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The lymphocyte-specific protein tyrosine kinase p56lck (LCK) is well documented with regard to its role in regulating T cell activation and thymocyte development through delivery of signals via the mature αβ TCR as well as the pre-TCR. Little is known, however, about the role of Lck in Th cell subset differentiation in the periphery. Here, we assess the requirement for tyrosine kinase activation of Lck in Th1 and Th2 cell differentiation by using a dominant-negative Lck (DLGKR) transgenic (Tg) mice under the control of a lck distal promoter that directs high expression in mature T cells, in which splenic CD4 T cells developed normally. This Tg mouse provides a good experimental model system to investigate the roles of Lck in mature T cell function in vivo. We show that the catalytically inactive Lck protein at about twice-normal concentrations inhibits Th2 subset differentiation in vivo and in vitro, whilst leaving the maturation of the other T cell subset, Th1, intact. These data indicate a requirement for Lck activity in Th2 cell differentiation, and a differential dependence for Lck activity between Th2 and Th1 cell differentiation. [ABSTRACT FROM PUBLISHER]

Regulatory mechanisms of T cell autoreactivity to MHC class II molecules were studied in transgenic (Tg) mice with auto-I-Ak-reactive TCR αβ transgenes (designated as MS Tg mice). Our previous study revealed that the T cell tolerance established in autoreactive MS Tg mice was not due to either clonal deletion in the thymus, anergy or an active suppression in the periphery. We proposed a novel form of self tolerance termed `clonal insufficiency', where autoreactive T cells were conditionally rendered unresponsiveness to self antigen in vivo, although retaining full potential reactivity in in vitro conditions. Here, we investigated the role of co-stimulatory molecules for the induction of self tolerance with `clonal insufficiency'. MS Tg mice were mated with CD80 (B7-1) Tg mice in which B cells exclusively and constitutively expressed CD80 molecules. Both MS Tg mice and CD80 Tg mice by themselves showed no evidence for activation of T cells and B cells, whereas MS x CD80 double-Tg mice with a H-2k background revealed an abnormal increase in the number of splenocytes and in the expression of activation markers (CD69 and CD25) on CD4 T cells in the spleen. These results indicated that the self tolerance established in MS Tg mice involved a down-regulation of CD80 molecules on B cells in vivo, resulting in a failure of sufficient T-B interactions. In addition, the serum concentration of IL-10, one of the down-regulators of CD80 expression, was found to be increased significantly in MS Tg mice. The autoreactivity of MS Tg T cells detected in vitro was significantly blocked by recombinant IL-10. Thus, IL-10-mediated down-regulation of CD80 on B cells was suggested to be involved in the clonal insufficiency in MS Tg mice in vivo. [ABSTRACT FROM PUBLISHER]

To meet the energetic requirements associated with activation, proliferation, and survival, T cells switch their metabolic signatures from energetically quiescent to activated. However, little is known about the role of metabolic pathway controlling the development of invariant natural killer T (iNKT) cells. In the present study, we found that acetyl-CoA carboxylase 1 (ACC1), a rate-limiting enzyme for the fatty acid biosynthesis pathway, plays an essential role in the development of iNKT cells in the thymus. Mice lacking T-cell specific ACC1 showed a reduced number of iNKT cells with an increased proportion of iNKT cells at immature stages 0 and 1. Furthermore, mixed bone marrow (BM) chimera experiments revealed that T-cell intrinsic ACC1 expression was selectively important for the development of thymic iNKT cells, especially for the differentiation of the NKT1 cell subset. Our single-cell RNA-sequencing (scRNA-seq) data and functional analysis demonstrated that ACC1 is responsible for survival of developing iNKT cells. Thus, these findings highlighted a novel role of ACC1 in controlling thymic iNKT cell development mediated by the control of cell survival., (© The Author(s) 2023. Published by Oxford University Press on behalf of The Japanese Society for Immunology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

