Mature Single-positive Thymocytes Research Articles

Patterns of Differentially Expressed circRNAs in Human Thymocytes.

Circular RNAs (circRNAs) are suggested to play a discriminative role between some stages of thymocyte differentiation. However, differential aspects of the stage of mature single-positive thymocytes remain to be explored. The purpose of this study is to investigate the differential expression pattern of circRNAs in three different development stages of human thymocytes, including mature single-positive cells, and perform predictions in silico regarding the ability of specific circRNAs when controlling the expression of genes involved in thymocyte differentiation. We isolate human thymocytes at three different stages of intrathymic differentiation and determine the expression of circRNAs and mRNA by RNASeq. We show that the differential expression pattern of 50 specific circRNAs serves to discriminate between the three human thymocyte populations. Interestingly, the downregulation of RAG2, a gene involved in T-cell differentiation in the thymus, could be simultaneously controlled by the downregulation of two circRNASs (hsa_circ_0031584 and hsa_circ_0019079) through the hypothetical liberation of hsa-miR-609. Our study provides, for the first time, significant insights into the usefulness of circRNAs in discriminating between different stages of thymocyte differentiation and provides new potential circRNA–miRNA–mRNA networks capable of controlling the expression of genes involved in T-cell differentiation in the thymus.

Alteration in Three-Dimensional Microenvironment of Themis-deficient Murine Thymus Using Image Cytometry Approach

Abstract Themis is a T-cell intrinsic protein involved in selection during thymocyte development in the thymus. Thus, Themis-deficient mice have gravely reduced numbers of T-cells. Despite extensive research on thymocyte signalling, the exact role of Themis remains a mystery. As the thymic 3-dimensional (3D) microenvironment is a critical element in supporting thymocyte development and vice versa, we were interested to investigate the effect of Themis deficiency on thymic components and processes. In this study, we paired 3D imaging with conventional flow cytometry to understand thymic development in normal and Themis-deficient condition. Current conventional 2-dimensional (2D) histology and 3D reconstruction from 2D sections are unsatisfactory for studying thymus morphology. We thus established a novel approach for whole thymus 3D imaging to visualize developing thymocytes and supporting stroma, such as epithelial cells, myeloid cells and vasculature. 3D imaging coupled with quantitative analysis of images using image cytometry allows for objective comparison of the thymic microenvironment between sample groups. Here, we show an altered medullary and vasculature organization in Themis−/− thymus, suggesting a suboptimal 3D niche supporting thymocyte development. Correspondingly, mature single-positive thymocytes which are dependent on the medullary environment are altered phenotypically. In addition, we detected higher levels of apoptosis in Themis−/− cortex and an increase in clonally deleted double-positive thymocytes. Our findings demonstrate how Themis-deficiency affects developing thymocytes and its supporting stromal components.

Immune Thymic Profile of the MOG-Induced Experimental Autoimmune Encephalomyelitis Mouse Model.

Multiple sclerosis (MS) is a chronic, immune-mediated, demyelinating disease that affects the neurons of the central nervous system. Activated T cells, specific for myelin epitopes, cross the brain barriers, and react against the myelin sheath, leading to demyelination. Since T cells are generated within the thymus, here we explored, in mice, the alterations occurring in this organ throughout the different phases of the disease. We induced experimental autoimmune encephalomyelitis (EAE) in C57BL/6 females and sacrifice them at the onset (day 16) and chronic phases of disease (day 23), along with non-induced controls. We observed thymic atrophy in EAE mice at the onset that remained until the chronic phase of disease. This atrophy was associated with a preferential loss of the CD4+CD8+ double positive thymocytes, an intermediate population between the more immature CD4−CD8− double negative and the most mature single positive thymocytes. This was accompanied by an increase in the thymic medullary/cortical ratio and by an altered expression levels of genes important for T cell survival. During the chronic phase, the thymi remained atrophic, but reacquired the normal proportion of the main four thymocyte populations and the normal medullary/cortical ratio. Importantly, at the onset phase, and accompanying these thymic alterations, EAE animals presented an increased percentage of demyelinating lesion area in the cerebellum, and an increased expression of interferon gamma (Ifng), interleukin (Il) 12a, and Il17a. This study suggests dynamic thymic alterations occurring in response to EAE, from the induction to the chronic phase, that might help to elucidate the MS pathophysiology.

Naïve helper T cells with high CD5 expression have increased calcium signaling.

