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Pemphigus vulgaris is an autoimmune blistering disease caused by IgG targeting desmoglein 3 (Dsg3), an adhesion molecule of keratinocytes. Anti-Dsg3 IgG production is prevented in healthy individuals, but it is unclear how Dsg3-specific B cells are regulated. To clarify the immunological condition regulating Dsg3-specific B cells, a pathogenic anti-Dsg3 Ig (AK23) knock-in mouse was generated. AK23 knock-in B cells developed normally without undergoing deletion or acquiring an anergic phenotype in vivo. The knock-in B cells showed Ca 2+ influx upon IgM cross-linking and differentiated into AK23-IgG + B cells after LPS and IL-4 stimulation in vitro that induced a pemphigus phenotype after adoptive transfer into Rag2 -/- mice. However, the knock-in mouse itself produced AK23-IgM but little IgG without blisters in vivo. Dsg3 immunization and skin inflammation caused AK23-IgG production and a pemphigus phenotype in vivo. Furthermore, Fcgr2b deficiency or haploinsufficiency spontaneously induced AK23-IgG production and a pemphigus phenotype with poor survival rates in AK23 knock-in mice. To assess Fcgr2b involvement in Ig class-switch efficiency, postswitch transcripts of B cells were quantified and significantly higher in Fcgr2b -/- and Fcgr2b +/- mice than wild-type mice in a gene dose-dependent manner. Finally, RNA sequencing revealed reduced expression of FCGR2B and FcγRIIB-related genes in patient B cells. These results indicated that Dsg3-specific B cells do not spontaneously perform pathogenic class switching in vivo, and pemphigus phenotype induction was prevented under normal conditions. Attenuated FcγRIIB signaling is also one of the drivers for pathogenic class switching and is consistent with immunological features identified from clinical samples. This study unveiled a characteristic immune state silencing autoreactive B cells in mice., (Copyright © 2022 by The American Association of Immunologists, Inc.)

Mast cells are major players in allergic responses. IgE-dependent activation through FcεR leads to degranulation and cytokine production, both of which require Gab2. To clarify how the signals diverge at Gab2, we established Gab2 knock-in mice that express Gab2 mutated at either the PI3K or SH2 domain-containing protein tyrosine phosphatase-2 (SHP2) binding sites. Examination of these mutants showed that both binding sites were required for the degranulation and anaphylaxis response but not for cytokine production or contact hypersensitivity. Furthermore, the PI3K, but not the SHP2, binding site was important for granule translocation during degranulation. We also identified a small GTPase, ADP-ribosylation factor (ARF)1, as the downstream target of PI3K that regulates granule translocation. FcεRI stimulation induced ARF1 activation, and this response was dependent on Fyn and the PI3K binding site of Gab2. ARF1 activity was required for FcεRI-mediated granule translocation. These data indicated that Fyn/Gab2/PI3K/ARF1-mediated signaling is specifically involved in granule translocation and the anaphylaxis response.

Polycomb group (PcG) gene products regulate the maintenance of homeobox gene expression in Drosophila and vertebrates. In the immune system, PcG molecules control cell cycle progression of thymocytes, Th2 cell differentiation, and the generation of memory CD4 T cells. In this paper, we extended the study of PcG molecules to the regulation of in vivo Th2 responses, especially allergic airway inflammation, by using conditional Ring1B-deficient mice with a CD4 T cell-specific deletion of the Ring1B gene (Ring1B(-/-) mice). In Ring1B(-/-) mice, CD4 T cell development appeared to be normal, whereas the differentiation of Th2 cells but not Th1 cells was moderately impaired. In an Ag-induced Th2-driven allergic airway inflammation model, eosinophilic inflammation was attenuated in Ring1B(-/-) mice. Interestingly, Ring1B(-/-) effector Th2 cells were highly susceptible to apoptosis in comparison with wild-type effector Th2 cells in vivo and in vitro. The in vitro experiments revealed that the expression of Bim was increased at both the transcriptional and protein levels in Ring1B(-/-) effector Th2 cells, and the enhanced apoptosis in Ring1B(-/-) Th2 cells was rescued by the knockdown of Bim but not the other proapoptotic genes, such as Perp, Noxa, or Bax. The enhanced apoptosis detected in the transferred Ring1B(-/-) Th2 cells in the lung of the recipient mice was also rescued by knockdown of Bim. Therefore, these results indicate that Ring1B plays an important role in Th2-driven allergic airway inflammation through the control of Bim-dependent apoptosis of effector Th2 cells in vivo.

