advisory board of Pandion Therapeutics and ArsenalBio. The J.S. lab receives research grants from Evive Biotech and Humanigen., (Copyright © 2021 Elsevier Inc. All rights reserved.)

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., (© 2021 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.)

Immune cells can metabolize glucose, amino acids, and fatty acids (FAs) to generate energy. The roles of different FA species and their impacts on humoral immunity remain poorly understood. Here, we report that proliferating B cells require monounsaturated FAs (MUFAs) to maintain mitochondrial metabolism and mTOR activity and to prevent excessive autophagy and endoplasmic reticulum (ER) stress. Furthermore, B cell-extrinsic stearoyl-CoA desaturase (SCD) activity generates MUFA to support early B cell development and germinal center (GC) formation in vivo during immunization and influenza infection. Thus, SCD-mediated MUFA production is critical for humoral immunity., Competing Interests: Declaration of Interests H.Z. and J.A.C. have a pending patent application regarding use of the SCD inhibitor SSI-4., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2020 by The American Association of Immunologists, Inc.)

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.

NKAP is a multi-functional nuclear protein that has been shown to be essential for hematopoiesis. Deletion of NKAP in hematopoietic stem cells (HSCs) was previously found to result in rapid lethality and hematopoietic failure. NKAP deficient cells also exhibited diminished proliferation and increased expression of the cyclin dependent kinase inhibitors (CDKIs) p19 Ink4d and p21 Cip1. To determine how dysregulation of CDKI expression contributes to the effects of NKAP deficiency, NKAP was deleted in mice also deficient in p19 Ink4d or p21 Cip1 using poly-IC treatment to induce Mx1-cre. Hematopoietic failure and lethality were not prevented by deficiency in either CDKI when NKAP was deleted. Inducible deletion of NKAP in cultured hematopoietic progenitors ex vivo resulted in a senescent phenotype and altered expression of numerous cell cycle regulators including the CDKI p16 INK4a. Interestingly, while combined deficiency in p16 INK4a and p21 Cip1 did not reverse the effect of NKAP deficiency on hematopoiesis in vivo , it did shift the consequence of NKAP deficiency from senescence to apoptosis in ex vivo cultures. These results suggest that NKAP may limit cellular stress that can trigger cell cycle withdrawal or cell death, a role critical for the maintenance of a viable pool of hematopoietic progenitors., (Copyright © 2019 Shapiro, Anderson, Lehrke, Chen, Nelson Holte and Shapiro.)

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., (Copyright © 2019 The Authors.)

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., (Copyright © 2019 by The American Association of Immunologists, Inc.)

Lymph nodes (LNs) play critical roles in adaptive immunity by concentrating in one location the antigens, antigen-presenting cells, and antigen-responsive lymphocytes involved in such responses. Recent studies have revealed nonrandom localization of innate and adaptive immune cells within these organs, suggesting that microanatomical positioning optimizes responses involving sparse cooperating cells. Here, we report that the peripheral localization of LN cDC2 dendritic cells specialized for MHC-II antigen presentation is matched by a similarly biased paracortical distribution of CD4 + T cells directed by the chemoattractant receptor Ebi2. In the absence of Ebi2, CD4 + T cells lose their location bias and are delayed in antigen recognition, proliferative expansion, differentiation, direct effector activity, and provision of help for CD8 + T cell-mediated memory responses, limiting host defense and vaccine responses. These findings demonstrate evolutionary selection for distinct niches within the LN that promote cellular responses, emphasizing the critical link between fine-grained tissue organization and host defense., (Published by Elsevier Inc.)

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., (Copyright © 2019 by The American Association of Immunologists, Inc.)

Intricate life-versus-death decisions are programmed during T cell development, and the regulatory mechanisms that coordinate their activation and repression are still under investigation. In this study, HDAC3-deficient double-positive (DP) thymocytes exhibit a severe decrease in numbers. The thymic cortex is rich in ATP, which is released by macrophages that clear apoptotic DP thymocytes that fail to undergo positive selection. We demonstrate that HDAC3 is required to repress expression of the purinergic receptor P2X7 to prevent DP cell death. HDAC3-deficient DP thymocytes upregulate the P2X7 receptor, increasing sensitivity to ATP-induced cell death. P2rx7/HDAC3-double knockout mice show a partial rescue in DP cell number. HDAC3 directly binds to the P2rx7 enhancer, which is hyperacetylated in the absence of HDAC3. In addition, RORγt binds to the P2rx7 enhancer and promotes P2X7 receptor expression in the absence of HDAC3. Therefore, HDAC3 is a critical regulator of DP thymocyte survival and is required to suppress P2X7 receptor expression., (Copyright © 2019 by The American Association of Immunologists, Inc.)

