The two major classes of antigen receptors on murine B lymphocytes, mIgM and mIgD, are both contained in a complex with two additional molecules, Ig‐alpha and Ig‐beta, which permit signal transduction. Accordingly, early biochemical events after antigen binding to either receptor are similar; biological effects, however, are different. Here, we describe three newly discovered intracellular proteins of 32, 37 and 41 kDa molecular mass, that are non‐covalently associated with mIgM, but not with mIgD. These proteins coprecipitate with mIgM in Triton X‐100 and Nonidet P‐40, but not in digitonin lysates. In addition, mIgM is to some extent associated with 29 and 31 kDa proteins that are predominantly associated with mIgD (see accompanying paper). Amino acid sequencing of p32 and p37 identified p32 as mouse prohibitin; this was corroborated by Western blot analysis with antibodies specific for rat prohibitin. p37 is a newly discovered protein. cDNA clones for both proteins were isolated and sequenced. The deduced amino acid sequence of p32 is identical to that of rat prohibitin. p37 is highly homologous to p32. Since prohibitin was identified as an inhibitor of cell proliferation, its association with mIgM, but not mIgD, could explain the different biological events elicited after engagement of each receptor.

CD26 is a T cell activation antigen known to bind adenosine deaminase and have dipeptidyl peptidase IV activity. Cross-linking of CD26 and CD3 with immobilized mAbs can deliver a costimulatory signal that contributes to T cell activation. Our earlier studies revealed that cross-linking of CD26 induces its internalization, the phosphorylation of a number of proteins involved in the signaling pathway, and subsequent T cell proliferation. Although these findings suggest the importance of internalization in the function of CD26, CD26 has only 6 aa residues in its cytoplasmic region with no known motif for endocytosis. In the present study, we have identified the mannose 6-phosphate͞insulin-like growth factor II receptor (M6P͞IGFIIR) as a binding protein for CD26 and that mannose 6-phosphate (M6P) residues in the carbohydrate moiety of CD26 are critical for this binding. Activation of peripheral blood T cells results in the mannose 6 phosphorylation of CD26. In addition, the cross-linking of CD26 with an anti-CD26 antibody induces not only capping and internalization of CD26 but also colocalization of CD26 with M6P͞IGFIIR. Finally, both internalization of CD26 and the T cell proliferative response induced by CD26-mediated costimulation were inhibited by the addition of M6P, but not by glucose 6-phosphate or mannose 1-phosphate. These results indicate that internalization of CD26 after crosslinking is mediated in part by M6P͞IGFIIR and that the interaction between mannose 6-phosphorylated CD26 and M6P͞IGFIIR may play an important role in CD26-mediated T cell costimulatory signaling.T cell activation antigen CD26 is a multifunctional, 110-kDa cell surface glycoprotein (1, 2). Although constitutively expressed in the liver, intestine, and kidney, the CD26 expression level is tightly regulated on T cells and its density is markedly enhanced after T cell activation. In the resting state, CD26 is expressed on a subset of CD4 ϩ memory T cells, and this CD4 ϩ CD26 high T cell population has been shown to respond maximally to recall antigens (1, 2).CD26 has a dipeptidyl peptidase IV (DPPIV) activity in its extracellular domain that can cleave amino-terminal dipeptides with either proline or alanine in the penultimate position (3, 4). Recently, it has been reported that an amino-terminal truncation of RANTES (regulated on activation, normal T cell expressed and secreted) by CD26͞DPPIV provides a mechanism for regulation of its activity and target cell specificity (5-7). On the other hand, CD26 interacts, presumably via its extracellular domain, with CD45, a protein tyrosine-phosphatase (8). In addition, the extracellular domain of CD26 on T cells forms a complex with adenosine deaminase, which reduces the immunosuppressive activity of local adenosine by its catalytic removal (9-12). The most striking evidence for the importance of adenosine deaminase for immune function is that a defect in adenosine deaminase activity results in severe combined immunodeficiency disease in humans (13,14).CD26 is not only highly expressed on act...

The IgM and IgD classes of antigen receptor can perform different functions on B cells. However, so far no class‐specific components communicating with the cytoplasm have been found in the two antigen receptors. We have employed a new biotinylation protocol to search for intracellular membrane Ig‐associated proteins. Here we describe two proteins of 29 and 31 kDa that are associated with membrane IgD and to some extent with membrane IgM. The membrane IgM molecule is associated specifically with three proteins of 32, 37 and 41 kDa. The purification and sequencing of the two mIgD‐associated proteins revealed that they are novel proteins which are related to each other. These proteins may be the missing link between the antigen receptor and the cytoskeleton and may contribute to functional differences between membrane IgM and membrane IgD.

