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Intestinal bacteria play a critical role in the regulation of the host immune system and an imbalance in intestinal bacterial composition induces various host diseases. Therefore, maintaining a balance in the intestinal bacterial composition is crucial for health. Immunoglobulin A (IgA), produced through T cell-dependent and T cell-independent (TI) pathways, is essential for host defense against pathogen invasion and maintaining the balance of intestinal symbiotic bacteria. Interleukin (IL)-5 is constitutively produced by group 2 innate lymphoid cells (ILC2s) and plays a critical role in the survival and proliferation of B cells and eosinophils. Here, we show that the role of IL-5-producing ILC2s in intestinal TI IgA production at steady state using TCRα deficient mice. In this mouse model, ILC2s increased fecal TI IgA levels in a non-inflammatory state in an IL-5-dependent manner. The administration of recombinant IL-33 (rIL-33) increased the amount of TI IgA production, accompanied by an increase in the number of IL-5-producing ILC2s in the large intestine. In addition, rIL-33 treatment increased IL-5-dependent IgA+ cells in isolated lymphoid follicles, the site of TI IgA production. Furthermore, eosinophils recruited by ILC2s were required for the maximal production of IgA in the TI pathway. Moreover, IL-5 increased the frequency of TI IgA-binding intestinal bacteria and was involved in the maintenance of intestinal bacterial composition. These findings indicate that IL-5-producing ILC2s together with eosinophils contribute to TI IgA production. In addition to their role in TI IgA production, IL-5-producing ILC2s may contribute to the homeostasis of intestinal commensal bacteria., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Japanese Society for Immunology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Interleukin-7 (IL-7) is a cytokine critical for the development and maintenance of group 2 innate lymphoid cells (ILC2s). ILC2s are resident in peripheral tissues such as the intestine and lung. However, whether IL-7 produced in the lung plays a role in the maintenance and function of lung ILC2s during airway inflammation remains unknown. IL-7 was expressed in bronchoalveolar epithelial cells and lymphatic endothelial cells (LECs). To investigate the role of local IL-7 in lung ILC2s, we generated two types of IL-7 conditional knockout (IL-7cKO) mice: Sftpc-Cre (SPC-Cre) IL-7cKO mice specific for bronchial epithelial cells and type 2 alveolar epithelial cells and Lyve1-Cre IL-7cKO mice specific for LECs. In steady state, ILC2s were located near airway epithelia, although lung ILC2s were unchanged in the two lines of IL-7cKO mice. In papain-induced airway inflammation dependent on innate immunity, lung ILC2s localized near bronchia via CCR4 expression, and eosinophil infiltration and type 2 cytokine production were reduced in SPC-Cre IL-7cKO mice. In contrast, in house dust mite (HDM)-induced airway inflammation dependent on adaptive immunity, lung ILC2s localized near lymphatic vessels via their CCR2 expression 2 weeks after the last challenge. Furthermore, lung ILC2s were decreased in Lyve1-Cre IL-7cKO mice in the HDM-induced inflammation because of decreased cell survival and proliferation. Finally, administration of anti-IL-7 antibody attenuated papain-induced inflammation by suppressing the activation of ILC2s. Thus, this study demonstrates that IL-7 produced by bronchoalveolar epithelial cells and LECs differentially controls the activation and maintenance of lung ILC2s, where they are localized in airway inflammation., (© 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.)

Radioprotective 105 (RP105) is a type I transmembrane protein, which associates with a glycoprotein, MD-1. Monoclonal antibody (mAb)-mediated ligation of RP105/MD-1 robustly activates B cells. RP105/MD-1 is structurally similar to Toll-like receptor 4 (TLR4)/MD-2. B-cell responses to TLR2 and TLR4/MD-2 ligands are impaired in the absence of RP105 or MD-1. In addition to RP105/MD-1, MD-1 alone is secreted. The structure of MD-1 shows that MD-1 has a hydrophobic cavity that directly binds to phospholipids. Little is known, however, about a ligand for MD-1 and the role of MD-1 in vivo To study the role of RP105/MD-1 and MD-1 alone, specific mAbs against MD-1 are needed. Here, we report the establishment and characterization of two anti-MD-1 mAbs (JR2G9, JR7G1). JR2G9 detects soluble MD-1, whereas JR7G1 binds both soluble MD-1 and the cell surface RP105/MD-1 complex. With these mAbs, soluble MD-1 was detected in the serum and urine. The MD-1 concentration was altered by infection, diet and reperfusion injury. Serum MD-1 was rapidly elevated by TLR ligand injection in mice. The quantitative PCR and supernatant-precipitated data indicate that macrophages are one of the sources of serum soluble MD-1. These results suggest that soluble MD-1 is a valuable biomarker for inflammatory diseases., (© The Japanese Society for Immunology. 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Stimulation of Toll-like receptor 4 (TLR4) induces not only innate but also adaptive immune responses, and has been suggested to exert adjuvant effects. Additional to such positive effects, pre-stimulation of TLR4 induces endotoxin tolerance where animals are unresponsive to subsequent lethal challenges with lipopolysaccharide (LPS). We examined the effects of pre-stimulation of TLR4 using an agonistic anti-TLR4 mAb (UT12) on antibody production in vivo. Pre-injection of UT12 prior to both primary and secondary immunization completely inhibited antigen-specific antibody responses. Cellular analysis revealed that the inhibition was not due to impairment of T-cell activation. Accordingly, T-helper activities in UT12 pre-injected mice were not impaired. In contrast, B-cell priming was defective in UT12 pre-injected mice. The observation that the expression of activation markers such as CD69 and CD86 on B cells was blocked by UT12 pre-injection supports this. Interestingly, UT12 pre-injection only showed inhibitory effects at the primary and not the secondary immunization. These results provide important information concerning the regulatory mechanisms of antibody production, especially in endotoxin-tolerant states.