CD4 and CD8 T cells are vital components of the immune system. We found that histone deacetylase 3 (HDAC3) is critical for the development of CD4 T cells, as HDAC3-deficient DP thymocytes generate only CD8SP thymocytes in mice. In the absence of HDAC3, MHC Class II-restricted OT-II thymocytes are redirected to the CD8 cytotoxic lineage, which occurs with accelerated kinetics. Analysis of histone acetylation and RNA-seq reveals that HDAC3-deficient DP thymocytes are biased towards the CD8 lineage prior to positive selection. Commitment to the CD4 or CD8 lineage is determined by whether persistent TCR signaling or cytokine signaling predominates, respectively. Despite elevated IL-21R/γc/STAT5 signaling in HDAC3-deficient DP thymocytes, blocking IL-21R does not restore CD4 lineage commitment. Instead, HDAC3 binds directly to CD8-lineage promoting genes. Thus, HDAC3 is required to restrain CD8-lineage genes in DP thymocytes for the generation of CD4 T cells., Competing Interests: RP, JL, KG, PC, JC, SR, AS, TO, VS No competing interests declared, (© 2019, Philips et al.)

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., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

iNKT cells are a unique lineage of T cells that recognize glycolipid presented by CD1d. In the thymus, they differentiate into iNKT1, iNKT2 and iNKT17 effector subsets, characterized by preferential expression of Tbet, Gata3 and ROR-γt and production of IFN-γ, IL-4 and IL-17, respectively. We demonstrate that the transcriptional regulator Runx1 is essential for the generation of ROR-γt expressing iNKT17 cells. PLZF-cre Runx1 cKO mice lack iNKT17 cells in the thymus, spleen and liver. Runx1-deficient iNKT cells have altered expression of several genes important for iNKT17 differentiation, including decreased expression of IL-7Rα, BATF and c-Maf and increased expression of Bcl11b and Lef1. However, reduction of Lef1 expression or introduction of an IL-7Rα transgene is not sufficient to correct the defect in iNKT17 differentiation, demonstrating that Runx1 is a key regulator of several genes required for iNKT17 differentiation. Loss of Runx1 leads to a severe decrease in iNKT cell numbers in the thymus, spleen and liver. The decrease in cell number is due to a combined decrease in proliferation at Stage 1 during thymic development and increased apoptosis. Thus, we describe a novel role of Runx1 in iNKT cell development and differentiation, particularly in orchestrating iNKT17 differentiation.

NKT cells are a distinct subset that have developmental requirements that often differ from conventional T cells. Here, we show that NKT-specific deletion of Hdac3 results in a severe reduction in the number of iNKT cells, particularly of NKT1 cells. In addition, there is decreased cytokine production by Hdac3-deficient NKT2 and NKT17 cells. Hdac3-deficient iNKT cells have increased cell death that is not rescued by transgenic expression of Bcl-2 or Bcl-xL. Hdac3-deficient iNKT cells have less Cyto-ID staining and lower LC3A/B expression, indicative of reduced autophagy. Interestingly, Hdac3-deficient iNKT cells also have lower expression of the nutrient receptors GLUT1, CD71 and CD98, which would increase the need for autophagy when nutrients are limiting. Therefore, Hdac3 is required for iNKT cell development and differentiation.

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., (Copyright © 2016 by The American Association of Immunologists, Inc.)