Studies of human asthma and of animal models of allergic airway inflammation revealed a crucial role for Th2 cells in the pathogenesis of allergic asthma. Kruppel-type zinc finger proteins are the largest family of a regulatory transcription factor for cellular development and function. Zinc finger protein (Zfp) 35 is an 18-zinc finger motif-containing Kruppel-type zinc finger protein, while its function remains largely unknown. The aim of this study was to clarify the role of Zfp35 in the pathogenesis of Th2-dependent allergic inflammation, such as allergic asthma. We examined airway eosinophilic inflammation and hyperresponsiveness in two mouse models, which use our newly generated Zfp35-deficient (Zfp35(-/-)) mice and adoptive transfer of cells. In Zfp35(-/-) mice, Th2 cell differentiation, Th2 cytokine production, eosinophilic inflammation, and airway hyperresponsiveness were substantially enhanced. Furthermore, adoptive transfer of Ag-sensitized Zfp35(-/-) CD4 T cells into the asthmatic mice resulted in enhanced airway inflammation and airway hyperresponsiveness. These results indicate that Zfp35 controls Th2 cell differentiation, allergic airway inflammation, and airway hyperresponsiveness in a negative manner. Thus, Zfp35 may control Th2-dependent diseases, such as allergic asthma.

In vivo immune response is triggered in the lymph node, where lymphocytes for entry into, retention at, and migration to effector sites are dynamically regulated. The molecular mechanism underlying retention regulation is the key to elucidating in vivo regulation of immune response. In this study, we describe the function of the adhesion molecule class I-restricted T cell-associated molecule (CRTAM) in regulating CD8+ T cell retention within the lymph node and eventually effector function. We previously identified CRTAM as a receptor predominantly expressed on activated CD8+ T cells, and nectin-like molecule-2 (Necl2) as its ligand. In vivo function of CRTAM-Necl2 interaction was analyzed by generating CRTAM(-/-) mice. CRTAM(-/-) mice exhibited reduced protective immunity against viral infection and impaired autoimmune diabetes induction in vivo. Although Ag-specific CRTAM(-/-) CD8+ T cells showed normal CTL functions in vitro, their number in the draining lymph node was reduced. Because CRTAM+ T cells bound efficiently to Necl2-expressing CD8+ dendritic cells (DCs) that reside in T cell area of lymph node, CRTAM may induce retention by binding to CD8+ DCs at the late stage of activation before proliferation. The CRTAM-mediated late interaction with DCs induced retention of activated CD8+ T cells in an Ag-independent fashion, and this possibly resulted in effective CTL development in the draining lymph node.

Macrophages have a wide variety of activities and it is largely unknown how the diverse phenotypes of macrophages contribute to pathological conditions in the different types of tissue injury in vivo. In this study we established a novel animal model of acute respiratory distress syndrome caused by the dysfunction of alveolar epithelial type II (AE2) cells and examined the roles of alveolar macrophages in the acute lung injury. The human diphtheria toxin (DT) receptor (DTR), heparin-binding epidermal growth factor-like growth factor (HB-EGF), was expressed under the control of the lysozyme M (LysM) gene promoter in the mice. When DT was administrated to the mice they suffered from acute lung injury and died within 4 days. Immunohistochemical examination revealed that AE2 cells as well as alveolar macrophages were deleted via apoptosis in the mice treated with DT. Consistent with the deletion of AE2 cells, the amount of surfactant proteins in bronchoalveolar lavage fluid was greatly reduced in the DT-treated transgenic mice. When bone marrow from wild-type mice was transplanted into irradiated LysM-DTR mice, the alveolar macrophages became resistant to DT but the mice still suffered from acute lung injury by DT administration. Compared with the mice in which both AE2 cells and macrophages were deleted by DT administration, the DT-treated LysM-DTR mice with DT-resistant macrophages showed less severe lung injury with a reduced amount of hepatocyte growth factor in bronchoalveolar lavage fluid. These results indicate that macrophages play a protective role in noninflammatory lung injury caused by the selective ablation of AE2 cells.