CCR4 is a major trafficking receptor for T-helper (Th) 2 cells and Th17 cells and is considered as a potential therapeutic target for atopic dermatitis (AD). The CCR4 ligands CCL17 and CCL22 have been reported to be upregulated in the skin lesions of AD patients. Of note, thymic stromal lymphopoietin (TSLP), a master regulator of the Th2 immune response, promotes the expression of CCL17 and CCL22 in AD skin lesions. Here, we investigated the role of CCR4 in an AD mouse model induced by MC903, a TSLP inducer. Topical application of MC903 to ear skin increased the expression of not only TSLP but also CCL17, CCL22, the Th2 cytokine IL-4, and the Th17 cytokine IL-17A. Consistently, MC903 induced AD-like skin lesions as shown by increased epidermal thickness; increased infiltration of eosinophils, mast cells, type 2 innate lymphoid cells, Th2 cells, and Th17 cells; and elevated serum levels of total IgE. We also found increased expansion of Th2 cells and Th17 cells in the regional lymph nodes (LNs) of AD mice. Compound 22, a CCR4 inhibitor, ameliorated AD-like skin lesions with reduction of Th2 cells and Th17 cells in the skin lesions and regional LNs. We further confirmed that compound 22 diminished the expansion of Th2 cells and Th17 cells in the coculture of CD11c+ dendritic cells (DCs) and CD4+ T cells derived from the regional LNs of AD mice. Collectively, CCR4 antagonists may exhibit anti-allergic effects by inhibiting both the recruitment and expansion of Th2 cells and Th17 cells in AD., (© The Author(s) 2023. Published by Oxford University Press on behalf of The Japanese Society for Immunology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

T helper 17 (Th17) cells express CC chemokine receptor 4 (CCR4) and secrete cytokines such as interleukin-17A (IL-17A) and granulocyte macrophage colony-stimulating factor (GM-CSF), while dendritic cells (DCs) produce CC chemokine ligand 22 (CCL22), a CCR4 ligand, upon stimulation with GM-CSF. Th17 cells are known to play a critical role in the pathogenesis of rheumatoid arthritis (RA). CCL22 has also been shown to be up-regulated in the synovial tissues of RA patients. Here, we investigated the role of CCR4 in collagen-induced arthritis (CIA), a mouse model of RA. DBA/1J mice efficiently developed CIA as shown by erythema, paw swelling, joint rigidity, and joint destruction. Th17 cells were increased in the arthritic joints and regional lymph nodes (LNs) of CIA mice. A fraction of Th17 cells were also shown to produce GM-CSF. On the other hand, we observed no significant increases of Th2 cells or Treg cells, the T cell subsets also known to express CCR4, in these tissues. We further observed clusters of CCR4-expressing memory Th17 cells and CCL22-producing DCs in the regional LNs of CIA mice, supporting the role of the CCR4-CCL22 axis in the expansion of Th17 cells in the regional LNs. Compound 22, a CCR4 inhibitor, ameliorated the disease severity with reduction of Th17 cells in the arthritic joints and regional LNs and Th17-DC clusters in the regional LNs. We further confirmed that CCR4-deficient mice in the C57BL/6J background were highly resistant to CIA induction compared with wild-type mice. Collectively, CCR4 contributes to the pathogenesis of CIA and may thus represent a new therapeutic target for RA., (© The Author(s) 2022. Published by Oxford University Press on behalf of The Japanese Society for Immunology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Cancer immunotherapy utilizes our immune system to attack cancer cells and is an extremely promising strategy for cancer treatment. Although immune-checkpoint blockade, such as anti-PD-1 (programmed cell death 1) antibody, has demonstrated significant enhancement of anti-tumor immunity and has induced notable clinical outcomes, its response rates remain low, and adverse effects are always a matter of concern; therefore, new targets for cancer immunotherapy are always desired. In this situation, new concepts are needed to fuel the investigation of new target molecules for cancer immunotherapy. We propose that CD69 is one such target molecule. CD69 is known to be an activation marker of leukocytes and is also considered a crucial regulator of various immune responses through its interacting proteins. CD69 promotes T-cell retention in lymphoid tissues via sphingosine-1-phosphate receptor 1 (S1P1) internalization and also plays roles in the pathogenesis of inflammatory disorders through interacting with its functional ligands Myl9/12 (myosin light chains 9, 12a and 12b). In anti-tumor immunity, CD69 is known to be expressed on T cells in the tumor microenvironment (TME) and tumor-draining lymph nodes (TDLNs). We revealed that CD69 negatively regulates the effector function of intratumoral T cells and importantly controls the 'exhaustion' of CD8 T cells. In addition, we and others showed that either CD69 deficiency or the administration of anti-CD69 monoclonal antibody enhances anti-tumor immunity. Thus, CD69 is an attractive target for cancer immunotherapy., (© The Author(s) 2022. Published by Oxford University Press on behalf of The Japanese Society for Immunology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