Marginal zone (MZ) B cells mount rapid T-cell-independent (T-I) immune responses against microbial components such as LPS. While Toll-like receptor 4 (TLR4) is essential for LPS responses, MZ B cells uniquely express high levels of another LPS sensor Radioprotective 105 (RP105). However, little is known about how RP105 is used by MZ B cells. In this study, we investigated TLR4- or RP105-dependent MZ B cell responses by utilizing agonistic monoclonal antibodies (mAbs) to each receptor. Cross-linking TLR4 and RP105 at the same time with the mAbs induced robust IgM-secreting plasma cell generation as lipid A moiety of LPS. In contrast, stimulation with either mAb alone did not elicit such responses. RP105-deficient MZ B cells failed to produce IgM-secreting plasma cells in response to lipid A. TLR4 or lipid A stimulation of MZ B cells up-regulated their B lymphocyte-induced maturation protein 1 (Blimp-1) and X-box-binding protein 1 (Xbp-1) mRNA expression. RP105 stimulation alone did not give these responses and in fact decreased TLR4-mediated their expression. Compared with wild-type (WT) MZ B cells, RP105-deficient MZ B cells exhibited increased levels of Blimp-1 and Xbp-1 mRNA expression in response to lipid A. Lipid A or TLR4 plus RP105 stimulation induced massive proliferation and expression of Bcl-xL and c-Myc in WT but not RP105-deficient MZ B cells. These responses contributed to TLR4-mediated anti-apoptotic responses in MZ B cells. Thus, RP105 contributes in a unique way to the TLR4-dependent survival, proliferation and plasma cell generation of MZ B cells.

Although the importance of T(h)1-type immune response in protection against mycobacterial infection is well recognized, its regulatory mechanism in the Mycobacterium tuberculosis (Mtb)-infected lung is not well characterized. To address this issue, we analyzed kinetics of induction of mycobacterial antigen-specific CD4(+) T(h)1 T cells after mycobacterial infection in P25 TCR-transgenic (Tg) mice which express TCR alpha and beta chains from a mycobacterial Ag85B-specific MHC class II A(b)-restricted CD4(+) T-cell clone. To supply normal regulatory T-cell repertoire, we transferred normal spleen T cells into the P25 TCR-Tg mice before infection. High dose subcutaneous infection with Mtb or Mycobacterium bovis bacillus Calmette-Guérin (BCG) induced P25 TCR-Tg CD4(+) T(h)1 cells within a week. In contrast, high-dose Mtb or BCG infection into the lung failed to induce P25 TCR-Tg CD4(+) T(h)1 cells at the early stage of the infection. Furthermore, low-dose Mtb infection into the lung induced P25 TCR-Tg CD4(+) T(h)1 cells on day 21 in the mediastinal lymph node but not in the lung. IL-10 was partially involved in the suppression of T(h)1 induction in the lung because pretreatment of mice with anti-IL-10 antibody resulted in increase of P25 TCR-Tg CD4(+) T(h)1 cells in the Mtb-infected lung on day 21 of the infection, whereas neutralization of transforming growth factor-beta, another important suppressive cytokine in the lung, showed no effects on the T(h)1 induction. Our data suggest that induction of anti-mycobacterial CD4(+) T(h)1 cells is suppressed in the mycobacteria-infected lung partially by IL-10.

IL-5 was originally defined as a T-cell-derived cytokine that triggers activated B cells for terminal differentiation into antibody-secreting plasma cells, at least in mice. Concurrently, IL-5 was recognized as the major maturation and differentiation factor for eosinophils in mice and humans. Over-expression of IL-5 significantly increases eosinophil numbers and antibody levels in vivo. Conversely, mice lacking a functional gene for IL-5 or the IL-5 receptor alpha chain (IL-5Ralpha) display a number of developmental and functional impairments in B-cell and eosinophil lineages. In addition to the Janus kinase-signal transducer and activator of transcription pathway, the tyrosine kinases Lyn and Btk (Bruton agammaglobulinemia tyrosine kinase) are involved, and Ras GTPase-extracellular signal-regulated kinase (Ras-ERK) signals are important for IL-5-dependent cell proliferation and survival. IL-5 critically regulates expression of genes involved in proliferation, cell survival and maturation and effector functions of B cells and eosinophils. Thus, IL-5 plays a pivotal role in innate and acquired immune responses and eosinophilia. In humans, the biologic effects of IL-5 are best characterized for eosinophils. The recent expansion in our understanding of the mechanisms of eosinophil development and activation in the context of IL-5 has led to advances in therapeutic options. A new therapy currently in clinical trials uses humanized mAbs against IL-5 or the IL-5R.

