B-cell proliferation initiated by Ia cross-linking and sustained by interleukins leads to class switching but not somatic mutation in vitro.

Somatic mutations that are acquired by antibody V genes of antigen-stimulated B cells ultimately provide the clonal diversity from which memory B cells are selected during immune responses to T-cell-dependent antigens. Somatic mutations apparently are not acquired when B cells are stimulated by mitogens nor when they participate in immune responses to T-cell-independent antigens. Since the basis of T-cell-dependent humoral immunity is T-cell recognition of processed antigen in the context of class II major histocompatibility glycoproteins (Ia) on the B-cell surface, we sought to determine whether the ligation of Ia on B cells induces somatic mutation. B cells were stimulated in vitro by a procedure in which their proliferation was dependent upon ligation of surface Ia with antibody. Sequences of hybridoma V genes derived from these B cells revealed no somatic mutations despite prolonged stimulation in vitro and the induction of immunoglobulin secretion and switching to isotypes characteristic of T cell-dependent humoral immunity. We infer that Ia-mediated signalling and isotype switching are not causally related to somatic mutation. The avenue of differentiation that leads to somatic mutation in memory B cells is apparently separable from that leading to proliferation, immunoglobulin secretion and switching.

Modeling of T cell contact-dependent B cell activation. IL-4 and antigen receptor ligation primes quiescent B cells to mobilize calcium in response to Ia cross-linking.

The generation of antibody secretory cells from resting B lymphocytes after immunization with most protein Ag requires B cell signaling by Ag, direct Th cell contact and lymphokines. Previous studies suggest that cell contact-mediated signals may be transduced by Ia after Ia binding by alpha beta TCR and/or CD4. Seemingly inconsistent with this concept are findings that cross-linking of Ia molecules on quiescent B cells leads to cAMP generation that is antagonistic for B cell mitogenesis. Here we show that ligand binding to IL-4 and Ag receptors on quiescent B cells induce transition of these cells into a competent state in which Ia molecules transduce signals via a distinct mechanism. This mechanism involves the tyrosine kinase-dependent activation of phospholipase C leading to Ca2+ mobilization from intracellular stores and the extracellular space. This competence, which is seen within 4 h of priming, is not simply a function of increased Ia expression by the B cell because the response can be induced by cross-linking of less than 5% of cell surface Ia molecules on primed cells. Finally, cross-linking of Ia molecules leads to more than fivefold greater increase in [Ca2+]i than is induced by membrane Ig ligation. These findings are consistent with alpha beta TCR/CD4 delivery via Ia of proliferative signals mediated by tyrosine kinase activation, phosphoinositide hydrolysis and Ca2+ mobilization.

Ia-mediated signal transduction leads to proliferation of primed B lymphocytes.

One of the most controversial questions in immunology is the molecular basis by which Th lymphocytes deliver activating signals to quiescent B lymphocytes during T cell-dependent immune responses. Recent studies suggest that T cell-dependent activation of quiescent B lymphocytes may involve signaling mediated by direct T helper cell-B cell contact. Since B cell membrane-associated MHC-encoded class II molecules (Ia) must be recognized by Th lymphocytes for generation of T cell-dependent humoral immune responses, they are obvious candidates for receptors of this signal. Here we report that stimulation of quiescent murine B cells with IL-4 and antibodies against the B cell antigen receptor for 12-16 h primes cells to proliferate in response to immobilized mIa binding ligands. In the presence of additional lymphokines, these B cells differentiate to secrete Ig of IgM and IgG classes. These results suggest that Ia molecules are receptors for direct, T helper cell-B cell contact mediated signaling that results in B cell proliferation.

The role of L3T4+ cells in the pathogenesis of lupus in lpr-bearing mice. I. Defects in the production of interleukins 2 and 3.

Mice which bear the lpr gene spontaneously develop autoimmune syndromes characterized by massive expansion of an unusual T cell subset which is phenotypically Thy-1+, L3T4-, Lyt-2-, B220+. The mutant T cells are refractory to stimulation with mitogenic lectins and, by implication, are thought to be solely responsible for the defects in lymphokine production manifested by lpr mice. The contribution of the remaining L3T4+ T cell subset to the latter derangements has not been previously examined and is the focus of this study. We found that abnormalities in concanavalin A-induced interleukin 2 and 3 production in the spleens of MRL-lpr/lpr and C57BL/6.lpr mice occurred in the presence of limited infiltration with B220+, L3T4- T cells. Mixing experiments indicated that B220+ T cells were not suppressive. Furthermore, lpr spleen cells enriched for L3T4+ cells and depleted of sIg+, B220+ and Lyt-2+ cells demonstrated reductions in lymphokine production which were comparable to those seen in unfractionated preparations. Spleen cells from C57BL/6.lpr mice, enriched for L3T4+ cells, were also markedly impaired in a mixed leukocyte reaction in response to stimulator cells from the class II major histocompatibility complex mutant bm12. The results indicate that the aberrations in lymphokine production and proliferation in the spleen cells of lpr mice involve not only B220+ T cells but also L3T4+ cells and suggest a potential role for the L3T4+ subset in the pathogenesis of lupus in lpr-bearing mice.

Interleukin 2 is a proliferative signal for B cells from autoimmune mice.

T cells from murine lupus strains manifest complex defects in interleukin 2 (IL 2) production and receptor expression. The capacity of B cells from such mice to utilize IL 2 as a growth factor has not been previously reported and is examined herein. Anti-Thy-1.2 plus complement-treated spleen cells from 6-8-week-old autoimmune MRL-lpr/lpr mice and from age and sex-matched immunologically normal CBA/J mice were cultured with lipopolysaccharide (LPS) for 36 h and analyzed for the expression of IL 2 receptors using the monoclonal antibody 7D4. The percentage of B cells expressing IL 2 receptors was comparable in MRL-lpr/lpr and CBA/J mice. In contrast to those from CBA/J, BALB/c and (BALB/c X NZW)F1 mice, LPS-stimulated B cells from MRL-lpr/lpr and from (NZB X NZW)F1 mice were capable of proliferating in response to IL 2. Fractionation of MRL-lpr/lpr B cells using Percoll gradient density separation demonstrated that the IL 2-responsive population consisted predominantly of large cells. In addition, unfractionated B cells from MRL-lpr/lpr mice were found to be substantially more responsive to IL 2 than those from CBA/J and BALB/c mice following activation with anti-immunoglobulin plus LPS. The hyper-responsiveness to IL 2 may be a consequence of the state of activation of autoimmune B cells and is of potential importance in the pathogenesis of systemic lupus erythematosus.