CD4+ T cells not only direct immune responses against infectious micro-organisms but are also involved in the pathogenesis of inflammatory diseases. In the last two to three decades, various researchers have identified and characterized several functional CD4+ T-cell subsets, including T-helper 1 (Th1), Th2, Th9 and Th17 cells and regulatory T (Treg) cells. In this mini-review, we introduce the concept of pathogenic Th cells that induce inflammatory diseases with a model of disease induction by a population of pathogenic Th cells: the 'pathogenic Th population disease-induction model'. We will focus on Th2 cells that induce allergic airway inflammation-pathogenic Th2 cells (Tpath2 cells)-and discuss the nature of Tpath2 cells that shape the pathology of chronic inflammatory diseases. Various Tpath2-cell subsets have been identified and their unique features are summarized in mouse and human systems. Second, we will discuss how Th cells migrate and are maintained in chronic inflammatory lesions. We propose a model known as the 'CD69-Myl9 system'. CD69 is a cell surface molecule expressed on activated T cells and interaction with its ligand myosin light chain 9 (Myl9) is required for the induction of inflammatory diseases. Myl9 molecules in the small vessels of inflamed lungs may play a crucial role in the migration of activated T cells into inflammatory lesions. Emerging evidence may provide new insight into the pathogenesis of chronic inflammatory diseases and contribute to the development of new therapeutic strategies for intractable inflammatory disorders., (© The Japanese Society for Immunology. 2021. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Extracellular ATP is known to promote Th17 cell differentiation in the intestinal lamina propria by stimulating CD70+CD11clow dendritic cells (DCs) via P2X receptors (P2XRs). Recent studies have also shown that Th17 cells enhance antitumor immunity by directly promoting proliferation of cytotoxic T lymphocytes (CTLs). These finding led us to test a P2XR agonist, αβ-methylene ATP (αβ-ATP), as a mucosal vaccine adjuvant to promote CTL responses through Th17 induction. We demonstrated that (i) CD70+CD11clow DCs were present in the nasal lamina propria and expressed P2X1R, P2X2R and P2X4R; (ii) CD70+CD11clow DCs isolated from the nasal lamina propria enhanced Th17 cell differentiation of cocultured splenic CD4+ T cells upon stimulation with αβ-ATP; (iii) mice intranasally immunized with ovalbumin (OVA) and αβ-ATP had increased OVA-specific Th17 cells and CTLs in the nasal lamina propria and regional lymph nodes; (iv) mice intranasally immunized with OVA and αβ-ATP also had elevated resistance to E.G7-OVA tumor growth compared with those intranasally immunized with OVA alone; (v) suramin, a broad-range inhibitor of P2 receptors, suppressed the increases of OVA-specific Th17 cells and CTLs in mice intranasally immunized with OVA and αβ-ATP; and (vi) suramin also abrogated the enhanced antitumor immunity of mice intranasally immunized with OVA and αβ-ATP against E.G7-OVA. Collectively, αβ-ATP may be a promising mucosal adjuvant that promotes antigen-specific CTL responses via CD70+CD11clow DC-mediated Th17 induction., (© The Japanese Society for Immunology. 2020. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The introduction of immune checkpoint inhibitors in cancer treatment highlights the negative regulation of anti-tumor immunity, such as effector T-cell exhaustion in the tumor microenvironment. However, the mechanisms underlying the induction and prevention of T-cell exhaustion remain largely unknown. We found that CD69, a type II glycoprotein known to regulate inflammation through T-cell migration and retention in tissues, plays an important role in inducing the exhaustion of tumor-infiltrating T cells. Cd69-/- mice showed reduced tumor growth and metastasis in a 4T1-luc2 murine breast cancer model, in which increased numbers of tumor-infiltrating lymphocytes, relatively little T-cell exhaustion, and enhanced IFNγ production were observed. Anti-CD69 monoclonal antibody treatment attenuated the T-cell exhaustion and tumor progression in tumor-bearing mice. These findings highlight a novel role of CD69 in controlling the tumor immune escape mediated by T-cell exhaustion and indicate that CD69 is a novel target for cancer immunotherapy.