CD4+ Th1 cells play a critical role in the induction of cell-mediated immune responses that are important for the eradication of intracellular pathogens. Peptide-25 is the major Th1 epitope for Ag85B of Mycobacterium tuberculosis and is immunogenic in I-Ab mice. To elucidate the role of the TCR and IFN-gamma/IL-12 signals in Th1 induction, we generated TCR transgenic mice (P25 TCR-Tg) expressing TCR alpha- and beta-chains of Peptide-25-reactive cloned T cells and analyzed Th1 development of CD4+ T cells from P25 TCR-Tg. Naive CD4+ T cells from P25 TCR-Tg differentiate into both Th1 and Th2 cells upon stimulation with anti-CD3. Naive CD4+ T cells from P25 TCR-Tg preferentially develop Th1 cells upon Peptide-25 stimulation in the presence of I-Ab splenic antigen-presenting cells under neutral conditions. In contrast, a mutant of Peptide-25 can induce solely Th2 differentiation. Peptide-25-induced Th1 differentiation is observed even in the presence of anti-IFN-gamma and anti-IL-12. Furthermore, naive CD4+ T cells from STAT1 deficient P25 TCR-Tg also differentiate into Th1 cells upon Peptide-25 stimulation. Moreover, Peptide-25-loaded I-Ab-transfected Chinese hamster ovary cells induce Th1 differentiation of naive CD4+ T cells from P25 TCR-Tg in the absence of IFN-gamma or IL-12. These results imply that interaction between Peptide-25/I-Ab and TCR may primarily influence determination of the fate of naive CD4+ T cells in their differentiation towards the Th1 subset.

Bruton's tyrosine kinase (Btk) is required for B cell development and signal transduction through cell-surface molecules such as BCR and IL-5 receptor. We have identified a Btk-associated molecule, BAM11 (hereafter referred to as BAM) that binds to the pleckstrin homology (PH) domain of Btk, and inhibits Btk activity both in vivo and in vitro. In this study, we demonstrate BAM's transcriptional co-activation activity and its functional interaction with Btk. By using transient transcription assays, we demonstrate that the enforced expression of BAM enhances transcriptional activity of the synthetic reporter gene. The C-terminus of BAM is essential for the transcriptional co-activation activity. The ectopic expression of Btk together with BAM enhances BAM's transcriptional co-activation activity. BAM's transcriptional co-activation activity is enhanced through interaction with Btk, and requires both its intact PH domain and functional kinase activity. We also show that enforced expression of TFII-I, another Btk-binding protein with transcriptional activity, together with BAM and Btk, further augments BAM- and Btk-dependent transcriptional co-activation. Furthermore, BAM can be co-immunoprecipitated with the INI1/SNF5 protein, a member of the SWI/SNF complex that remodels chromatin and activates transcription. We propose a model in which Btk regulates gene transcription in B cells by activating BAM and the SWI/SNF transcriptional complex via TFII-I activation.

Ag85B (also known as alpha antigen or MPT59) is immunogenic, and induces expansion and differentiation of TCRVbeta11(+)CD4(+) T cells to IFN-gamma-producing cells in C57BL/6 (I-A(b)) mice. We reported that Peptide-25 (amino acids 240-254) of Ag85B is a major T cell epitope, and its amino acid residues at position 244, 247, 249 and 252 are I-A(b) contact residues. Here we examined roles of IFN-gamma in the generation of Peptide-25-reactive CD4(+) TCRVbeta11(+) T cells and the efficacy of mutant peptides of Peptide-25 for T(h)1 development in mice other than C57BL/6 mice. Immunization of C57BL/6 mice with Peptide-25 included in incomplete Freund's adjuvant led to preferential induction of CD4(+) TCRVbeta11(+) IFN-gamma- and tumor necrosis factor-alpha-producing T cells. Compared with other I-A(b)-binding peptides such as Peptide-9 of Ag85B, 50V of pigeon cytochrome c and ovalbumin (OVA)(265-280) peptide, only Peptide-25 was capable of inducing enormous expansion of TCRVbeta11(+) IFN-gamma-producing T cells. Treatment of C57BL/6 mice with anti-Vbeta11 antibody before Peptide-25 immunization reduced the development of CD4(+) IFN-gamma-producing T cells. Furthermore, B10.A(3R) mice, I-A(b)-positive and TCRVbeta11(-) strain, showed remarkably lower response to Peptide-25 immunization than C57BL/6 mice. Peptide-25-primed IFN-gamma(-/-) cells showed significantly decreased expansion of CD4(+) TCRVbeta11(+) T cells as compared with wild-type cells. Interestingly, Peptide-25-primed cells from MyD88-deficient mice responded to Peptide-25 and differentiated into IFN-gamma-producing cells to a similar extent as wild-type mice, indicating Toll-like receptor-independent IFN-gamma production. These results imply that IFN-gamma plays important roles for the generation and expansion of CD4(+) TCRVbeta11(+) T cells in response to Peptide-25. Although Peptide-25 was non-immunogenic in C3H/HeN mice, a substituted mutant of Peptide-25, 244D247V, capable of binding to I-A(k), induced T(h)1 development. These results clearly demonstrate important roles of IFN-gamma in the expansion of CD4(+) TCRVbeta11(+) T cells, and will provide useful information for delineating the regulatory mechanisms of T(h)1-cell development and for analyzing mechanisms on T(h)1-dominant immune responses.