The Polycomb group (PcG) gene products regulate the maintenance of the homeobox gene expression in Drosophila and vertebrates and also the cell cycle progression in thymocytes and Th2 cell differentiation in mature T cells. We herein studied the role of PcG gene bmi-1 product in Th1/Th2 cell differentiation and found that Bmi-1 facilitates Th2 cell differentiation in a Ring finger-dependent manner. Biochemical studies indicate that Bmi-1 interacts with GATA3 in T cells, which is dependent on the Ring finger of Bmi-1. The overexpression of Bmi-1 resulted in a decreased ubiquitination and an increased protein stability of GATA3. In bmi-1-deficient Th cells, the levels of Th2 cell differentiation decreased as the degradation and ubiquitination on GATA3 increased. Therefore, Bmi-1 plays a crucial role in the control of Th2 cell differentiation in a Ring finger-dependent manner by regulating GATA3 protein stability.

During development in the thymus, mature CD4+ or CD8+ cells are derived from immature CD4+CD8+ cells through a series of selection events. One of the hallmarks of this maturation process is the expression of CD69, which first appears on thymocytes as they begin positive selection. We have used blockade and overexpression of CD69 to determine the role of CD69 in thymocyte development. Blockade of CD69 led to a reduction in single-positive cells and a concomitant increase in double-positive cells in the thymus. Overexpression of a CD69 transgene in the thymus resulted in a dramatic increase in both CD8SP and CD4SP cells. Coexpression with a TCR transgene demonstrated that both positive and negative selection were enhanced by the increased levels of CD69 on thymocytes. Finally, mice overexpressing CD69 displayed a sharp reduction in the number of T cells in the spleen and lymph node. Taken as a whole, these data suggest the involvement of CD69 in the process of selection and maturation during the trafficking of thymocytes to the medulla.

The B cell-specific activator protein (BSAP) is a DNA-binding transcription factor expressed in pro-B, pre-B, and mature B cells but not in plasma cells. We explored the role of BSAP in B cell function by creating clones in a late B cell and a plasma cell line transfected with a BSAP expression plasmid. We found that the plasma cell line MPC11, which does not produce BSAP, is still permissive to BSAP production driven by heterologous promoter. Overexpression of BSAP in a late B cell line (CH12.LX.A2) and a plasma cell line augmented cell proliferation and led to greater suppression of Ig synthesis in a late B cell line than in the plasma cell line. The reduction was seen mostly in synthesis of a secretory form of Ig. Overexpression of BSAP reduced Blimp-1 expression in CH12.LX.A2 clones but not in MPC11 clones. In addition, overexpression of BSAP in CH12.LX.A2 cells suppressed spontaneous appearance of cells with high Syndecan-1 expression and high amounts of intracytosolic as well as secreted Ig synthesis. To corroborate the above findings, we cloned nontransfected CH12.LX.A2 cells and found reduced BSAP mRNA expression in the high Ig-secreting clones, which produced more Blimp-1 mRNA with greater Syndecan-1 expression than the low Ig-secreting clones. Taken together, these results indicate that BSAP expression is sufficient to reduce Ig production in late B cells; this effect is mediated in part by suppression of differentiation to cells of plasma-cell phenotype.

A novel subset of peripheral T cells, peripheral NK T cells, is found to be a major population comprising 5% of splenic T and 40% of bone marrow T cells. The majority of peripheral NK T cells are characterized by the expression of an invariant TCR-alpha encoded by V alpha 14/J alpha 281 with a one nucleotide N region. Moreover, a specific reduction of V alpha 14+ NK T cells has been demonstrated to be tightly associated with various autoimmune diseases, indicating their decisive role in autoimmune disease development. In this study, we investigated the phenotypes of peripheral V alpha 14+ NK T cells and their TCR-beta repertoire. Peripheral V alpha 14+ NK T cells, comprise two populations, i.e., small and large sized cells, at an equal frequency, belonged to the CD4- CD8- fraction, and are heat stable antigen(bright), macrophage-1bright, B220bright, CD45RBdim, and Mel-14dim, but CD5-, distinct from thymic NK T cells. TCR-beta analysis clearly showed that peripheral V alpha 14+ NK T cells utilized two to three dominant invariant TCR-beta, such as V beta 8.2 D beta J beta 2.5/V beta 7 D beta J beta 2.1 in the spleen and liver, V beta 8.2 D beta J beta 2.5/V beta 8.3 D beta J beta 2.2/V beta 7 D beta J beta 2.6 in the bone marrow, and V beta 7 D beta J beta 2.1/V beta 3 D beta J beta 1.2 in intestinal intraepithelial lymphocytes. Judging from the unusual surface phenotypes, such as heat stable antigen, macrophage-1, B220, CD45RBdim, and Mel-14dim, which are known to be T cell activation markers, peripheral V alpha 14+ NK T cells may always be activated under physiologic conditions, resulting in the oligoclonal expansion of V alpha 14+ NK T cells with different invariant TCR-beta in different peripheral organs. The unique features of V alpha 14+ NK T cells are discussed.