Mycobacterium bovis Bacille Calmette-Guérin (BCG) has been shown to possess potent anti-tumor activity particularly in various animal models, while the cellular and molecular mechanisms underlying its activity are not well understood. We found that lipomannan (BCG-LM), a lipophilic component of the mycobacterial cell envelope, specifically inhibits tumor growth and induces the infiltration of eosinophils at local tumor invasion sites. In contrast, neither lipoarabinomannan (BCG-LAM) nor the cell wall of Mycobacterium bovis BCG (BCG-CW) exerted anti-tumor immunity. BCG-LM enhances cytotoxic activity of eosinophils via the increased production of superoxide. Global transcriptomic analyses of BCG-LM-pulsed dendritic cells identified C-C motif ligand (CCL) 5 as a crucial chemokine for the anti-tumor immunity induced by BCG-LM, indicating that CCL5 plays an important role for the accumulation of eosinophils in the tumor microenvironment. Furthermore, BCG-LM and memory Th2 cells exerted a synergetic effect on tumor progression by cooperatively enhancing the eosinophil function. Thus, this study revealed an un-identified BCG-LM-mediated anti-tumor mechanism via superoxide produced by infiltrated eosinophils in the tumor microenvironment. Since BCG-LM activates this unique pathway, it may have potent therapeutic potential as immune cell therapy for cancer patients., (© The Japanese Society for Immunology. 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

CD4(+)T cells are crucial for directing appropriate immune responses during host defense and for the pathogenesis of inflammatory diseases. In addition to the classical biphasic model of differentiation of T-helper 1 (Th1) and Th2 cells, unexpected increases in the numbers of CD4(+)T-cell subsets, including Th17, Th9, T follicular-helper (Tfh) and T-regulatory (Treg) cells, have been recognized. In the present review, we focus on how these various T-helper cell subsets contribute to the pathogenesis of immune-mediated inflammatory diseases. In particular, we focus on multiple sclerosis, psoriasis and asthma as typical model diseases in which multiple T-helper cell subsets have recently been suggested to play a role. We will also discuss various unique sub-populations of T-helper cells that have been identified. First, we will introduce the heterogeneous T-helper cell subsets, which are classified by their simultaneous expression of multiple key transcription factors. We will also introduce different kinds of memory-type Th2 cells, which are involved in the pathogenesis of chronic type-2 immune-related diseases. Finally, we will discuss the molecular mechanisms underlying the generation of the plasticity and heterogeneity of T-helper cell subsets. The latest progress in the study of T-helper cell subsets has forced us to reconsider the etiology of immune-mediated inflammatory diseases beyond the model based on the Th1/Th2 balance. To this end, we propose another model--the pathogenic T-helper population disease-induction model--as a possible mechanism for the induction and/or persistence of immune-mediated inflammatory diseases., (© The Japanese Society for Immunology. 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Dendritic cells (DCs) play critical roles in determining the fate of CD4⁺ T cells. Among DC sub-populations, monocyte-derived inflammatory DCs (iDCs) have been shown to play an important role in the induction of adaptive immune responses under inflammatory conditions. Although previous studies have shown that DCs have an indispensable role in the induction of allergic airway inflammation and airway hyperreactivity (AHR) in murine asthma models, the precise roles of iDCs in the asthmatic responses remain largely unknown. We show here that T(h)2 cell-mediated inflammation in murine asthma models induces the expression of some markers of alternatively activated macrophage such as arginase 1 and resistin-like molecule-α in iDCs by a mechanism depending on the intrinsic expression of STAT6. In contrast, T(h)1 cell-mediated inflammation induces iDCs to express TNF-α and inducible nitric oxide synthase (iNOS), markers of TNF-α- and iNOS-producing DCs. Moreover, we show that iDCs under a T(h)2 environment play an important role in the induction of AHR, independently of allergic airway inflammation. Our results thus indicate the importance of iDCs in the induction of AHR as downstream effector cells in T(h)2 cell-mediated asthmatic responses.

The viral protein Nef is a key element for the progression of HIV disease. Previous in vitro studies suggested that Nef expression in T-cell lines enhanced TCR signaling pathways upon stimulation with TCR cross-linking, leading to the proposal that Nef lowers the threshold of T-cell activation, thus increasing susceptibility to viral replication in immune response. Likewise, the in vivo effects of Nef transgenic mouse models supported T-cell hyperresponse by Nef. However, the interpretation is complicated by Nef expression early in the development of T cells in these animal models. Here, we analyzed the consequence of Nef expression in ovalbumin-specific/CD4(+) peripheral T cells by using a novel mouse model and demonstrate that Nef inhibits antigen-specific T-cell proliferation and multiple functions required for immune response in vivo, which includes T-cell helper activity for the primary and memory B-cell response. However, Nef does not completely abrogate T-cell activity, as defined by low levels of cytokine production, which may afford the virus a replicative advantage. These results support a model, in which Nef expression does not cause T-cell hyperresponse in immune reaction, but instead reduces the T-cell activity, that may contribute to a low level of virus spread without viral cytopathic effects.