The adaptive immune response is orchestrated by T helper cells and their function is dependent upon interactions between the T cell receptor (TCR), peptide MHC (pMHC) and co-receptors. TCR-pMHC interactions initiate calcium signaling cascades which determine T cell activation, survival, proliferation and differentiation. CD5 is a co-receptor that plays an important role in regulating T cell signaling and fate during thymocyte education. CD5 surface expression on mature single positive thymocytes correlates with the TCR signal strength for positive selecting self-ligands. CD5 also plays a role in T cell function after thymic development is complete. Peripheral T cells with higher CD5 expression respond better to foreign antigen than those with lower CD5 expression and CD5-high T cells are enriched in memory populations. In our study, we examined the role of CD5 expression and calcium signaling in the primary response of T cells using two Listeria monocytogenes specific T helper cells (LLO118 and LLO56). These T cells recognize the same immunodominant epitope (LLO190-205) of L. monocytogenes and have divergent primary and secondary responses and different levels of CD5 expression. We found that each T cell has unique calcium mobilization in response to in vitro stimulation with LLO190-205 and that CD5 expression levels in these cells changed over time following stimulation. LLO56 naïve T helper cells, which expresses higher levels of CD5, have higher calcium mobilization than naïve LLO118 T cells. Three days after in vitro stimulation, LLO118 T cells had more robust calcium mobilization than LLO56 and there were no differences in calcium mobilization 8 days after in vitro stimulation. To further evaluate the role of CD5, we measured calcium signaling in CD5 knockout LLO118 and LLO56 T cells at these three time points and found that CD5 plays a significant role in promoting the calcium signaling of naïve CD5-high LLO56 T cells.

T-cell development is regulated by the coordinated function of proximal and distal Lck promoters active at different developmental stages.

Expression of Lck, a T-cell lineage-specific tyrosine kinase critical for T-cell development and activation, can be mediated by either proximal or distal lck promoter. We generated BAC transgenic mice in which BAC lck promoter was deleted and bred these transgenes to an Lck knockout background. Lck-PROX mice, in which only the proximal promoter is functional, have maximal Lck protein and normal thymic development through CD4- CD8- double negative (DN) and CD4+ CD8+ double positive (DP) stages, but undetectable Lck later in development and reduced mature single positive thymocytes. In contrast, Lck-DIST mice, in which only distal promoter was functional, are deficient in Lck protein in DN and DP thymocytes and severely defective in early T-cell development, with a block at the DN3-DN4 beta checkpoint equivalent to complete Lck knockouts. The ability of the proximal lck promoter to support thymic development is independent of Fyn; while, in contrast, the distal lck promoter alone is completely unable to support development in the absence of Fyn. Notably, normal thymocyte development is restored by presence of both proximal and distal promoters, even when independently expressed on different lck genes. These results define distinct and complementary requirements for proximal and distal lck promoters during T-cell development.

Development of thymic Foxp3(+) regulatory T cells: TGF-β matters.

CD4(+) regulatory T cells expressing the transcription factor Foxp3 can be generated in the thymus (tTreg cells), but the cellular and molecular pathways driving their development remain incompletely understood. TGF-β is essential for the generation of Foxp3(+) Treg cells converted from peripheral naïve CD4(+) T cells (pTreg cells), yet a role for TGF-β in tTreg-cell development was initially refuted. Nevertheless, recent studies have unmasked a requirement for TGF-β in the generation of tTreg cells. Experimental evidence reveals that TGF-βin the context of TCR stimulation induces Foxp3 gene transcription in thymic Treg precursors, CD4(+) CD8(-) CD25(-) semimature and mature single-positive thymocytes. Intriguingly, thymic apoptosis was found to be intrinsically linked to the generation of tTreg cells, as apoptosis induced expression of TGF-β intrathymically. In this short review, we will highlight key data, discuss the experimental evidence and propose a modified model of tTreg-cell development involving TGF-β. We will also outline the remaining unresolved questions concerning generation of thymic Foxp3(+) Treg cells and provide our personal perspectives on the mechanisms controlling tTreg-cell development.

T cell-B cell thymic cross-talk: maintenance and function of thymic B cells requires cognate CD40-CD40 ligand interaction.