Ligation of CD38 on murine B cells with agonistic anti-CD38 mAb induces B cell proliferation, expression of germline gamma1 transcripts and enhances IL-5 receptor expression. This leads to Ig class switch recombination from the micro to gamma1 heavy chain gene, and high levels of IgM and lgG1 production, particularly in response to anti-CD38 and IL-5 co-stimulation. Although some of the post-receptor signaling events initiated by CD38 ligation have been characterized, signaling pathways involved in CD38-mediated germline gamma1 transcript expression in B cells are poorly understood. Here we show that CD38 ligation of murine splenic B cells activates members of the NF-kappaB/Rel family of proteins including c-Rel, p65 and p50. The activation patterns and kinetics of NF-kappaB-like proteins in CD38-stimulated B cells differ somewhat from those seen in CD40-stimulated B cells. Activation of NF-kappaB-like proteins by CD38 ligation is not observed in splenic B cells from Bruton's tyrosine kinase (Btk)-deficient (Btk(-/-)) mice, with inhibitors of protein kinase C (PKC) and phosphatidylinositol (PI)-3 kinase also suppressing NF-kappaB activation in CD38-activated B cells. We infer from these results that activation of Btk, PI-3 kinase and PKC play, at least in part, important roles in the induction of NF-kappaB in CD38-stimulated murine B cells. Consistent with a role for NF-kappaB/Rel signaling in CD38-mediated germline gamma1 transcript expression, p50(-/-) B cells show significant impairment of germline gamma1 transcript expression in response to CD38 ligation, whereas the CD40-induced response was not altered. In contrast, c-Rel(-/-) B cells show a severe impairment of germline gamma1 transcript expression in response to CD38 or CD40 ligation. These results indicate an essential role for NF-kappaB proteins in the induction of germline gamma1 transcripts by CD38-ligated murine B cells giving rise to IL-5-induced IgG1 production.

Formation of the pre-BCR complex is a critical check point during B cell development and induces the transition of pro-B to pre-B cells. CD79b (Igbeta) is a signaling component in the pre-BCR complex, since differentiation to the pre-B phenotype is induced by cross-linking the CD79b expressed on developmentally arrested pro-B cells from recombination-activating gene (RAG)-2-deficient mice. Bruton's tyrosine kinase (BTK) plays important roles in B cell development. However, its molecular mechanisms in early B cell development are not fully understood. To examine whether BTK functions in CD79b-mediated signaling for the pro-B/pre-B transition, we utilized RAG2/BTK double-knockout (DKO) mice. Pro-B cells from RAG2/BTK-DKO mice did not differentiate into pre-B cells following CD79b cross-linking, although tyrosine phosphorylation of cellular proteins including Erk1/2 and phospholipase C-gamma2 was induced in the same manner as RAG2-KO mice. BTK is phosphorylated after cross-linking of CD79b on RAG2-deficient pro-B cells. These findings suggest that BTK-dependent pathways downstream of CD79b are critical for the pro-B/pre-B transition and BTK-independent signaling pathways are also activated via the pre-BCR complex.

Bruton's tyrosine kinase (Btk) is required for normal B cell development and signal transduction through cell surface molecules, and its defects lead to X-linked immune deficiency in mice and X-linked agammaglobulinemia in humans. In this report, we will describe the identification and characterization of a molecule, BAM11, which binds to the pleckstrin homology domain of Btk. A sequence homology search revealed that BAM11 has 89% homology, at the amino acid level, to human LTG19/ENL, that was originally identified as one of the fusion partners involved in chromosomal translocations of 11q23, MLL/ALL-1/HRX, in leukemia cells. Deletion mutants demonstrated that the region of BAM11 required for binding to Btk was localized between amino acid residues 240 and 256. Forced expression of a truncated form of BAM11 (amino acids 246-368) inhibited IL-5-induced proliferation by 50%, whereas forced expression of full-length BAM11 in Y16 cells did not affect the IL-5 responsiveness. We have also shown that BAM11 (amino acids 246-368) inhibited the kinase activity of Btk. These results suggest that the binding of BAM11 to Btk plays a regulatory role in the Btk signal transduction pathway. A cell fractionation study and analysis using EGFP-fused Btk protein demonstrated that a proportion of Btk is present within the nucleus.

Src family kinases Fyn and Lyn associate with the B cell antigen receptor (BCR). Accumulating data show that Lyn plays important roles in BCR-mediated signaling, while the role of Fyn remains obscure. Here we dissected the role of Fyn and Lyn in BCR signaling using B cells from fyn(-/-), lyn(-/-) and fyn/lyn double-deficient (fyn(-/-)lyn(-/-)) mice. In contrast to previous reports, fyn(-/-) B cells were slightly hyporeactive to both anti-IgM and anti-IgD-dextran. Although lyn(-/-) B cells were hyper-reactive to anti-IgM, anti-IgD-induced proliferation was impaired in lyn(-/-) B cells. Most of the other phenotypes of fyn(-/-)lyn(-/-) mice were similar to that of lyn(-/-) mice, except that proliferative responses of B cells to various stimuli, such as BCR cross-linking and lipopolysaccharide, were significantly lower in fyn(-/-)lyn(-/-) mice than in lyn(-/-) mice. Finally, immune responses to thymus-independent type 2 antigen were affected in these mutant mice. These observations suggest that Fyn and Lyn are involved in B cell functions, and play similar, but partly distinct, roles in BCR signaling.