Cyclin A2 activates the cyclin-dependent kinases Cdk1 and Cdk2 and is expressed at elevated levels from S phase until early mitosis. We found that mutant mice that cannot elevate cyclin A2 are chromosomally unstable and tumor-prone. Underlying the chromosomal instability is a failure to up-regulate the meiotic recombination 11 (Mre11) nuclease in S phase, which leads to impaired resolution of stalled replication forks, insufficient repair of double-stranded DNA breaks, and improper segregation of sister chromosomes. Unexpectedly, cyclin A2 controlled Mre11 abundance through a C-terminal RNA binding domain that selectively and directly binds Mre11 transcripts to mediate polysome loading and translation. These data reveal cyclin A2 as a mechanistically diverse regulator of DNA replication combining multifaceted kinase-dependent functions with a kinase-independent, RNA binding-dependent role that ensures adequate repair of common replication errors., (Copyright © 2016, American Association for the Advancement of Science.)

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., (Copyright © 2016 by The American Association of Immunologists, Inc.)

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., (Copyright © 2016 by The American Association of Immunologists, Inc.)

The transcription factor Runx1 has essential roles throughout hematopoiesis. Here, we demonstrate that Runx1 is critical for T cell maturation. Peripheral naïve CD4(+) T cells from CD4-cre Runx1 cKO mice are phenotypically and functionally immature as shown by decreased production of TNF-α upon TCR stimulation. The loss of peripheral CD4(+) T cells in CD4-cre Runx1 cKO mice is not due to defects in homeostasis or decreased expression of IL-7Rα, as transgenic expression of IL-7Rα does not rescue the loss of CD4(+) T cells. Rather, immature Runx1-deficient CD4(+) T cells are eliminated in the periphery by the activation and fixation of the classical complement pathway. In the thymus, there is a severe block in all aspects of intrathymic T cell maturation, although both positive and negative selection are unaltered. Thus, loss of Runx1 leads to the earliest characterized block in post-positive selection intrathymic maturation of CD4 T cells.

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., (Copyright © 2015 by The American Association of Immunologists, Inc.)

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., (Copyright © 2015 by The American Association of Immunologists, Inc.)

B lymphopoiesis in bone marrow (BM) is critical for maintaining a diverse peripheral B cell pool to fight infection and establish lifelong immunity. The generation of immature B cells is reduced in Flt3-ligand (FL-/-) mice leading to deficiencies in splenic B cells. Here, we sought to understand the cellular basis of the spleen B cell deficiency in FL-/- mice. Significant reductions in transitional (TS) and follicular (FO) B cells were found in FL-/- mice, and increased frequencies, but not absolute numbers, of marginal zone (MZ) B cells. BAFF-R expression on splenic B cells and serum levels of B cell activating factor (BAFF) was comparable to wildtype (WT) mice. Mixed BM chimeras revealed that the reductions in TS and FO B cells were cell extrinsic. FL administration into FL-/- mice restored the deficiency in TS B cells and normalized the MZ compartment. Ki67 analysis revealed a significant decrease in the proliferative capacity of TS B cells in FL-/- mice. A Bcl2 transgene did not rescue TS cells in FL-/- mice, uncoupling FL-deficiency to Bcl2-dependent survival pathways. Upregulation of CD1d expression and adoptive transfer experiments suggested MZ skewing in FL-/- mice. These findings support an integral role for Flt3 signaling in peripheral B cell maturation.

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., (Copyright © 2014 by The American Association of Immunologists, Inc.)

Actin depolymerizing factor-homology (ADF-H) family proteins regulate actin filament dynamics at multiple cellular locations. Herein, we have investigated the function of the ADF-H family member coactosin-like 1 (COTL1) in the regulation of actin dynamics at the T cell immune synapse (IS). We initially identified COTL1 in a genetic screen to identify novel regulators of T cell activation, and subsequently found that it associates with F-actin and localizes at the IS in response to TCR+CD28 stimulation. Live cell microscopy showed that depletion of COTL1 protein impaired T cell spreading in response to TCR ligation and abrogated lamellipodial protrusion at the T cell - B cell contact site, producing only a band of F-actin. Significantly, re-expression of wild type COTL1, but not a mutant deficient in F-actin binding could rescue these defects. In addition, COTL1 depletion reduced T cell migration. In vitro studies showed that COTL1 and cofilin compete with each other for binding to F-actin, and COTL1 protects F-actin from cofilin-mediated depolymerization. While depletion of cofilin enhanced F-actin assembly and lamellipodial protrusion at the IS, concurrent depletion of both COTL1 and cofilin restored lamellipodia formation. Taken together, our results suggest that COTL1 regulates lamellipodia dynamics in part by protecting F-actin from cofilin-mediated disassembly.

