CD19 regulation of human B cell responses. B cell proliferation and antibody secretion are inhibited or enhanced by ligation of the CD19 surface glycoprotein depending on the stimulating signal used.

The regulation of human B cell proliferation and differentiation by the CD19 surface glycoprotein was investigated. As expected, proliferation induced by costimulation with anti-IgM plus IL-4 or IL-2, or with G28.8 antibody plus IL-4 was inhibited by antibody ligation of CD19. In contrast, proliferation of tonsillar B cells to mitogenic doses of PMA (5 ng/ml) or to EBV were enhanced, and proliferation of B cell lines to BCGF(low) was unaffected. Similarly, specific antibody responses by tonsillar B cells to influenza virus, and Ig secretion by the CESS lymphoblastoid cell line in response to IL-6 were inhibited, whereas polyclonal Ig production in response to EBV was enhanced. These results show that human B cell responses may be inhibited or enhanced by CD19 depending on the stimulating signal used. The difference in response to CD19 ligation did not depend on whether proliferation or differentiation was being measured, or whether stimulation was by surface Ig. In experiments using PMA as a T cell independent mitogen, it was found that ligation of CD19 inhibited proliferation of B cells costimulated with low doses of PMA plus G28.5 (CD40) antibody, but enhanced the response to higher (mitogenic) doses with or without costimulation with G28.5. The change from inhibition to enhancement occurred over a very small increase in PMA dose (0.5-1.0 ng/ml) that corresponded exactly to the lowest dose required for mitogenic activity. Finally, we showed that CD19 ligation inhibited the increase in surface expression of CD23, but not IgM, induced by IL-4, showing that CD19 ligation can have opposed effects on different responses to the same signal. Together our results suggest that CD19 activation of human B cells interacts with other signaling events to enhance or inhibit the subsequent response.     

CD19 monoclonal antibody HD37 inhibits anti-immunoglobulin-induced B cell activation and proliferation

The 95 Kd CD19 antigen is the broadest lineage specific surface marker for B cells: it is present on the surface of virtually all B lymphocytes, including early B progenitor cells. In this study we have evaluated the function of the CD19 antigen by using the CD19 mAb HD37. Binding of HD37 mAb to B cells at low doses (0.5 microgram/ml) induced a strong inhibition of the proliferative response to anti-Ig. This inhibition was not mediated by the Fc portion of the antibody, since F(ab')2 fragments were as effective as the whole antibody. Both dose-response curve analysis and experiments in which a cross-linking second step anti-mouse antibody was added suggested that cross-linking of CD19 antigens was necessary for optimal inhibition. Early phases in B cell activation were affected by the HD37 mAb: it significantly reduced the number of cells that left G0 and entered the G1 phase of the cell cycle upon triggering with anti-mu. The increase in free intracellular ionized calcium [Ca2+]i that is induced by anti-mu was also consistently reduced by CD19 mAb. Cross-linking was also crucial for this effect, suggesting that a causal relationship may exist between the inhibition of anti-Ig-mediated [Ca2+]i fluxes and inhibition of proliferation. A variable but clear increase in [Ca2+]i levels followed cross-linking of CD19 antigens by specific mAb. This evidence suggests that CD19 molecules may function in the downregulation of B cell growth and proliferation.     

IgE enhances antibody and T cell responses in vivo via CD23+ B cells

IgE Abs, passively administered together with their specific Ag, can enhance the production of Abs recognizing this Ag by >100-fold. IgE-mediated feedback enhancement requires the low affinity receptor for IgE, CD23. One possible mechanism is that B cells take up IgE-Ag via CD23 and efficiently present Ag to Th cells, resulting in better Ab responses. To test whether IgE Abs have an effect on Th cells in vivo, mice were adoptively transferred with CD4+ T cells expressing a transgenic OVA-specific TCR, before immunization with IgE anti-TNP (2,4,6-trinitrophenyl) plus OVA-TNP or with OVA-TNP alone. IgE induced a 6- to 21-fold increase in the number of OVA-specific T cells. These cells acquired an activated phenotype and were visible in splenic T cell zones. The T cell response peaked 3 days after immunization and preceded the OVA-specific Ab response by a few days. Transfer of CD23+ B cells to CD23-deficient mice rescued their ability to respond to IgE-Ag. Interestingly, in this situation also CD23-negative B cells produce enhanced levels of OVA-specific Abs. The data are compatible with the Ag presentation model and suggest that B cells can take up Ag via "unspecific" receptors and activate naive T cells in vivo.     

I-kappa B kinase beta is critical for B cell proliferation and antibody response.

The NF-kappaB proteins are critical in the regulation of the immune and inflammatory response. Stimulation of the NF-kappaB pathway leads to increases in I-kappaB kinase beta (IKKbeta) kinase activity to result in the enhanced phosphorylation and degradation of I-kappaB and the translocation of the NF-kappaB proteins from the cytoplasm to the nucleus. In this study, a dominant-negative IKKbeta mutant expressed from the IgH promoter was used to generate transgenic mice to address the role of IKKbeta on B cell function. Although these transgenic mice were defective in activating the NF-kappaB pathway in B cells, they exhibited no defects in B lymphocyte development or basal Ig levels. However, they exhibited defects in the cell cycle progression and proliferation of B cells in response to treatment with LPS, anti-CD40, and anti-IgM. Furthermore, selective defects in the production of specific Ig subclasses in response to both T-dependent and T-independent Ags were noted. These results suggest that IKKbeta is critical for the proliferation of B cells and the control of some aspects of the humoral response.     