IL-5, IL-10 and lipopolysaccharide (LPS) are known to activate B-1 cells in vivo in normal mice and anti-red blood cell autoantibody transgenic mice (HL mice). To assess the exact role of IL-5 in proliferation and activation of peritoneal B-1 cells, we analyzed IL-5 receptor alpha chain-deficient HL (IL-5Ralpha-/- x HL) mice generated by the cross between IL-5Ralpha-/- and HL mice. In IL-5Ralpha-/- x HL mice, Ig-producing B-1 cells in the peritoneal cavity were negligible, although the total number of B-1 cells in the peritoneal cavity were as many as 30% of that in HL mice. Moreover, LPS- or IL-10-induced differentiation of B-1 cells into antibody-producing cells was severely impaired in IL-5Ralpha-/- x HL mice. We also used in vivo 5-bromo-2'-deoxyuridine labeling to estimate the proliferation of B-1 cells in IL-5Ralpha-/- mice. The absence of IL-5Ralpha did not affect spontaneous proliferation of peritoneal B-1 cells. However, induced proliferation of peritoreal B-1 cells by oral administration of LPS was markedly impaired in IL-5Ralpha-/- mice. These results suggest that IL-5 is required for activation-associated proliferation of B-1 cells but not for their spontaneous proliferation and support the idea that IL-5 plays an important role on the induction of autoantibody production from B-1 cells.

CD38 ligation on mouse B cells by CS/2, an anti-mouse CD38 mAb, induces proliferation, IL-5 receptor alpha chain expression and tyrosine phosphorylation of Bruton's tyrosine kinase. Furthermore, stimulation of splenic B cells with IL-5 together with CS/2 induces Blimp-1 expression and differentiation into Ig-producing cells. Here we examined the role of IL-5 in IgG1 and IgA production by B cells isolated from the spleen and peritoneal cavity. CD38 recognized by CS/2 was expressed in the follicular mantle B cells surrounding the germinal center, sIgD+ splenic B cells and peritoneal B cells. IL-5 induced IgG1 production in splenic sIgD+ B cells stimulated with CS/2, while it was ineffective to induce IgA production. Among the various cytokines tested, only IL-5 had a synergistic effect on IgG1 production with CS/2. IL-5 could induce the generation of S micro-Sgamma1 reciprocal recombination DNA products in CS/2-stimulated B cells. IL-4 was ineffective to induce either micro-gamma1 switch recombination or IgG1 secretion with CS/2, demonstrating that IL-5 promotes both micro-gamma1 switch recombination and IgG1 secretion in an IL-4-independent manner. The peritoneal B-2 cells exhibited both IgG1 and IgA production in response to IL-5 plus CS/2, while B-1 cells produced IgG1. These results imply that the pattern of differentiation to Ig-producing cells seen with peritoneal B cells is not identical to the pattern seen with splenic B cells and that peritoneal B-2 cells contain precursors of IgA-producing cells responding to IL-5 plus CS/2.

The germinal center (GC) develops in secondary lymphoid tissues in response to thymus-dependent (TD) antigens. To investigate the molecular mechanism of B cell differentiation in GC, we enriched GC B cells from spleen of TD antigen-immunized wild-type and X-linked immunodeficient (XID) mice, and examined the differentiation of GC B cells into antigen-specific IgG1 antibody-forming cells (AFC) in response to anti-CD40 mAb and cytokines. A significant proportion of freshly purified GC B cells expressed receptors for IL-4 and IL-5. Anti-CD40 mAb sustained the viability of GC B cells and IL-4 co-operated with anti-CD40 mAb for further enhancement of the cell viability. Anti-CD40 mAb and IL-4 were essential for inducing differentiation of GC B cells into antigen-specific IgG1-AFC and IL-5 efficiently enhanced their differentiation. GC B cells with the xid mutation responded for proliferation to CD40 ligation to a lesser extent and for the IgG1-AFC response to anti-CD40 mAb together with IL-4, but they showed impaired responsiveness to IL-5, regardless of enhanced expression of IL-5R in response to anti-CD40 mAb and IL-4. These results suggest that anti-CD40 mAb, IL-4 and IL-5 play a critical role in the differentiation of mouse GC B cells. The GC B cells from XID mice show a functional defect with respect to IL-5-mediated differentiation.

Antigenic epitopes for Mycobacterium tuberculosis-reactive T cell immune responses have been mapped using the purified Mycobacterium protein antigen. Lymph node cells from C57BL/6 mice that had been immunized with heat-killed M. tuberculosis were cultured with various Mycobacterium protein antigens and their reactivity was monitored by proliferative response. Usage of the TCR beta chain repertoire was analyzed by flow cytometry. Stimulation of M. tuberculosis-primed lymph node cells with MPT59 (antigen 85B, alpha antigen) induced proliferative response, production of IL-2 and IFN-gamma, and the expansion of V beta 11+ CD4+ T cells in conjunction with antigen-presenting cells in an I-Ab-restricted manner. Lymph node cells from non-primed mice failed to proliferate in response to MPT59. Using peptides covering the complete mature 285 amino acids long MPT59 protein as 15-mer molecules overlapping by five amino acids, we identified the antigenic epitope for MPT59-specific V beta 11+ T cells. The 15-mer peptide, covering amino acid residues 240-254 of MPT59 [peptide-25 (amino acids 240-254)], contains the motif that is conserved for I-Ab and requires processing by antigen-presenting cells to trigger peptide-25-specific V beta 11+ CD4+ T cells. We conclude from these results that MPT59 and peptide-25 (amino acids 240-254) are not superantigens and require antigen processing in order to stimulate V beta 11+ Th1 cells. This experimental system will provide us with a useful tool for delineating the regulation of T cell development in a particular subset of M. tuberculosis infection and for developing antigenic peptides for Th1-dominant immune responses.

