T cell-induced expression of membrane IgG by 70Z/3 B cells

To study T cell regulation of B cell isotype differentiation, we determined the capacity of clonal T cell populations (hybridomas derived by fusing BW5147 with Con A-activated Peyer's patch (PP) and spleen T cells) to induce "downstream" isotype expression by the pre-B cell lymphoma 70Z/3. In initial studies, we found that 70Z/3 B cells cultured in the presence of LPS (1 microgram/ml) expressed membrane IgM (mIgM) but not membrane IgG (mIgG). In contrast, 70Z/3 B cells cultured with HAJ-3 T cells, a PP-derived T cell hybridoma (as well as other similarly derived PP and spleen hybridomas), or with HAJ-3 T cells plus LPS do express mIgG. Such expression occurred in spite of mitomycin C-induced blockage of cell proliferation, and is observed in 70Z/3 B cell subclones cultured with HAJ-3 T cells. For these reasons, it is not due to selective expansion of a small pre-switched mIgG-bearing 70Z/3 B cell subpopulation. In other studies it was shown that 70Z/3 B cells expressing mIgG after induction by HAJ-3 T cells continue to express mIgM and do not secrete IgG. Finally, exposure of 70Z/3 B cells to the macrophage factor IL 1 and the T cell factors IL 2, BSF-pl, and BCGF-II present in EL-4 cell supernatants did not result in mIgG expression. On the basis of these studies, we conclude that a clonal B cell population expressing mIgM can be induced by T cells to co-express mIgG. Because the B cells do not express mIgG unless exposed to T cells, this represents a T cell-induced isotype switch.     

Expression of CD14 corrects the slow response to lipopolysaccharide in the 1B8 mutant of the B cell lymphoma 70Z/3

 B cells and macrophages both activate NF-κB/Rel in response to lipopolysaccharide (LPS), but differ in sensitivity to LPS and in downstream genes that are activated. CD14 is a high-affinity receptor for LPS found on macrophages, but not B cells. We expressed human CD14 (hCD14) in the mouse B lymphoma, 70Z/3, and a mutant, 1B8, which responds slowly to LPS, to test whether expression of hCD14 could correct or bypass the defect in 1B8 cells. We compared the timing and extent of known responses to LPS in 70Z/3 cells and the 1B8 mutants. The hCD14⁺ 1B8 and 70Z/3 cells responded more rapidly and were sensitive to 100-fold lower levels of LPS than their untransfected counterparts. Degradation of the IκB-α and -β molecules and translocation of the NF-κB/Rel complexes into the nucleus were more rapid and the steady-state levels of Igk mRNA and mIgM on the cell surface were markedly increased in cells that expressed hCD14. The LPS response of the hCD14⁺ 1B8 and 70Z/3 cells showed subtle differences. In the 1B8 hCD14 cells, the p50/p50 complexes were never abundant in nuclear extracts, and degradation of IκB-β was slower than in hCD14 70Z/3 cells. This partial correction of the 1B8 phenotype suggests that the defective component in 1B8 participates in the CD14 signaling pathway and could include the B-cell LPS receptor itself. Received: 3 June 1997 / Revised: 26 June 1997     

Lipopolysaccharide-unresponsive mutant pre-B-cell lines blocked in NF-kappa B activation.

NF-kappa B activation is a crucial late step in the induction of immunoglobulin kappa light-chain gene expression in pre-B cells by lipopolysaccharide (LPS). We have analyzed NF-kappa B activation in three independent mutant lines of 70Z/3 pre-B cells which are unresponsive to LPS. All three variant cell lines failed to activate NF-kappa B when induced with LPS or the phorbol ester 12-O-tetradecanoylphorbol 13-acetate. However, all three cell lines contained functional NF-kappa B, as revealed by detergent treatment of cytoplasmic extracts. Moreover, cycloheximide induced limited activation of NF-kappa B comparable to that in wild-type 70Z/3 pre-B cells in two of the three variant lines. These results indicate that the mutations blocking kappa gene induction in these variant 70Z/3 pre-B-cell lines affect NF-kappa B activation.     

Up-regulation and down-regulation of cell surface and mRNA expression of CD5 antigen by various humoral factors on murine 70Z/3 pre-B cell leukemia cell line: IL-4 down-regulates CD5 antigen expression.