A novel peripheral T cell subset characterized by the expression of a NK marker and invariant TCR encoded by V alpha 14 J alpha 281 gene segments with a 1-base N-region was investigated in relation to autoimmune disease development. First, we observed that invariant V alpha 14+ NK T cells are specifically reduced with aging in C57BL/6 lpr/lpr or MRL lpr/lpr mice, whereas no change was observed in age-matched control C57BL/6 or MRL +/+ mice as determined by FACS analysis and RNase protection assay. This reduction precedes the disease development and could also be detected in other autoimmune disease-prone mice, such as C3H gld/gld and (NZB x NZW)F1 mice. These results suggest that the specific decrease in invariant V alpha 14+ NK T cells correlates strongly with the development of autoimmunity. Second, injection of MRL lpr/lpr mice with anti-V alpha 14 mAb resulted in the early onset and exacerbation of lymphosplenomegaly due to the accumulation of abnormal CD3+ B220+ CD4-CD8- T cells as well as an increase in the titers of anti-dsDNA autoantibodies. These results indicate that V alpha 14+ NK T cells regulate autoimmune responses and play a crucial role in controlling the development of autoimmune diseases.

We have isolated a cDNA (H52) of 2.8-kb-long encoding an 80-kDa mouse melanoma Ag that is defined by a syngeneic anti-B16 melanoma mAb with an ability to block anti-melanoma cytotoxic T cell responses. H52 transfectants were brightly stained with the antibody, and the 80-kDa molecule was immunoprecipitated from the transfectants. Northern blot analysis showed that this transcript was detected in mouse melanoma cells of C57BL/6 and DBA/2 origin, C1300 A/J neuroblastoma, L cell (C3H) and EL-4 T lymphoma (C57BL/6), faintly in BW5147 (AKR) T lymphoma, but not in other tumors, such as S913 fibrosarcoma (C57BL/10), NIH3T3, 70 Z/3 pre-B lymphoma, and P3U1 plasmacytoma (BALB/c). Since the transcripts were not found in normal C57BL/6 tissues of fetus, newborn, and adult origin, the H52 expression is associated with transforming phenotypes. However, no tissue- or cell type-specific expression was observed. Nucleotide sequence analysis has clearly demonstrated that H52 cDNA encodes the full length of the env gene and long terminal repeat region of endogenous ecotropic murine leukemia provirus of AKV-type, which is defective in C57BL/6. The H52 envelope protein has several amino acid changes compared to those of AKV, one of which is in the env 14 peptide region preferentially associated with MHC molecule, suggesting the possible reason for the difference of antibody reactivity even in H52-positive tumors. We also demonstrate that CTL against H52 transfectant kills B16 melanoma. Thus, the above results are direct evidence that even the endogenous self molecule, when constitutively expressed, does act as a tumor Ag.

Heterogeneity of V alpha 1+ and V beta 10+ TCR alpha beta-chains, which are predominantly used in anti-FBL-3 CTL clones established in vitro, was investigated at a nucleotide level in FBL-3 tumor-infiltrating lymphocytes (TIL) in vivo. The majority (90%) of V beta 10+ beta-chains dominated in TIL used homogeneous V beta 10D beta 2.1 sequences identical to that used in the T cell clones with cytotoxic functions. The homogeneous TCR beta-chain expression was dominant and found to be about 10% of the total TCR beta-chains in the TIL population, which was a greater than 300-to 900-fold increase than in the regional lymph nodes. This is in good agreement with the in vitro data showing that about 11% CTL clones used the homogeneous V beta 10D beta 2.1+ beta-chain. However, the J beta segment does not seem to contribute greatly to the recognition and selection of this TCR because some of homogeneous VD+ beta-chains were associated with J beta segments other than J beta 2.7 of the CTL clones. The frequency of the V alpha 1J alpha 112-2+ alpha-chain expression of the CTL type was much less (3- to 80-fold increase compared to that of lymph node) and also varied in sample materials, indicating the lower contribution of the alpha-chain for the oligoclonality of the TCR. The results were also confirmed by quantitative PCR and RNase protection assays. This suggests that the dominant expression of the homogeneous TCR beta-chain is due to the expansion of the particular anti-FBL-3 CTL in the tumor in situ. Also, the TCR beta-chain, especially the V beta D beta region, rather than alpha-chain is more important for the recognition and selection of the anti-FBL-3 TIL with cytotoxic functions.