Thymic development requires bidirectional interaction or cross-talk between developing T cells and thymic stromal cells, a relationship that has been best characterized for the interaction between thymocytes and thymic epithelial cells. We have characterized in this article the requirement for similar cross-talk in the maintenance and function of thymic B cells, another population that plays a role in selection of developing thymic T cells. We found that maintenance of thymic B cells is strongly dependent on the presence of mature single-positive thymocytes and on the interactions of these T cells with specific Ag ligand. Maintenance of thymic B cell number is strongly dependent on B cell-autonomous expression of CD40, but not MHC class II, indicating that direct engagement of CD40 on thymic B cells is necessary to support their maintenance and proliferation. Thymic B cells can mediate negative selection of superantigen-specific, self-reactive, single-positive thymocytes, and we show that CD40 expression on B cells is critical for this negative selection. Cross-talk with thymic T cells is thus required to support the thymic B cell population through a pathway that requires cell-autonomous expression of CD40, and that reciprocally functions in negative selection of autoreactive T cells.

Thymic medullary epithelium and thymocyte self-tolerance require cooperation between CD28-CD80/86 and CD40-CD40L costimulatory pathways.

A critical process during thymic development of the T cell repertoire is the induction of self-tolerance. Tolerance in developing T cells is highly dependent on medullary thymic epithelial cells (mTEC), and mTEC development in turn requires signals from mature single-positive thymocytes, a bidirectional relationship termed thymus crosstalk. We show that CD28-CD80/86 and CD40-CD40L costimulatory interactions, which mediate negative selection and self-tolerance, upregulate expression of LTα, LTβ, and receptor activator for NF-κB in the thymus and are necessary for medullary development. Combined absence of CD28-CD80/86 and CD40-CD40L results in profound deficiency in mTEC development comparable to that observed in the absence of single-positive thymocytes. This requirement for costimulatory signaling is maintained even in a TCR transgenic model of high-affinity TCR-ligand interactions. CD4 thymocytes maturing in the altered thymic epithelial environment of CD40/CD80/86 knockout mice are highly autoreactive in vitro and are lethal in congenic adoptive transfer in vivo, demonstrating a critical role for these costimulatory pathways in self-tolerance as well as thymic epithelial development. These findings demonstrate that cooperativity between CD28-CD80/86 and CD40-CD40L pathways is required for normal medullary epithelium and for maintenance of self-tolerance in thymocyte development.

S1P lyase in thymic perivascular spaces promotes egress of mature thymocytes via up-regulation of S1P receptor 1

Sphingosine 1-phosphate (S1P) and S1P receptor 1 (S1P1) play an important role in the egress of mature CD4 or CD8 single-positive (SP) thymocytes from the thymus. Fingolimod hydrochloride (FTY720), an S1P1 functional antagonist, induced significant accumulation of CD62L(high)CD69(low) mature SP thymocytes in the thymic medulla. Immunohistochemical staining using anti-S1P1 antibody revealed that S1P1 is predominantly expressed on thymocytes in the thymic medulla and is strongly down-regulated even at 3h after FTY720 administration. 2-Acetyl-4-tetrahydroxybutylimidazole (THI), an S1P lyase inhibitor, also induced accumulation of mature SP thymocytes in the thymic medulla with an enlargement of the perivascular spaces (PVS). At 6h after THI administration, S1P1-expressing thymocytes reduced partially as if to form clusters and hardly existed in the proximity of CD31-expressing blood vessels in the thymic medulla, suggesting S1P lyase expression in the cells constructing thymic medullary PVS. To determine the cells expressing S1P lyase in the thymus, we newly established a mAb (YK19-2) specific for mouse S1P lyase. Immunohistochemical staining with YK19-2 revealed that S1P lyase is predominantly expressed in non-lymphoid thymic stromal cells in the thymic medulla. In the thymic medullary PVS, S1P lyase was expressed in ER-TR7-positive cells (reticular fibroblasts and pericytes) and CD31-positive vascular endothelial cells. Our findings suggest that S1P lyase expressed in the thymic medullary PVS keeps the tissue S1P concentration low around the vessels and promotes thymic egress via up-regulation of S1P1.