CD5, a pan-T cell antigen, is expressed on a minor subset of normal B lymphocytes and on cells of most B lineage tumors or transformed B cells in both man and animal models. In the present study, the effects of various humoral factors on CD5 expression by cells of a subcloned 70Z/3 murine pre-B leukemia cell line were investigated. Among the humoral factors studied, only LPS up-regulated CD5 expression on 70Z/3 cells (three- to fourfold) in a dose-dependent manner. However, this up-regulatory effect of LPS was not observed when cells were cultured in serum-free medium. NZB-serum factor (NZB-SF), a cytokine we have identified and shown to enhance the maturation and proliferation of immature B cells, synergistically enhanced CD5 expression in the presence of suboptimal doses of LPS. IL-4 down-regulated CD5 expression by 70Z/3 cells induced by LPS or LPS plus NZB-SF in a dose-dependent manner. IL-4 also suppressed spontaneous CD5 expression by 70Z/3 cells. No other cytokine tested showed an inhibitory effect. LPS, IFN-gamma, NZB-SF, and IL-1 enhanced sIg expression on 70Z/3 cells and their action on sIg expression was not inhibited by IL-4. Thus, the down-regulatory action of IL-4 on CD5 expression appeared specific for this antigen. IFN-gamma, which inhibits IL-4 induced CD23 and DR expression on B cells, does not abolish the down-regulatory action of IL-4 on CD5 expression by 70Z/3 cells. Changes in mRNA levels on coding CD5 were also examined following the incubation of 70Z/3 cells (24 hr) in the presence of humoral factors which can influence CD5 Ag expression. The levels of mRNA for CD5 Ag were moderately increased in the presence of LPS and NZB-SF. IL-4 appeared to suppress the actions of NZB-SF and LPS at least in part by reducing the levels of mRNA encoding CD5.     

Murine model of immune-mediated rejection of the acute lymphoblastic leukemia 70Z/3

70Z/3 is a murine pre-B cell leukemia line derived from BDF(1) mice and has been used in the study of signaling pathways in B cells. 70Z/3 cells were initially found to cause widespread disease upon injections in animals. We have isolated 70Z/3 variants divergent in their capacity to lead to morbidity after injections. One variant, 70Z/3-NL, elicits an immune response protecting the animal from tumor growth. Another variant, 70Z/3-L, does not induce an effective immune response and causes morbidity. We demonstrated that both CD4(+) and CD8(+) T cells are required for the rejection of 70Z/3-NL cells. Interestingly, the immune response generated against 70Z/3-NL cells was found to protect against a challenge with the lethal variant, 70Z/3-L. This indicates that although both lines can be recognized and killed by the immune system, only 70Z/3-NL is capable of inducing a protective response. Further observations, using subclones isolated from 70Z/3-NL, demonstrated that immune recognition of a portion of the cells was sufficient for protection. Depletion of CD4(+) and CD8(+) T cells in animals injected previously with 70Z/3-NL cells showed that T cells, and not Abs, were required for the maintenance of the protection initiated by 70Z/3-NL. We tested the capacity of 70Z/3-NL cells to treat mice challenged with 70Z/3-L. We can delay injections of 70Z/3-NL and still provide protection for the animals. We have a model of immune-mediated rejection which will allow us to dissect the requirements for the initiation of immune responses against an ALL tumor cell line.     

Differential effect of antigen-nonspecific T-cell factors and lipopolysaccharide on the Ia antigens and surface immunoglobulins of BALB/c lymphoma cell lines

In order to study the mechanism of B-cell differentiation using B lymphoid tumor cells as models, we investigated the effects of antigen-nonspecific T-cell factors in combination with lipopolysaccharide (LPS) on the expression of surface markers on B lymphoid cell lines. This study demonstrated that culture supernatant from concanavalin A-activated spleen cells (CAS) gave 2- to 3.5-fold enhancement of the expression of Ia antigens. The effect of CAS was dose dependent and as little as 2% CAS gave maximum enhancement of Ia antigen expression. The CAS effect was due to concanavalin A-activated cell products and was not due to the concanavalin A. The effects of allogeneic effect factor (AEF) on Ia antigens were similar to those of CAS. In contrast to CAS and AEF, LPS did not affect the expression of Ia antigens on ×16C 8.5. LPS enhanced 1.5- to 3-fold the expression of sIgM on this cell line. The expression of sIgM was minimally affected by T-cell factors; CAS induced 20 to 70% enhancement of sIgM expression while AEF induced no significant effects. This study showed that antigen-nonspecific factors (CAS and AEF) influenced mainly Ia expression on the B-cell lymphoma, ×16C 8.5, while LPS selectively affected sIgM expression. Therefore, it was concluded that the mechanisms by which B cells are activated by T-cell factors and mitogens are different.     