Infection of C57BL/6 mice with LP-BM5 murine leukemia virus (MuLV) leads to the development of murine acquired immunodeficiency syndrome (MAIDS) characterized by abnormal lymphoproliferation, hypergammaglobulinemia and severe immunodeficiency. Progression of MAIDS is delayed in X chromosome-linked immunodeficient (XID) mice, which have an abnormality of Bruton's tyrosine kinase (Btk) and lack functionally mature B cells including CD5+ B cells. In this study, we report the following four major findings. (i) Susceptibility to disease induction is not reconstituted by transfer of CD5+ B cells to XID mice. (ii) Spleen cells from asymptomatic XID mice are able to transmit MAIDS to wild-type mice. (iii) MAIDS can be transmitted to XID mice with the transfer of B cells, but not T cells, from C57BL/6 mice with MAIDS. (iv) Cells which undergo massive lymphoproliferation in XID mice with MAIDS by cell transfer are of host origin, but are not from the donor. We suggest from these results that a B cell subpopulation that is impaired in XID mice plays an important role in the initiation of MAIDS.

We have examined phosphorylation mediated by cross-talk between growth signal pathways induced by IL-2 and IL-5. To analyze the phosphorylation process in the same cells, we established two sublines, T88-Mbeta1, which is a subline of a murine IL-5-dependent cell line, T88-M, by introduction of the human IL-2 receptor beta chain (IL-2Rbeta), and secondly CTLL-5Ralphabeta, which is a subline of a murine IL-2-dependent cell line, CTLL-2, by introduction of the murine IL-5 receptor alpha chain (IL-5Ralpha) and IL-5 receptor beta chain (IL-5Rbeta, betac) genes. Both T88-Mbeta1 and CTLL-5Ralphabeta expressed high-affinity receptors for IL-2 and IL-5, and proliferated in response to both factors. Tyrosine phosphorylation of IL-2Rbeta was induced by stimulation of T88-Mbeta1 with not only IL-2 but also IL-5. Anti-IL-2Rbeta-directed immune complexes from T88-Mbeta1 stimulated with IL-5 as well as with IL-2 contained an activated tyrosine kinase. However, stimulation with IL-5 but not IL-2 induced the tyrosine phosphorylation of IL-5Rbeta, betac, suggesting that IL-2 does not activate a tyrosine kinase which efficiently catalyzes the IL-5Rbeta molecule in response to IL-5. On the other hand, the detection of JAK1 and the other common set of phosphotyrosine-containing proteins after stimulation with either IL-5 or IL-2 suggests the existence of the same tyrosine phosphorylation pathways.

The high-affinity receptor (R) for IL-5 consists of a unique alpha chain (IL-5R alpha) and a beta chain (beta c) that is shared with the receptors for IL-3 and granulocyte macrophage colony stimulating factor (GM-CSF). We defined two regions of IL-5R alpha for the IL-5-induced proliferative response, the expression of nuclear proto-oncogenes, and the tyrosine phosphorylation of cellular proteins including beta c, SH2/SH3-containing proteins and JAK2 kinase. In the studies described here, we demonstrate that IL-5, IL-3 or GM-CSF stimulation induces the tyrosine phosphorylation of JAK2, and to a lesser extent JAK1, and of STAT5. Mutational analysis revealed that one of the proline residues, particularly Pro352 and Pro355, in the membrane-proximal proline-rich sequence (Pro352-Pro353-X-Pro355) of the cytoplasmic domain of IL-5R alpha is required for cell proliferation, and for both JAK1 and JAK2 activation. In addition, transfectants expressing chimeric receptors which consist of the extracellular domain of IL-5R alpha and the cytoplasmic domain of beta c responded to IL-5 for proliferation and tyrosine phosphorylation of JAK1. Intriguingly, electrophoretic mobility shift assay analysis revealed that STAT5 was activated in cells showing either JAK1 or JAK2 tyrosine phosphorylation. These results indicate that activation of JAK1, JAK2 and STAT5 is critical to coupling IL-5-induced tyrosine phosphorylation and ultimately mitogenesis, and that Pro352 and Pro355 in the proline-rich sequence appear to play more essential roles in cell growth and in both JAK1/STAT5 and JAK2/STAT5 activation than Pro353 does.