B-cell-biased lymphoid progenitors (BLPs) and Pre-pro B cells lie at a critical juncture between B cell specification and commitment. However, both of these populations are heterogenous, which hampers investigation into the molecular changes that occur as lymphoid progenitors commit to the B cell lineage. Here, we demonstrate that there are PDCA-1(+)Siglec H(+) plasmacytoid dendritic cells (pDCs) that co-purify with BLPs and Pre-pro B cells, which express little or no CD11c or Ly6C. Removal of PDCA-1(+) pDCs separates B cell progenitors that express high levels of a Rag1-GFP reporter from Rag1-GFP(low/neg) pDCs within the BLP and Pre-pro B populations. Analysis of Flt3-ligand knockout and IL-7Rα knockout mice revealed that there is a block in B cell development at the all-lymphoid progenitor (ALP) stage, as the majority of cells within the BLP or Pre-pro B gates were PDCA-1(+) pDCs. Thus, removal of PDCA-1(+) pDCs is critical for analysis of BLP and Pre-pro B cell populations. Analysis of B cell potential within the B220(+)CD19(-) fraction demonstrated that AA4.1(+)Ly6D(+)PDCA-1(-) Pre-pro B cells gave rise to CD19(+) B cells at high frequency, while PDCA-1(+) pDCs in this fraction did not. Interestingly, the presence of PDCA-1(+) pDCs within CLPs may help to explain the conflicting results regarding the origin of these cells.

Invariant natural killer T cells have a distinct developmental pathway from conventional αβ T cells. Here we demonstrate that the transcriptional repressor NKAP is required for invariant natural killer T cell but not conventional T cell development. In CD4-cre NKAP conditional knockout mice, invariant natural killer T cell development is blocked at the double-positive stage. This cell-intrinsic block is not due to decreased survival or failure to rearrange the invariant Vα14-Jα18 T cell receptor-α chain, but is rescued by overexpression of a rec-Vα14-Jα18 transgene at the double-positive stage, thus defining a role for NKAP in selection into the invariant natural killer T cell lineage. Importantly, deletion of the NKAP-associated protein histone deacetylase 3 causes a similar block in the invariant natural killer T cell development, indicating that NKAP and histone deacetylase 3 functionally interact to control invariant natural killer T cell development.

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)β(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.

Newly generated T cells are unable to respond to antigen/MHC. Rather, post-selection single-positive thymocytes must undergo T cell maturation to gain functional competency and enter the long-lived naive peripheral T cell pool. This process is poorly understood, as no gene specifically required for T cell maturation has been identified. Here, we demonstrate that loss of the transcriptional repressor NKAP results in a complete block in T cell maturation. In CD4-cre NKAP conditional knockout mice, thymic development including positive selection occurs normally, but there is a cell-intrinsic defect in the peripheral T cell pool. All peripheral naive CD4-cre NKAP conditional knockout T cells were found to be functionally immature recent thymic emigrants. This defect is not simply in cell survival, as the T cell maturation defect was not rescued by a Bcl-2 transgene. Thus, NKAP is required for T cell maturation and the acquisition of functional competency.

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., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

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.

The activation of T cells and the initiation of an immune response is tightly controlled by both positive and negative regulators. Two adaptors which function as negative regulators of T cell activation are ALX and LAX. ALX constitutively associates with LAX in T cells, and T cells from mice deficient in ALX and LAX display similar hyper-responsiveness upon T cell receptor (TCR)/CD28 stimulation, including increased production of interleukin-2. During T cell activation, ALX is inducibly phosphorylated, however the site of ALX phosphorylation had not been previously identified and the role of phosphorylation in the inhibitory function of ALX was not known. Here, using mass spectrometry, we demonstrate that ALX is phosphorylated on a serine at position 318. Substitution of alanine for serine at this position (ALX S318A) leads to an abrogation of the mobility shift in ALX induced upon TCR/CD28 stimulation. However, ALX S318A retained the ability to bind to and stimulate tyrosine phosphorylation of LAX. In addition, overexpression of ALX S318A inhibited RE/AP activation upon TCR/CD28 stimulation to a similar extent as wild-type ALX. Therefore, although ALX is inducibly phosphorylated upon TCR/CD28 stimulation, this phosphorylation is not required for ALX to inhibit T cell activation., ((c) 2010 Elsevier Inc. All rights reserved.)