The transcription factor GATA-3 has been shown to play an important role for the in vitro induction of T(h)2 cells. To clarify how the in vivo immune response is governed under GATA-3 function, we generated double-transgenic mice by crossing GATA-3 transgenic mice with ovalbumin (OVA)-specific TCR transgenic mice. After immunization with OVA, the double-transgenic mice showed increased expression of GATA-3 in antigen-reactive fresh CD4(+) T cells, and higher production of IL-5 and IL-13 in cultured spleen cells in the presence of cognate antigen without any polarizing conditions for T(h)2 cells. Moreover, the immunized double-transgenic mice showed a higher increase of in vivo secretion of IL-5 and IL-13 in bronchoalveolar lavage fluid after OVA aerosol challenging. The serum levels of OVA-specific IgG1, IgE and IgA antibodies were much higher in the immunized double-transgenic mice than TCR transgenic mice. These findings provide direct evidence that antigen-stimulated CD4(+) T cells in the immunized mice have already been committed into T(h)2 cells producing IL-5 and IL-13 selectively through enhanced GATA-3 expression in vivo, thereby inducing higher production of antigen-specific antibody for three isotypes other than IgM.

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The core region of Pseudomonas aeruginosa lipopolysaccharide (LPS) was analysed by four LPS-core-specific human monoclonal antibodies (mAbs; FK-2E7, MH-4H7,OM-lD6 and NM-3G8). Reactivity of these mAbs to about 180 P. aeruginosa strains was tested. FK-2E7 bound to strains of Homma serotype E and I at a frequency of about 90%, to strains of serotype M at about 50%, and to strains of serotype A and G at about 30%. MH-4H7 bound to P. aeruginosa strains of serotype A, F, G, H, K and M at a high frequency (45-87%), but did not bind to any strains of serotype B, C, E and I. OM-1D6 and NM-3G8 bound to P. aeruginosa strains in a nearly serotypespecific manner. OM-1D6 reacted with all strains of serotype G so far tested, and a few strains of serotype M.Furthermore, L-rhamnose in the LPS core of serotype G was an immunodominant sugar recognized by OM-1D6 as an epitope. NM-3G8 bound to only a few strains of serotype B and M. The variable reactivity of these mAbs suggests that antigenic heterogeneity of the LPS core is somewhat related with (O-polysaccharide-based) serotype. Among these mAbs, MH-4H7 and OM-1D6 showed a high level of protective activity against P.aeruginosa in an experimental infection model using normal mice. In vitto protective activity was shown to be closely related to in vitro binding activity to whole cells as determined by agglutination and flow cytometry, but not ELISA.

Background:The precise roles of T helper (Th)1-type and Th2-type cytokine responses in nickel (Ni)-induced allergic contact dermatitis have not yet been clearly defined. We investigated the involvement of Th2 cytokines in Ni-induced contact hypersensitivity reaction using GATA-3 transgenic (Tg) mice. Methods: A Ni-titanium (Ti) alloy was implanted under the skin of GATA-3 Tg mice. A Ni solution was then injected 1 month after sensitization. The ear swelling response was measured at several time points after the injection; the cytokine levels in the skin were measured at 48 h after injection, and the serum levels of IgE were measured 1 month after injection. In addition, purified CD4+ splenic cells obtained from the GATA-3 Tg mice sensitized with the Ni-Ti alloy were infused into Rag-2^–/– mice, and the ear swelling response of these mice after a further challenge with Ni solution was also measured. Results:Marked ear swelling and elevated serum IgE levels and skin tissue levels of IL-4 were observed in Ni-Ti-sensitized GATA-3 Tg mice. The Rag-2^–/– mice transfused with the CD4+ splenic cells from the Ni-Ti alloy sensitized GATA-3 Tg mice showed a significantly more pronounced ear swelling response than the control mice. Conclusion: We confirmed the participation of Th2-type immune reactions in Ni-induced allergy using GATA-3 Tg mice.

. Involvement of GATA-3-dependent Th2 lymphocyte activation in airway hyperresponsiveness.