Invariant NK T (iNKT) cells are known to play a critical role in the regulation of inflammatory responses in various clinical settings. In the present study, we assessed the contribution of iNKT cells to the development of acute lung injury (ALI), which was caused by intra-tracheal administration of LPS. Jα18 gene-disrupted mice lacking these cells underwent neutrophilic inflammatory responses in lungs at an equivalent level as control mice. Next, mice were sensitized intra-tracheally with α-galactosylceramide, an activator of iNKT cells, followed by challenge with LPS. In this model, mice showed severe lung injury, and all mice were killed within 72 h after LPS injection. IFN-γ and tumor necrosis factor (TNF)-α were strikingly elevated in the lungs of these mice. Administration of neutralizing mAb against IFN-γ and TNF-α attenuated lung injury in a histopathological analysis and improved their survival rate. Flow cytometric analysis revealed that IFN-γ was expressed in NK cells, iNKT cells and also Gr-1(dull+)Ly-6C(+) monocytes and TNF-α was detected mainly in Gr-1(bright+)Ly-6G(+) neutrophils and Gr-1(dull+)Ly-6C(+) monocytes. Otherwise, in mice treated with LPS alone, IFN-γ was not detected in the lungs and Gr-1(bright+)Ly-6G(+) neutrophil was a main cellular source of TNF-α production. Anti-Gr-1 mAb resulted in the attenuation of ALI and decrease in the level of these cytokines. These results indicated that activation of iNKT cells led to striking exacerbation of ALI caused by LPS and that Gr-1(+) monocytes were recruited in the lungs with expressing IFN-γ and TNF-α and played an important role in the development of these responses.

CTLs are thought to be major effectors for clearing viruses in acute infections including hepatitis B virus (HBV). Persistent HBV infection is characterized by a lack of or a weak CTL response to HBV, which is thought to reflect tolerance to HBV antigens. In the present study, we found that alpha-galactosylceramide (alpha-GalCer), a ligand for Valpha14-positive NKT cells, strongly enhanced the induction and proliferation of HBV-specific CTLs by HBsAg. In HBsAg transgenic mice, which are thought to be tolerant to HBV-encoded antigens, administration of HBsAg or alpha-GalCer alone failed to induce HBsAg-specific CTLs, but they were induced by co-administration of both compounds. Furthermore, by limiting dilution analysis, we confirmed the existence of HBsAg-specific CTL precursors in the HBsAg transgenic mice immunized with HBsAg and alpha-GalCer. A blocking experiment using antibodies to cytokines and CD40 ligand showed that IL-2 and CD40-CD40L interaction mediate the enhancement of CTL induction caused by alpha-GalCer through NKT cell activation. Our results may open up a new method for clearing the virus from patients with persistent HBV infection.

Mycobacterium tuberculosis (tubercle bacilli) and the related acid-fast bacteria including Mycobacterium bovis Bacille Calmett-Guerin (BCG) have a characteristic cell wall (CW) containing various lipoglycans and glycolipids. Such lipoglycans have been reported to activate type-I inflammatory responses via dendritic cells (DCs) through Toll-like receptor 2. In this study, lipoglycans, lipoarabinomannan (LAM), lipomannan (LM) and phosphatidylinositol mannoside (PIM), were purified from the CW fractions of M. bovis BCG Tokyo-172, and the effect on the differentiation of human peripheral blood naive CD4 T cells into T(h)1 and T(h)2 was examined. LAM/LM molecules enhanced T(h)1 differentiation under both T(h)1 and T(h)2 conditions, whereas some other glycolipids and phospholipid enhanced T(h)2 differentiation under T(h)2 conditions. Other components had little effect under the given conditions. Even in highly purified CD4 T cell cultures, LAM/LM enhanced T(h)1 generation only under T(h)1 culture conditions. These results indicate that LAM/LM possesses a potent augmenting activity in T(h)1 differentiation in human CD4 T cells. LAM/LM appeared to act directly on naive CD4 T cells to enhance T(h)1 differentiation under T(h)1 culture conditions, while acting indirectly to up-regulate the generation of T(h)1 cells via IL-12/DCs under T(h)1 and T(h)2 conditions. Therefore, these results provide the first evidence indicating that LAM/LM from M. bovis BCG may possess a potent modulating activity in the human system, and thus supporting the strategy for the use of BCG components in the vaccine development for such T(h)2 diseases as allergic asthma and rhinitis.