NADPH oxidase-2 derived ROS dictates murine DC cytokine-mediated cell fate decisions during CD4 T helper-cell commitment

NADPH oxidase-2 (Nox2)/gp91(phox) and p47(phox) deficient mice are prone to hyper-inflammatory responses suggesting a paradoxical role for Nox2-derived reactive oxygen species (ROS) as anti-inflammatory mediators. The molecular basis for this mode of control remains unclear. Here we demonstrate that IFNγ/LPS matured p47(phox-/-)-ROS deficient mouse dendritic cells (DC) secrete more IL-12p70 than similarly treated wild type DC, and in an in vitro co-culture model IFNγ/LPS matured p47(phox-/-) DC bias more ovalbumin-specific CD4(+) T lymphocytes toward a Th1 phenotype than wild type (WT) DC through a ROS-dependent mechanism linking IL-12p70 expression to regulation of p38-MAPK activation. The Nox2-dependent ROS production in DC negatively regulates proinflammatory IL-12 expression in DC by constraining p38-MAPK activity. Increasing endogenous H(2)O(2) attenuates p38-MAPK activity in IFNγ/LPS stimulated WT and p47(phox-/-) DC, which suggests that endogenous Nox 2-derived ROS functions as a secondary messenger in the activated p38-MAPK signaling pathway during IL-12 expression. These findings indicate that ROS, generated endogenously by innate and adaptive immune cells, can function as important secondary messengers that can regulate cytokine production and immune cell cross-talk to control during the inflammatory response.     

Aberrant trafficking of the B cell receptor Ig-alpha beta subunit in a B lymphoma cell line

The B cell Ag receptor (BCR) has two important functions: first, it binds and takes up Ag for presentation to T lymphocytes; and second, it transmits signals that regulate B cell development. Normal expression of the BCR requires the association of the Ag binding subunit, membrane IgM (mIgM), with the signaling component, the Ig-alpha beta heterodimer. After assembly in the endoplasmic reticulum, the intact BCR travels through the secretory pathway to the cell surface. In this paper, we report two variants of the B lymphoma cell lines, WEHI 279 and WEHI 231, that have both lost the ability to express mu heavy chain and consequently do not express mIgM. However, these variants do express the Ig-alpha beta heterodimer. In one variant, WEHI 279*, the Ig-alpha beta remained trapped intracellularly in the absence of mIgM. The other variant, 303.1.5.LM, expressed an aberrantly glycosylated Ig-alpha beta on the cell surface that was capable of signaling after cross-linking with anti-Ig-beta Abs. Further characterization uncovered a point mutation in the 303.1.5.LM mb1 gene that would change a proline for a leucine in the extracellular domain of Ig-alpha. The 303.1.5.LM Ig-alpha beta could not associate with a wild-type mIgM after mu heavy chain was reconstituted by DNA transfection. Thus, this mutation could define a region of the Ig-alpha polypeptide that is important for recognition by the endoplasmic reticulum quality control system, for association with glycosylating enzymes, and for the association of Ig-alpha beta subunits with mIgM subunits to create a complete BCR complex.     

Induction and regulation of immunoglobulin expression in a murine pre-B cell line, 70Z/3. I. Cell cycle-associated induction of sIgM expression and kappa-chain synthesis in 70Z/3 cells by LPS stimulation

Stimulation of a murine pre-B cell line, 70Z/3, with LPS induced sIgM expression and an increase of L kappa-chain mRNA within 12 hr. In synchronized 70Z/3 cells, the cell division cycle was 10 to 11 hr and LSP-stimulation did not affect the cell division cycle. LPS-signals given at G1/S boundary induced sIgM expression in the G2 to M-phase. On the other hand, LPS-signals given after M-phase did not induce sIgM expression. Inhibition of cell division with demecorcin did not affect sIgM expression in the M-phase. Induction of intracellular kappa-chain synthesis was also observed in the G2 to M-phase when the LPS-stimulation was provided at G1/S boundary, but LPS-signals given after the M-phase did not induce de novo synthesis of kappa-chain. These results showed that LPS-induced sIgM expression and synthesis of kappa-chain were cell cycle-related events and sIgM expression was associated wih de novo synthesis of kappa-chain.     