IL-7 Produced by Thymic Epithelial Cells Plays a Major Role in the Development of Thymocytes and TCRγδ+ Intraepithelial Lymphocytes

IL-7 is a cytokine essential for T cell development and survival. However, the local function of IL-7 produced by thymic epithelial cells (TECs) is poorly understood. To address this question, we generated IL-7-floxed mice and crossed them with FoxN1 promoter-driven Cre (FoxN1-Cre) mice to establish knockout mice conditionally deficient for the expression of IL-7 by TECs. We found that αβ and γδ T cells were significantly reduced in the thymus of IL-7(f/f) FoxN1-Cre mice. Proportion of mature single-positive thymocytes was increased. In lymph nodes and the spleen, the numbers of T cells were partially restored in IL-7(f/f) FoxN1-Cre mice. In addition, γδ T cells were absent from the fetal thymus and epidermis of IL-7(f/f) FoxN1-Cre mice. Furthermore, TCRγδ(+) intraepithelial lymphocytes (IELs) were significantly decreased in the small intestines of IL-7(f/f) FoxN1-Cre mice. To evaluate the function of IL-7 produced in the intestine, we crossed the IL-7(f/f) mice with villin promoter-driven Cre (Vil-Cre) mice to obtain the mice deficient in IL-7 production from intestinal epithelial cells. We observed that αβ and γδ IELs of IL-7(f/f) Vil-Cre mice were comparable to control mice. Collectively, our results suggest that TEC-derived IL-7 plays a major role in proliferation, survival, and maturation of thymocytes and is indispensable for γδ T cell development. This study also demonstrates that IL-7 produced in the thymus is essential for the development of γδ IELs and indicates the thymic origin of γδ IELs.

Introduction of Exogenous T-cell Receptors Into Human Hematopoietic Progenitors Results in Exclusion of Endogenous T-cell Receptor Expression

Current tumor immunotherapy approaches include the genetic modification of peripheral T cells to express tumor antigen-specific T-cell receptors (TCRs). The approach, tested in melanoma, has led to some limited success of tumor regression in patients. Yet, the introduction of exogenous TCRs into mature T cells entails an underlying risk; the generation of autoreactive clones due to potential TCR mispairing, and the lack of effective negative selection, as these peripheral cells do not undergo thymic selection following introduction of the exogenous TCR. We have successfully generated MART-1-specific CD8 T cells from genetically modified human hematopoietic stem cells (hHSC) in a humanized mouse model. The advantages of this approach include a long-term source of antigen specific T cells and proper T-cell selection due to thymopoiesis following expression of the TCR. In this report, we examine the molecular processes occurring on endogenous TCR expression and demonstrate that this approach results in exclusive cell surface expression of the newly introduced TCR, and the exclusion of endogenous TCR cell surface expression. This suggests that this stem cell based approach can provide a potentially safer approach for anticancer immunotherapy due to the involvement of thymic selection.

FTY720 Markedly Increases Alloengraftment but Does Not Eliminate Host Anti-Donor T Cells that Cause Graft Rejection on Its Withdrawal

The immunomodulator FTY720 (FTY) is beneficial in models of graft-versus-host disease, solid organ transplantation, and autoimmunity and has been approved for use in patients with multiple sclerosis. FTY modifies the homing and migration of many cell types. We report that FTY has profound positive and negative effects on allogeneic bone marrow (BM) engraftment in sublethally irradiated recipients. FTY increased donor hematopoietic progenitors in the BM, resulting in high donor engraftment in the B cell, myeloid cell, and natural killer cell, but not T cell, lineages. Donor T cell progenitors within the thymus of FTY-treated recipients were dramatically reduced, resulting in a lack of donor T cell reconstitution. In addition to preventing the ingress of donor (and host) T cell progenitors, FTY prevented the egress of fully functional host CD4+CD8- and CD4-CD8+ thymocytes that on cessation of FTY administration were able to exit from the thymus and contribute to a rapid and complete rejection of a well-established donor BM graft. When used in combination with anti-CD40L mAbs to block the CD40L:CD40 costimulatory pathway, FTY markedly enhanced anti-CD40L mAb-mediated alloengraftment promotion. In contrast to FTY alone, the combination of anti-CD40L mAb and FTY resulted in a surprisingly stable, multilineage, long-term donor chimerism. These data illustrate FTY's profound migration modulating effects and suggest a use in combinatorial therapy in achieving stable alloengraftment under nonmyeloablative conditions.