Both CD4 and CD8 T cells play crucial roles in immune responses in transplantation. Immunosuppressive drugs, such as FK506 and cyclosporin A, block the priming of alloreactive CD4 T(h) cells and the subsequent induction of allospecific CD8 cytotoxic effector T cells and inhibit allograft rejection. However, the desire to minimize chronic complications that may arise from the use of immunosuppressive agents drives the search for additional strategies for immunosuppression of allograft rejection. In this study, CD4 or CD8 T cells into which the IL-10 gene is introduced using an adenovirus vector containing human IL-10 (hIL-10) cDNA (Ad-hIL-10) and into mouse T cells transgenic for the Coxsackie virus and adenovirus receptor form a model system to study the effect of administration of IL-10-secreting T cells on the survival of the allogenic skin grafts. Ad-hIL-10-infected CD4 and CD8 T cells secreted a large amount of hIL-10 for 3-4 days in culture in vitro. Ad-hIL-10-infected CD4 T cells administered in vivo could be detected in the spleen for 7 days post-transfer. Significantly prolonged survival of grafts was observed in animals that received either Ad-hIL-10-infected activated CD4 T cells or T(h)2-skewed CD4 T cells as compared with controls. Furthermore, substantial enhancement of the effect was observed in B6.C-H2(bm1)/ByJ transplants. Thus, a direct manipulation of T cells through the introduction of the immunosuppressive cytokine gene IL-10 may be a novel strategy for the control of allograft rejection.

Sarcoidosis is a systemic disorder associated with granuloma characterized by an abnormal T(h)1-type cytokine production and accumulation of T(h)1 CD4 T cells in the granuloma lesions, suggesting an importance of T(h)1 responses in sarcoidosis. However, the pathogenesis of sarcoidosis remains to be solved. Here, we investigated the nature of V(alpha)24 NKT cells with immunoregulatory functions in sarcoidosis. Patients with non-remitting sarcoidosis displayed a decrease in the number of V(alpha)24 NKT cells in peripheral blood, but an accumulation of these cells in granulomatous lesions. When stimulated with the specific glycolipid ligand, alpha-galactosylceramide, peripheral blood V(alpha)24 NKT cells from patients with non-remitting disease produced significantly less IFN-gamma than those from healthy volunteers, but normal levels of IL-4. The reduced IFN-gamma production was observed only in V(alpha)24 NKT cells and not conventional CD4 T cells, but was normal in patients with remitting disease, suggesting that non-remitting sarcoidosis involves an insufficient IFN-gamma production of V(alpha)24 NKT cells which is well correlated with disease activity. Thus, these results suggest that V(alpha)24 NKT cells play a crucial role in the disease status of sarcoidosis.

NKT cells expressing the invariant Valpha14 antigen receptor constitute a novel lymphocyte subpopulation with immunoregulatory functions. Stimulation via their invariant Valpha14 receptor with anti-CD3 or a ligand, alpha-galactosylceramide (alpha-GalCer), triggers activation of Valpha14 NKT cells, resulting in a rapid cytokine production such as IFN-gamma and IL-4. Soon after their receptor activation, Valpha14 NKT cells disappeared as judged by staining with CD1d tetramer loaded with alpha-GalCer (alpha-GalCer/CD1d tetramer), which has been believed to be due to apoptotic cell death. Here we show that such a disappearance was largely attributed to down-regulation of the Valpha14 receptor. In fact, Valpha14 NKT cells were relatively resistant to apoptosis compared to the conventional T cells as evidenced by less staining with Annexin-V, a limited DNA fragmentation, and their preferential expression of anti-apoptotic genes such as NAIP and MyD118. Furthermore, they did not become tolerant, and maintained their proliferative capacity and cytokine production even after their receptor down-regulation. These as yet unrecognized facets of Valpha14 NKT cells are discussed in relation to their regulatory functions.

CD69, known as an early activation marker antigen on T and B cells, is also expressed on platelets and activated neutrophils, suggesting certain roles in inflammatory diseases. In order to address the role of CD69 in the pathogenesis of arthritis, we established CD69-null mice. CD69-null mice displayed a markedly attenuated arthritic inflammatory response when injected with anti-type II collagen antibodies. Cell transfer experiments with neutrophils, but not T cells or spleen cells, from wild-type mice into CD69-null mice restored the induction of arthritis. These results indicate a critical role for CD69 in neutrophil function in arthritis induction during the effector phase. Thus, CD69 would be a possible therapeutic target for arthritis in human patients.