Interleukin 1-mediated induction of kappa-light chain synthesis and surface immunoglobulin expression on pre-B cells

In this study we have examined the effect of interleukin 1 (IL 1) on the maturation of normal and neoplastic pre-B cells. We have found that IL 1 can enhance the in vitro functional maturation of surface immunoglobulin negative (sIg-) pre-B cells from normal bone marrow. In addition, IL 1 specifically induced sIg expression on an established pre-B cell line, 70Z/3. These effects of IL 1 were obtained with the same concentrations of IL 1 that are effective in assays for T cell proliferation and functional activation. Previous studies by other investigators have demonstrated that LPS can also induce the expression of sIg on 70Z/3 cells. The stimulatory effect of LPS was dependent on the stimulation of kappa-light chain synthesis, the synthesis of mu-chains being constitutive. Our results indicate that IL 1 may also enhance sIg expression via the induction of kappa-light chain synthesis. The stimulatory effect of IL 1 was not due to contaminating LPS in the IL 1 preparations, because removal of the IL 1 by using specific antibodies against IL 1 and fixed Staphylococcus aureus cells resulted in the disappearance of kappa-chain inducing activity. In addition to IL 1, a pH 2-sensitive mediator(s) present in concanavalin A (Con A)-stimulated spleen cell supernatants was also shown to induce kappa-chain synthesis and the appearance of sIg on 70Z/3 cells. Removal of IL 1 or the inhibition of any contaminating LPS activity with polymyxin B did not diminish the activity of the pH 2-sensitive Con A supernatant factor(s). On the basis of our findings, we have concluded that IL 1 may enhance antibody responses by not only increasing the number of helper T cells but also by stimulating the maturation of B cell precursors.     

Elevated tumor-associated antigen expression suppresses variant peptide vaccine responses

Variant peptide vaccines are used clinically to expand T cells that cross-react with tumor-associated Ags (TAA). To investigate the effects of elevated endogenous TAA expression on variant peptide-induced responses, we used the GP70 TAA model. Although young BALB/c mice display T cell tolerance to the TAA GP70(423-431) (AH1), expression of GP70 and suppression of AH1-specific responses increases with age. We hypothesized that as TAA expression increases, the AH1 cross-reactivity of variant peptide-elicited T cell responses diminishes. Controlling for immunosenescence, we showed that elevated GP70 expression suppressed AH1 cross-reactive responses elicited by two AH1 peptide variants. A variant that elicited almost exclusively AH1 cross-reactive T cells in young mice elicited few or no T cells in aging mice with Ab-detectable GP70 expression. In contrast, a variant that elicited a less AH1 cross-reactive T cell response in young mice successfully expanded AH1 cross-reactive T cells in all aging mice tested. However, these T cells bound the AH1/MHC complex with a relatively short half-life and responded poorly to ex vivo stimulation with the AH1 peptide. Variant peptide vaccine responses were also suppressed when AH1 peptide is administered tolerogenically to young mice before vaccination. Analyses of variant-specific precursor T cells from naive mice with Ab-detectable GP70 expression determined that these T cells expressed PD-1 and had downregulated IL-7Rα expression, suggesting they were anergic or undergoing deletion. Although variant peptide vaccines were less effective as TAA expression increases, data presented in this article also suggest that complementary immunotherapies may induce the expansion of T cells with functional TAA recognition.     

Cholera toxin promotes B cell isotype differentiation

Cholera toxin (CT) is a powerful oral immunogen and adjuvant that elicits strong IgG and IgA antibody responses. In our study we investigated whether this property of CT was associated with an effect on B cell isotype differentiation. Initially, we determined the effect of CT on normal LPS-induced Peyer's patch B cells and found that whereas CT is strongly inhibitory of IgM production, it increases by approximately three-fold the number and frequency of IgG- and IgA-producing cells. Subsequently, using cell sorting technology, we demonstrated that CT acts on membrane (m)IgM+, mIgG/mIgA- B cells rather than mIgG/mIgA+ B cells. In addition, we showed that CT does not cause selective inhibition of mIgM, or enhancement of mIgG/mIgA B cell proliferation. In parallel studies we determined the effect of CT on the differentiation of a clonal B cell population, CH12.LX cells, i.e., a population comprised mainly of mIgM+ cells (98%) admixed with a small subpopulation of mIgA+ cells (2%). Here we found that CT (in the absence of LPS) causes a rapid decrease (24 h) in the intensity of mIgM expression as well as a marked increase in the size of the subpopulation expressing mIgA. In addition, we found that CT (in the presence of LPS), inhibits CH12.LX IgM production while increasing the absolute number and frequency of IgA-producing cells. In contrast, CT inhibits IgA production by CH12.LX.A2 cells, a subclone of CH12.LX cells that bears only IgA. Finally, we demonstrated that CT is equally inhibitory of the proliferation of CH12.LX cells and CH12.LX.A2 cells. Taken together, these effects of CT on normal B cells and a clonal B cell line indicate that CT induces substantial numbers of mIgM+ cells to undergo isotype differentiation into mIgG+ or mIgA+ B cells. In a final series of studies we showed that the effect of CT on isotype differentiation was mimicked by the B subunit of CT, i.e., the subunit that does not activate intracellular adenylate cyclase; thus the induction of isotype differentiation by CT is not mediated by a perturbation in cAMP level.