Expression of Notch receptors and ligands on immature and mature T cells

Notch plays multiple roles in T cell development in the thymus and T cell differentiation in the periphery. In order to systematically examine the role of Notch in T cell biology, we determined the cell surface expression of all Notch receptors and ligands on various populations of T cells by using a panel of specific monoclonal antibodies we recently established. Notch1 and Notch3 were upregulated at double-negative (DN) 2-DN4 stages of immature thymocytes, then downregulated on mature single-positive thymocytes and peripheral T cells, but were rapidly upregulated again upon activation. Notch2 was consistently expressed on T cells while Notch4 was not. Jagged1 and Jagged2 were expressed at double-positive stage of immature T cells. Jagged2 was also inducible on mature T cells upon activation. In contrast, no Delta-like (Dll) 1 or Dll4 expression was observed on T cells. These comprehensive profiling of the expression of Notch receptors and ligands would be informative to fully understand the role of individual Notch receptors and ligands in T cell development and differentiation.

LKB1 regulates TCR-mediated PLCγ1 activation and thymocyte positive selection

The serine/threonine kinase LKB1 is a tumour suppressor that regulates cell growth, polarity, and proliferation in many different cell types. We previously demonstrated that LKB1 controls thymocyte survival via regulation of AMPK activation. In this study, we show that LKB1 was also involved in thymocyte positive selection through regulation of T cell receptor (TCR) signalling. Both Lck-Cre- and CD4-Cre-mediated deletion of LKB1 impaired the generation of mature CD4 and CD8 single positive (SP) thymocytes that might have resulted from the attenuated tyrosine phosphorylation of phospholipase C-γ 1 (PLCγ1) in the absence of LKB1. We found that LKB1 was directly phosphorylated by Lck at tyrosine residues 36, 261, and 365 and predominately interacted with LAT and PLCγ1 following TCR stimulation. Loss of LKB1 led to impaired recruitment of PLCγ1 to the LAT signalosome. Correlatively, LKB1-deficient thymocytes failed to upregulate lineage-specifying factors, and to differentiate into SP thymocytes even if their impaired survival was rescued. These observations indicated that LKB1 is a critical component involved in TCR signalling, and our studies provide novel insights into the mechanisms of LKB1-mediated thymocyte development.

Fine Tuning of the Threshold of T Cell Selection by the Nck Adapters

Thymic selection shapes the T cell repertoire to ensure maximal antigenic coverage against pathogens while preventing autoimmunity. Recognition of self-peptides in the context of peptide-MHC complexes by the TCR is central to this process, which remains partially understood at the molecular level. In this study we provide genetic evidence that the Nck adapter proteins are essential for thymic selection. In vivo Nck deletion resulted in a reduction of the thymic cellularity, defective positive selection of low-avidity T cells, and impaired deletion of thymocytes engaged by low-potency stimuli. Nck-deficient thymocytes were characterized by reduced ERK activation, particularly pronounced in mature single positive thymocytes. Taken together, our findings identify a crucial role for the Nck adapters in enhancing TCR signal strength, thereby fine-tuning the threshold of thymocyte selection and shaping the preimmune T cell repertoire.

Lymphotoxin Signals from Positively Selected Thymocytes Regulate the Terminal Differentiation of Medullary Thymic Epithelial Cells

The thymic medulla represents a key site for the induction of T cell tolerance. In particular, autoimmune regulator (Aire)-expressing medullary thymic epithelial cells (mTECs) provide a spectrum of tissue-restricted Ags that, through both direct presentation and cross-presentation by dendritic cells, purge the developing T cell repertoire of autoimmune specificities. Despite this role, the mechanisms of Aire(+) mTEC development remain unclear, particularly those stages that occur post-Aire expression and represent mTEC terminal differentiation. In this study, in mouse thymus, we analyze late-stage mTEC development in relation to the timing and requirements for Aire and involucrin expression, the latter a marker of terminally differentiated epithelium including Hassall's corpuscles. We show that Aire expression and terminal differentiation within the mTEC lineage are temporally separable events that are controlled by distinct mechanisms. We find that whereas mature thymocytes are not essential for Aire(+) mTEC development, use of an inducible ZAP70 transgenic mouse line--in which positive selection can be temporally controlled--demonstrates that the emergence of involucrin(+) mTECs critically depends upon the presence of mature single positive thymocytes. Finally, although initial formation of Aire(+) mTECs depends upon RANK signaling, continued mTEC development to the involucrin(+) stage maps to activation of the LTα-LTβR axis by mature thymocytes. Collectively, our results reveal further complexity in the mechanisms regulating thymus medulla development and highlight the role of distinct TNFRs in initial and terminal differentiation stages in mTECs.