Recombination-activating gene (RAG) 1 and 2 are essential for the gene rearrangement of antigen receptors of both T and B cells. To investigate RAG gene expression in peripheral lymphoid organs other than the thymus and bone marrow, we established mice in which a green fluorescent protein (GFP) gene is knocked-in the RAG2 gene locus (RAG2-GFP mice). In the thymus and bone marrow of heterozygous RAG2-GFP mice, as expected, GFP expression was detected in the appropriate stages of developing T and B cells. Interestingly, only a fraction of Thy-1.2(+) cells in the Peyer's patch were found to be GFP(+) amongst the peripheral lymphoid organs. The GFP(+) cells expressed high levels of surface TCRbeta and CD3, suggesting mature T cells with rearranged TCRalphabeta. However, they showed activated/memory phenotypes, i.e. CD45RB(low), CD69(high), CD44(high) and CD62L(low), and belonged to a CD4(+)CD8(+) population expressing c-kit, IL-7R and pTalpha characteristic of immature developing lymphocytes. Moreover, RAG(+) Peyer's patch T cells seem to be of thymic origin as judged by their expression of CD8alphabeta. These results show that there exists a fraction of mature T cells expressing RAG genes in the Peyer's patch, implying a potential for a secondary rearrangement of TCR in extrathymic tissues.

In this study we investigate the stage at which developing T cells in the thymus acquire the ability to differentiate into T(h)1 and T(h)2 cells. We addressed this question by using sorted heat-stable antigen (HSA)(+) and HSA(-) CD4 single-positive (SP) thymocytes prepared from ovalbumin-specific TCRalphabeta transgenic mice and an in vitro T(h)1/T(h)2 differentiation culture system. HSA(-) CD4 SP thymocytes show nearly full functional capacity to differentiate into either T(h)1 or T(h)2 cells. A dramatic difference was observed, however, between HSA(+) and HSA(-) CD4 SP thymocytes in the efficiency for T(h)1 cell differentiation. TCR function of HSA(+) CD4 SP thymocytes appeared to be fully developed because antigen-induced proliferation and IL-2 production were essentially equivalent to that of HSA(-) CD4 SP thymocytes. However, the levels in IL-12 receptor (IL-12R) beta2 chain expression following anti-TCR stimulation were dramatically low in the HSA(+) CD4 SP thymocytes. Decreased IL-12-induced STAT4 phosphorylation was also observed. Moreover, IL-12-dependent transcriptional up-regulation of T-bet and STAT4 was deficient in the HSA(+) CD4 SP thymocytes. Thus, the poor capacity of HSA(+) CD4 SP thymocytes to proceed to T(h)1 cell differentiation appears to be at least partly due to underdeveloped capacity in IL-12R expression and function.

Thymus and activation-regulated chemokine (TARC)/CCL17 and macrophage-derived chemokine (MDC)/CCL22 are a pair of CC chemokines known to selectively attract T(h)2 type memory T cells via CCR4. Here we examined circulating levels of TARC and MDC in patients with atopic dermatitis (AD) and control subjects by using plasma samples, which reflect blood contents of chemokines more accurately than serum samples. The plasma levels of TARC and MDC were significantly elevated in AD patients. These values also strongly correlated with disease severity and serum lactate dehydrogenase levels, and weakly correlated with serum total IgE levels and blood eosinophilia. Previous studies demonstrated TARC immunoreactivity in the epidermal layer of AD lesional skin and production of TARC by a human keratinocytic cell line HaCaT upon stimulation with IFN-gamma. Here we demonstrated MDC immunoreactivity in the epidermal layer of AD skin at levels stronger than that of TARC. Furthermore, primary epidermal keratinocytes expressed both TARC and MDC mRNA upon stimulation with IFN-gamma, but efficiently secreted only MDC. These results suggest a post-transcriptional regulation in TARC production. IFN-gamma also induced TARC and MDC mRNA in mouse skin. Collectively, both TARC and MDC play important roles in the local accumulation of T(h)2 cells in AD lesional skin. Production of T(h)2-attracting chemokines by epidermal keratinocytes upon treatment with IFN-gamma, which is also the potent inducer of T(h)1-attracting chemokines, may underline the pivotal role of IFN-gamma in the chronic phase of AD where both T(h)1 and T(h)2 responses are mixed.