Murine (m) IL-5 induces proliferation and differentiation of both Ly-1+ B cells and activated conventional B cells. X-linked immunodeficient (XID) mice do not respond to thymus-independent type II antigens, and have an abnormal response to a variety of activation signals through Ig receptors, CD40 and cytokine receptors. Furthermore, XID mice show a B cell specific defect, reflected in decreased numbers of IL-5R alpha+ B cells and reduced responsiveness of IL-5R alpha+ B cells to mIL-5. We generated IL-5R alpha transgenic (5R alpha-Tg) mice in which B cells expressed recombinant IL-5R alpha. We crossed male 5R alpha-Tg mice with female XID mice and used their offspring to determine the IL-5 responsiveness of these B cells. All B cells of F1 male mice carrying the xid gene together with the transgene expressed the recombinant IL-5R alpha. However, those mice lacked Ly-1 B cells and their B cells acquired responsiveness to mIL-5. Interestingly, XID-5R alpha-Tg B cells, but not XID B cells, acquired mIL-5 proliferative and Ig-secretory responsiveness only in the presence of suboptimal doses of lipopolysaccharide. Stimulation of these B cells with mIL-5 plus phorbol myristate acetate induced proliferation, but not Ig secretion. These results indicate that the impaired mIL-5 responsiveness of B cells in XID mice is due to an abnormality of IL-5R-mediated signaling which may correlate with the xid gene mutation, alteration of a single amino acid of Bruton's tyrosine kinase.

The murine acquired immunodeficiency syndrome (MAIDS) caused by a defective murine leukemia virus produces severe immunodeficiency with abnormal lymphoproliferation and hypergammaglobulinemia. The presence of both CD4+ T cells and B cells is critical for the development of this disease. Remarkably elevated mRNA expression for IFN-gamma and IL-10 was observed in spleen cells of C57BL/6 mice starting from the early phase of viral infection. IFN-gamma production was induced by spleen cells from virus-infected mice upon stimulation with concanavalin A or lipopolysaccharide in both the early and late phases of MAIDS progression. When mice that had been passively administered anti-IFN-gamma mAb were infected with the virus, the development and progression of lymphadenopathy, immunodeficiency and elevated levels of serum IgG2a associated with MAIDS were delayed. Treatment with anti-IL-4 or anti-IL-10 mAb in place of anti-IFN-gamma mAb did not induce the delayed progression of MAIDS. These data support the concept that IFN-gamma-dependent pathway may be involved in the development of MAIDS.

Mouse IL-5 (mIL-5) acts on B cells and eosinophils to induce growth and differentiation through the mIL-5 specific receptor (mIL-5R). The functional high-affinity mIL-5R is a heterodimer composed of alpha and beta chains. We investigated the expression of mIL-5R and the responsiveness of B cells and eosinophils to mIL-5 in X-linked immunodeficient (xid) mice. mIL-5R expression analyzed by using mAbs specific for alpha and beta chains revealed that xid B cells had fewer mIL-5R alpha +mIL-5R beta + than BALB/c B cells. In particular, a decrease in the number of peritoneal mIL-5R+ B cells among Ly-1 B cells (known as B-1 cells) was remarkable. Furthermore, the frequency of precursors of mIL-5 responsive B cells in xid mice was approximately 100-fold lower than that of BALB/c mice. Interestingly, sorted mIL-5R+ peritoneal B cells from xid mice displayed a low response to mIL-5. Intraperitoneal injection of mIL-5 into BALB/c mice induced polyclonal IgM production and an increase in the number of eosinophils. The same regimen failed to induce an increase in the same parameters in xid mice. However, xid mice showed mIL-5-induced eosinophilia in peripheral blood to a similar extent as BALB/c mice. Eosinophils from mIL-5-injected xid mice expressed both alpha and beta chains of mIL-5, and responded to mIL-5 with prolonged in vitro survival.

The murine interleukin 5 receptor (IL-5R) was identified by utilizing an immobilized IL-5 and an immobilized monoclonal antibody against the murein IL-5R (designated H7 mAb). The H7 mAb immunoaffinity-purified materials from the extract of cell-surface radioiodinated T88-M cells (an IL-5-dependent early B cell line) using 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) were reacted with an immobilized IL-5 matrix. SDS-PAGE of the adsorbed fraction revealed a single band at approximately 60 kDa. The binding of the 60 kDa protein to the immobilized IL-5 matrix was inhibited by the excess IL-5. The CHAPS-extract depleted of the 60 kDa protein by the absorption with H7 mAb did not contain any IL-5 binding proteins. Immunoaffinity procedure provided a final 7400-fold purification, based on an estimation of the content of the 60 kDa protein (approximate purity: 20%) from the silver-stained pattern of SDS-PAGE. Actin was copurified with the 60 kDa protein at an approximate ratio of 1:1, suggesting that the intracytoplasmic domain of the IL-5R may interact with actin. Furthermore, soluble IL-5R (molecular mass: 50 kDa) was purified by the H7 mAb-immunoaffinity chromatography. The purified soluble IL-5R was capable of inhibiting the binding of IL-5 to T88-M cells. Preparative SDS-PAGE followed by electroblotting onto a membrane permitted the determination of the N-terminal sequence of the IL-5R. The determined N-terminal sequence of the IL-5R and the deduced primary sequence from recently isolated cDNA were compared.