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.

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.

T cell receptor signaling processes are controlled by the integrated actions of families of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPases). Several distinct cytosolic protein tyrosine phosphatases have been described that are able to negatively regulate TCR signaling pathways, including SHP-1, SHP-2, PTPH1, and PEP. Using PTPase substrate-trapping mutants and wild type enzymes, we determined that PTPN4/PTP-MEG1, a PTPH1-family member, could complex and dephosphorylate the ITAMs of the TCR zeta subunit. In addition, the substrate-trapping derivative augmented basal and TCR-induced activation of NF-kappaB in T cells. To characterize the contribution of this PTPase in T cells, we developed PTPN4-deficient mice. T cell development and TCR signaling events were comparable between wild type and PTPN4-deficient animals. The magnitude and duration of TCR-regulated ITAM phosphorylation, as well as overall protein phosphorylation, was unaltered in the absence of PTPN4. Finally, Th1- and Th2-derived cytokines and in vivo immune responses to Listeria monocytogenes were equivalent between wild type and PTPN4-deficient mice. These findings suggest that additional PTPases are involved in controlling ITAM phosphorylations.

The T-cell specific adapter protein (TSAd) encoded by the SH2D2A gene is up-regulated in activated human CD4+ T-cells in a cAMP-dependent manner. Expression of SH2D2A is important for proper activation of T-cells. Here, we show that SH2D2A expression is regulated both at the transcriptional and translational level. cAMP signaling alone induces TSAd-mRNA expression but fails to induce increased TSAd protein levels. By contrast, TCR engagement provides signals for both TSAd transcription and translation. We further show that cAMP signaling can prime T-cells for a more prompt expression of TSAd protein upon TCR stimulation. Our study thus points to a novel mechanism for how cAMP signaling may modulate T-cell activation through transcriptional priming of resting cells.

Costimulatory signals are critical to T cell activation, but how their effects are mediated remains incompletely characterized. Here, we demonstrate that locally produced C5a and C3a anaphylatoxins interacting with their G protein-coupled receptors (GPCRs), C5aR and C3aR, on APCs and T cells both upstream and downstream of CD28 and CD40L signaling are integrally involved in T cell proliferation and differentiation. Disabling these interactions reduced MHC class II and costimulatory-molecule expression and dramatically diminished T cell responses. Importantly, impaired T cell activation by Cd80-/-Cd86-/- and Cd40-/- APCs was reconstituted by added C5a or C3a. C5aR and C3aR mediated their effects via PI-3 kinase-gamma-dependent AKT phosphorylation, providing a link between GPCR signaling, CD28 costimulation, and T cell survival. These local paracrine and autocrine interactions thus operate constitutively in naive T cells to maintain viability, and their amplification by cognate APC partners thus is critical to T cell costimulation.

Adaptors play a critical role in regulating signaling pathways that control lymphocyte development and activation. Adaptor in lymphocytes of unknown function X (ALX) and Rlk/Itk-binding protein (RIBP) are adaptors related by structure and sequence, coexpressed in T cells. Mice deficient for each adaptor demonstrated that ALX and RIBP, respectively, negatively and positively regulate T cell activation in response to TCR/CD28 stimulation. However, these results did not preclude that they may function redundantly in other cell populations, or in response to other stimuli. Therefore, to understand the relationship between these related adaptors, ALX/RIBP-deficient mice were generated. We demonstrate that although ALX and RIBP are expressed throughout T cell development, T cell development occurs normally in these mice. Using the H-Y TCR transgenic model, positive and negative selection were found to proceed unimpeded in the absence of ALX and RIBP. We demonstrate that RIBP is also expressed in B cells; however, RIBP- and ALX/RIBP-deficient mice had normal B cell development, and responded equivalently to wild type in response to IgM, CD40, B cell-activating factor/B lymphocyte stimulator, CpG, and LPS. Interestingly, T cells deficient in both ALX and RIBP behaved similarly to those deficient in ALX alone during T cell activation in response to TCR/CD28, exhibiting increased IL-2 production, CD25 expression, and proliferation, thus showing that ALX deficiency masked the effect of RIBP deficiency. ALX/RIBP-deficient T cells did not have any alterations in either activation-induced cell death or Th1/2 polarization. Therefore, we did not find any functional redundancy or synergy during lymphocyte development, selection, activation, or survival in ALX/RIBP-deficient mice, demonstrating that these molecules function independently.