Transgenic (TG) mice with TCR alpha and beta chain genes from a CD4-dependent auto-I-Ak reactive T cell clone were generated. H-2k TG mice had a large number of thymic and splenic CD4 T cells expressing the autoreactive TCR without manifestation of autoimmunity. The cells were not anergic, as they could respond to autologous antigen presenting cells and anti-TCR antibodies in vitro to proliferate and to produce interleukins. Various degrees of down-regulation of CD2 and CD44 was observed in TG mice, indicating the presence of a defective co-stimulatory process in TG T cells. These features indicate that the self tolerance in autoreactive TCR TG mice is due not to clonal deletion and anergy but to a novel mechanism where T cells cannot sufficiently respond to normally existing self ligand in vivo. That such an in vivo unresponsiveness of autoreactive T cells is dictated in the thymus during CD4 T cell differentiation atypical form of positive selection of autoreactive T cells was suggested by the abnormal surface expression of CD69 and HSA.

CD69, an 'activation marker' that is rapidly induced on mature T cells after stimulation through the T cell antigen receptor (TCR) was found to be expressed on approximately 10% of normal thymocytes. All of these CD69+ thymocytes express alpha beta TCR, and they include both TCRlowCD4+CD8+ and TCRhighCD4+CD8- or CD4-CD8+ thymocytes. The CD69+ cells can be further segregated into heat-stable antigen (HSA)+TCRlow, HSA+TCRhigh and HSA-TCRhigh thymocyte populations. None of CD69+ cells express the mature T cell marker Qa-2. Thus CD69+ cells present in vivo appear phenotypically to represent transitional cell populations between immature TCRlowHSA+Qa-2-double-positive cells and mature TCRhighHSA-QA-2+ single-positive cells. In addition, TCR engagement by MHC molecules is required for CD69 expression in the thymus. Taken together, the CD69+ thymocytes appear to represent the cells auditioning in positive selection process or they are the cells that have been positively selected recently. Analysis of a TCR transgenic mouse model revealed an increased number of CD69+ thymocytes in a positively selecting thymus, whereas no CD69+ transgenic TCR+ thymocytes were observed in the non-selecting thymus. Based on the results of this study, we suggest that the surface expression of CD69 serves as a useful marker to identify and trace those thymocytes that are engaged in the TCR-mediated positive selection process in the thymus.

We cloned and sequenced T cell receptor (TCR) alpha and beta chain cDNA from a lambda gt10 library obtained from a murine I-Ak autoreactive helper T cell clone MS202. Two types of cDNA clones for the alpha chain and one for the beta chain were obtained. The two alpha chain transcripts used two different V alpha genes: V alpha 4, joined to J alpha 11.2; and V alpha 5, J alpha TA13. The four V alpha 4 cDNA clones obtained did not have a complete sequences, lacking the leader portion. The V alpha 4 genomic gene segment of MS202 was revealed to contain two exons corresponding to the V alpha 4.MD13 cDNA sequence, and the potential RNA splicing signals between the two exons were intact. Both of the alpha chain cDNA clones showed in-frame rearrangements. Immunoprecipitation of 125I-surface-labeled lysate of MS202 with anti-TCR antiserum and subsequent electrophonetic analyses indicated that only one of the alpha chain polypeptides was expressed on the cell surface. Thus, allelic exclusion of the alpha chain in MS202 is achieved by post-transcriptional regulation rather than rearrangements.

A monoclonal anti-I-Jk antibody JK10-23 was capable of precipitating the putative I-Jk molecule from NP-40 lysates of 125I-surface labelled mouse T cell clones with either helper or suppressor functions. The I-J molecule detected by specific immunoprecipitation and subsequent one- or two-dimensional gel analysis was a Mr 84-90 K dimer composed of 42-46 K glycopeptide subunits having isoelectric point pH 5.3 to 6.4. A monomeric form of I-J also existed in some of the T cell clones. The I-J subunit was a glycosylated polypeptide with a 41 K backbone having at least two glycosylation sites. I-J was distinguishable from other known dimeric T cell surface molecules with comparable molecular size, that is, T cell receptor alpha beta heterodimer, A1 and YE molecules expressed on a T cell leukemia EL4, and mouse CD28. The I-Jk molecule was precipitable from T cell clones with I-Ak and I-Ek restriction specificities including a clone derived from an H-2b----H-2bxkF1 radiation bone marrow chimera. None of the H-2b-restricted T cell clones from H-2b and its F1 showed the I-Jk immunoreactivity. T cell clones having either I-Ab or I-Ek restriction specificities derived from intra-H-2 recombinant mouse B10.A(5R) were positive for the I-Jk, while an I-Ab-restricted T cell clone from B10.A(3R) was negative in the I-Jk immunoprecipitation. The results indicate that I-J is a novel dimeric surface molecule, most likely to be a homodimer, expressed on T cells according to the major histocompatibility complex.