Interleukin 5 (IL-5) is a multifunctional cytokine that regulates the proliferation and differentiation of hematopoietic cells including B cells and eosinophils. The murine IL-5 acts on target cells via an IL-5 specific high-affinity receptor (Kd approximately 150 pM) that has been proposed to be composed of at least two membrane polypeptide chains. The p60 component recognized by anti-murine IL-5 receptor mAbs H7 and T21 binds IL-5 with low affinity (Kd approximately 10 nM). The other component is p130, detectable by following cross-linking experiments with IL-5. Using H7, T21, and R52.120 mAbs specific to murine IL-5 receptor, we characterized the molecular nature of the p130 of the high affinity receptor for murine IL-5. R52.120 mAb did not recognize the IL-5 binding recombinant p60 expressed on COS7 cells, but reacted with p130/140 on IL-5-dependent cell lines. R52.120 mAb showed partial inhibition of the IL-5-induced proliferation of the IL-5-dependent early B cell line Y16 at high IL-5 concentrations. Addition of R52.120 mAb together with H7 or T21 mAb caused more striking inhibition of the IL-5-dependent proliferation than that caused by either of them alone. R52.120 mAb down-regulated the number and dissociation constant of IL-5 binding sites with high affinity without affecting the levels of these with low-affinity. It also preferentially inhibited the formation of the cross-linked complex of p130 with radiolabeled IL-5. These results indicate that p130/p140, recognized by R52.120 mAb, is indispensable, together with p60, for the formation of high affinity IL-5 receptor. We propose to designate p60 and p130/p140 as the alpha and beta chain of IL-5 receptor, respectively.

We investigated the in vivo role of interleukin 5 (IL-5) and its receptor (IL-5R) in eosinophil growth and differentiation. When mice were administered IL-5 i.p., an increase in the number of eosinophils was observed within 5 days in peripheral blood and the peritoneal cavity. Hypereosinophilia was observed in IL-5 transgenic mice who displayed constitutive production of IL-5. A binding assay with 35S-labeled IL-5 revealed the presence of two classes of IL-5 binding sites (low and high affinity) on the surface of eosinophils. IL-5Rs on eosinophils were recognized using mAbs against murine IL-5R. When the IL-5 transgenic mice were passively administered with anti-murine IL-5R mAbs, the number of recognizable eosinophils in peripheral blood dropped within 5 days to normal levels. The antibody treatment also prevented the increase in the number of eosinophils in IL-5-injected mice. The inhibition of the above experimental eosinophilia was also observed by the passive administration of anti-IL-5 mAb. The results of the in vivo experiments clearly demonstrate that IL-5 plays an essential role in in vivo eosinophilopoiesis and may be acting on eosinophils or their precursors directly through IL-5Rs, resulting in preferential growth of this lineage of hematopoietic cells. It can also be stressed that IL-5 regulates a specific lineage of hematopoietic cells (eosinophils).

Murine interleukin 5 (IL-5), a lymphokine produced by helper T cells, is involved in the regulation of growth and differentiation of B cells and other hematopoietic cells. The receptor for IL-5 has been identified as two cross-linked complexes on T88-M cells (a murine IL-5-dependent early B cell line). In this study the IL-5 receptor was directly characterized by utilizing an immobilized IL-5 column and a rat monoclonal antibody, designated H7, directed against the IL-5 receptor. H7 completely inhibited specific binding of 35S-labeled IL-5 to T88-M cells, and bound to IL-5-responsive cells, e.g. T88-M, BCL1-B20 (a chronic B-cell leukemia), and MOPC104E (a myeloma), whereas H7 did not bind to IL-5-non-responsive cells, e.g. X5563 (a myeloma), FDC-P1 (an IL-3-dependent line), and MTH (an IL-2-dependent CTLL). H7 could barely bind to T88-M cells in the presence of IL-5, and immunoprecipitated a major band with an Mr of approximately 60 kd from the extract of surface-radioiodinated T88-M cells. The precipitation of this 60 kd molecule was inhibited by the addition of IL-5. Analysis with immobilized IL-5 also revealed that a 60 kd molecule bound specifically to IL-5-coupled beads compared with control beads. Furthermore, no additional molecule with a higher Mr that was recognized by H7 appeared under non-reducing, compared with reducing, conditions. The 60 kd molecule recognized by H7 could be digested with N-glycanase to yield a protein band of approximately 55 kd.(ABSTRACT TRUNCATED AT 250 WORDS)

Interferon-gamma (IFN-gamma) is supposed to be produced by type 1 helper T cells (TH1) and inhibits IL-4-dependent B cell growth and differentiation. IL-5 (T cell-replacing factor, TRF), is a T cell-derived lymphokine which is predominantly produced by type 2 helper T cells (TH2) and regulates proliferation and differentiation of activated B cells. In this study, the effect of IFN-gamma on IL-5-dependent B cell growth and differentiation has been studied using murine chronic B cell leukemic cells (BCL1), normal splenic B cells, and cloned early B cell line. IFN-gamma selectively inhibits the IL-5-mediated proliferation of activated B cells as well as cloned early B cell lines at a low concentration (2 U/ml) in which polyclonal IgM production was not affected. This inhibitory effect of IFN-gamma occurs within 24 h after the onset of culture, as demonstrated by the inability of antibody to IFN-gamma to reverse totally the IFN-gamma-mediated suppressive effects if it was added later than 24 h after the onset of the culture. On the contrary, IL-5-mediated IgM secretion of BCL1 and IgA formation of LPS-stimulated normal B cells were relatively resistant to the suppressive effect of IFN-gamma. IFN-gamma does not affect the receptor expression for IL-5. Interestingly, IL-4-mediated IgG1 formation of LPS-stimulated B cells was markedly suppressed by IFN-gamma at 10 U/ml. These results strongly suggest that IFN-gamma may have differential effects on IL-5-mediated B cell triggering.