Activation of naïve T cells requires synergistic signals produced by the T-cell receptor (TCR) and by CD28. We previously identified the novel adaptor ALX, which, upon overexpression in Jurkat T cells, inhibited activation of the interleukin-2 (IL-2) promoter by TCR/CD28, suggesting that it is a negative regulator of T-cell activation. To further understand the physiological role of ALX, ALX-deficient mice were generated. Purified T cells from ALX-deficient mice demonstrated increased IL-2 production, CD25 expression, and proliferation in response to TCR/CD28 stimulation. Enhanced IL-2 production and proliferation were also observed when ALX-deficient mice were primed in vivo with ovalbumin-complete Freund's adjuvant and then restimulated ex vivo. Consistent with our initial overexpression studies, these data demonstrate that ALX is a negative regulator of T-cell activation. While TCR/CD28-mediated activations of phosphotyrosine induction, extracellular signal-regulated kinase 1/2, Jun N-terminal protein kinase, IkappaB kinase alpha/beta, and Akt were unaltered, constitutive activation of p38 mitogen-activated protein kinase and its upstream regulators MKK3/6 were observed for ALX-deficient splenocytes. The phenotype of ALX-deficient mice resembled the phenotype of those deficient in the transmembrane adaptor LAX, and an association between ALX and LAX proteins was demonstrated. These results suggest that ALX, in association with LAX, negatively regulates T-cell activation through inhibition of p38.

The adaptor protein ALX acts downstream of CD28 to regulate the interleukin-2 (IL-2) promoter during T cell activation. Whereas ALX is predominantly localized to the cytoplasm, ALX partially resides in the nucleus, and the nuclear pool is rapidly depleted in response to T cell receptor (TCR)/CD28 signaling. Here it is shown that this depletion occurs via nuclear export of ALX, which depends on a leucine-rich nuclear export signal (NES) in its carboxyl segment and on the CRM-1 transport protein. Nuclear import of ALX also depends on its carboxyl-terminal segment. Blocking nuclear export of ALX, either pharmacologically, by leptomycin B, or by site-directed mutation of the ALX NES, impairs CD28-mediated phosphorylation of ALX. Additionally, upon overexpression, the ALX NES mutant was found to be impaired in inhibiting TCR/CD28-induced transcriptional up-regulation of the RE/AP composite element from the IL-2 promoter, whereas a truncated form of ALX that is a potent inhibitor of RE/AP activation was found to reside entirely in the cytoplasm. Together, these results show that ALX exerts its effect on IL-2 up-regulation in the cytoplasm and suggest an intricate relationship between the nuclear localization/export, phosphorylation, and activity of ALX in response to TCR and CD28 signaling.

T cell-specific adapter protein (TSAd), encoded by the SH2D2A gene, is expressed in activated T cells. The function of TSAd is as yet unknown. We previously showed that TSAd may modulate T cell receptor-triggered signaling events. TSAd contains a Src homology (SH)2 domain, ten tyrosines and a C-terminal proline-rich region. Here, we show that human TSAd interacts with Lck through the Lck SH2 and SH3 domains and is a substrate for Lck. The TSAd C terminus, including the proline-rich region and five tyrosines, is both necessary and sufficient for TSAd interaction with and phosphorylation by Lck. Expression of TSAd in Jurkat TAg cells results in hyperphosphorylation of endogenous Lck on Y394 and to an even larger extent on Y505, resulting in a reduced Y394/Y505 phosphorylation ratio in these cells. Furthermore, full-length TSAd, but not TSAd lacking the C terminus, inhibits the hyperactive Lck Y505F mutant when both are expressed in Jurkat T cells. In contrast, expression of the TSAd C terminus alone is sufficient to inhibit Lck Y505F in phosphorylating its substrates in Jurkat T cells. Our results indicate that the TSAd C terminus is essential for inhibition of Lck activity by TSAd, and suggest a mechanism for how TSAd may inhibit early T cell activation events.