CCR7-mediated migration of developing thymocytes to the medulla is essential for negative selection to tissue-restricted antigens

Immature double-positive thymocytes are generated in the thymic cortex, and on positive selection, are induced to differentiate into mature single-positive thymocytes and relocate to the medulla. CCR7 is pivotal for cortex-to-medulla migration of positively selected thymocytes, and CCR7-mediated migration to the medulla is essential for establishing central tolerance, thereby, preventing tissue-specific autoimmunity. However, it was unclear how CCR7-mediated migration to the medulla affects the establishment of self-tolerance. Here, we show that the deletion of thymocytes specific for insulin-promoter-driven tissue-restricted antigens (TRAs) is significantly impaired in CCR7- or CCR7-ligand-deficient mice. These results indicate that CCR7-mediated migration to the medulla contributes to the negative selection of TRA-reactive thymocytes.

Loss of GIMAP5 (GTPase of immunity-associated nucleotide binding protein 5) impairs calcium signaling in rat T lymphocytes

The recessive lyp allele, which harbors a defective gimap5 (GTPase of immunity-associated nucleotide binding protein 5) gene, causes spontaneous apoptosis of T lymphocytes in the biobreeding diabetes-prone strain of rats. Mechanisms underlying the pro-survival function of GIMAP5 remain unclear. In this study, we show that gimap5lyp/lyp T cells display diminished calcium flux in response to thapsigargin or signaling via the T cell antigen receptor. This defect is manifested in mature single positive thymocytes, where the survival defect first occurs. We also show that GIMAP5 deficiency does not affect the thapsigargin-induced calcium release from the intracellular stores but impairs subsequent calcium entry across the plasma membrane. Our findings suggest that GIMAP5 is an important regulator of calcium response in T lymphocytes and impaired calcium signaling might underlie spontaneous apoptosis of gimap5lyp/lyp T cells.

Metalloproteinase‐Dependent Control of Thymocyte Differentiation and Proliferation

The development of T cells in the thymus is dependent on interactions between thymocytes and thymic stromal cells, on stimulation by growth factors, and on the binding to and migration along extracellular matrix (ECM) components. As metalloproteinases (MP) are involved in processes such as growth factor release and ECM modelling, we assessed the effect of MP inhibitors on T-cell development using fetal thymic organ culture systems. MP inhibitors significantly reduced the numbers of CD4/CD8 double-positive (DP) and mature single-positive thymocytes generated, correlated with a reduced number of cell cycles between the double-negative (DN)3 and DP stages. The progression of early thymocyte progenitors through the DN1-4 stages of development was also severely affected, including incomplete upregulation of CD25, decreased DN3 cell numbers, reduced rearrangement of the T-cell receptor (TCR)-beta locus and expression of intracellular TCR-beta by fewer DN3 cells. When purified DN1 cells were utilized as donor cells in reaggregate thymic organ cultures, essentially no DP thymocytes were produced in the presence of MP inhibitors. The results suggest that MP inhibitors affect the differentiation of developing thymocytes before, and reduce proliferation after, pre-TCR-mediated selection.

Patterns of expression, membrane localization, and effects of ectopic expression suggest a function for MS4a4B, a CD20 homolog in Th1 T cells

AbstractThe membrane-spanning 4A (MS4A) family of proteins includes CD20, FcϵRIβ, and HTm4, whose genes are grouped in a chromosomal location that is associated with increased susceptibility to allergy and atopic asthma. One family member, Chandra/MS4a4B, was reported to be expressed in T helper 1 (Th1) T cells but not Th2 T cells. In the present study, Ms4a4b was isolated in a screen of genes differentially expressed during thymocyte development. MS4a4B was detected in immature CD4-CD8-CD44+CD25- thymocytes, turned off during further stages of thymocyte development and reexpressed in mature single-positive thymocytes. MS4a4B expression was found in naive CD8+ and CD4+ peripheral T cells and natural killer (NK) cells but not in B cells. MS4a4B is expressed at the cell surface with its C-terminus located in the cytoplasm. When expressed in a T-cell hybridoma by retroviral vector, MS4a4B protein constitutively associated with lipid raft microdomains, whereas in primary T cells endogenous MS4a4B protein became enriched in rafts after T-cell activation. Overexpression of MS4a4B in primary CD4+ T-cell blasts enhanced T-cell receptor (TCR)-induced Th1 cytokine production. These results suggest that MS4a4B expression is tightly regulated during T-cell development and that MS4a4B expression promotes Th1 function and/or differentiation. (Blood. 2006;107:2400-2408)