Inverse relationship between proliferation and differentiation in a human TNP-specific B cell line. Cell cycle dependence of antibody secretion

Studies of an EBV-transformed and TNP-specific human B cell line revealed that, unlike myeloma or hybridoma cell lines that consist mainly of fully differentiated cells, most of the cloned EBV-transformed cells were not fully differentiated, as judged by inability to bind TNP-SRBC and to secrete anti-TNP antibody. The minority of more differentiated cells were selected by TNP-SRBC rosetting. They were found to proliferate to a lesser extent than nonrosetting cells and to contain increased numbers of antibody-secreting cells. This inverse relationship between proliferation and differentiation was also shown to be cell cycle related in that the TNP-SRBC rosetting cells resided, to a greater extent than the nonrosetting cells, in the G1 phase of the cell cycle. The finding that the G1 phase of the cell cycle was associated with differentiation into anti-TNP secreting cells was confirmed by demonstrating that treatment with hydroxyurea, which arrests the cells in G1, resulted in decreased proliferation and an increased proportion of antibody-secreting cells. Similarly, addition of phorbol ester resulted in increased antibody secretion and decreased proliferation, suggesting a role for protein kinase C in this differentiation pathway. The strategy of increasing the number of antibody-producing cells in this human EBV line, by promoting differentiation of the cells in G1, may be relevant to the large scale production of specific human mAb for the treatment and diagnosis of human diseases.     

Regulation of an idiotype+ B cell lymphoma. Effects of antigen and anti-idiotopic antibodies on proliferation and Ig secretion

The B cell surface Ig molecule plays an important regulatory role in delivering inductive/tolerogenic signals to the cell. In this paper, the effect of Ag and anti-idiotopic antibodies on the in vitro proliferation and Ig secretion of a B cell tumor was studied. The tumor (BCL1), which had been transfected with the TEPC-15 VH and VL Ig genes, expresses surface Ig and secretes antibody that binds the hapten phosphorylcholine. We found that Ag (C polysaccharide and phosphorylcholine carrier Ag) and two different anti-idiotopic antibodies, in the absence of T cells, all inhibited the proliferation of the T15+ transfectant cell line. The anti-idiotopic antibodies, but not Ag, also inhibited the secretion of T15 Ig by this cell line, suggesting different functional roles for Ag vs anti-Id in the regulation of B cell inactivation. The inhibition of secretion and proliferation appears to be cell cycle phase related. In addition, mouse rIL-4 could override the inhibition of proliferation induced in these studies. These phenomena, demonstrating that binding of surface Ig can result in the transduction of negative growth signals to a B cell tumor, can be viewed as a manifestation of immunologic tolerance. These findings collectively demonstrate that Ag and anti-Id mediate different signals to B cells via interaction with the surface Ig. Because of the monoclonal nature of the T15 transfectant and the anti-idiotypic antibodies, this system can be used to investigate the underlying molecular reactions involved in the B cell response and induction of tolerance.     

Cutting edge: BAFF regulates CD21/35 and CD23 expression independent of its B cell survival function

Herein we demonstrate that B cell-activating factor of the TNF family (BAFF), a B cell survival factor, also regulates CD21/35 and CD23 expression. BAFF blockade in wild-type mice down-modulates CD21/35 and CD23 on B cells while survival remains intact, and BAFF exposure causes elevated CD21/35 and CD23 expression. Similar down-modulation is observed in bcl-2-transgenic mice treated with a BAFF inhibitor. This is the first evidence that BAFF has a function independent of B cell survival. Reports using CD21/35 and CD23 expression to assess splenic B cell subsets in BAFF-null mice concluded a lack of B cells beyond the immature stage. Since CD21/35 and CD23 are inadequate for delineating B cell subpopulations in BAFF-null mice, we used expression of BAFF-R and several B cell markers to identify more mature splenic B cells in these mice. These data broaden our understanding of BAFF function and correct the view that BAFF-null mice lack mature B cells.     

Induction of human T cell proliferation by a monoclonal antibody to CD5.

A mouse mAb, TS 43, which recognized the human CD5 molecule, was found to induce the proliferation of human peripheral blood T cells. TS 43 mAb precipitated from 125I-radiolabeled T cells a 67-kDa band, which comigrated with the 67-kDa band precipitated by the anti-CD5 mAb OKT1. Preclearing of cell lysates with OKT1 mAb abolished the capacity of TS 43 mAb to precipitate radiolabeled material from T cell lysates. Furthermore, a mouse T cell hybridoma transfected with human CD5 was stained by TS 43 mAb. T cell proliferation mediated by TS 43 mAb was monocyte dependent, and was accompanied by IL-2R expression and by IL-2 synthesis. T cell activation by TS 43 mAb involved a rise in intracellular calcium level (CA2+)i and was dependent on the expression of the TCR/CD3 complex because no rise in (Ca2+)i was observed in a TCR-beta-deficient Jurkat T cell mutant. This study indicates that CD5 should be added to the list of surface molecules that can signal T cells to proliferate.     

Identification of the B cell derived T cell colony promoting activity to soluble CD23.

We previously reported that supernatants of phytohemagglutinin (PHA)-stimulated normal human B cells (NBCsup) contain a T cell colony promoting activity. NBCsup were able to (a) increase the number of secondary colonies generated under PHA and interleukin-2 (IL-2) stimulation by peripheral blood-derived primary T colony cells, (b) enhance the ability of CD4+ but not CD8+ peripheral blood T cells to form agar colonies in the presence of PHA and IL-2 and (c) support in vitro differentiation of CD2-3-4-8- prothymocytes into CD2+3+4+ T cells. This activity was therefore refered to as Prothymocyte Differentiating Activity (PTDA). Subsequent studies pointed to striking biochemical and cell source homologies between this B cell derived factor and the 25-kDa soluble CD23 (sCD23). sCD23 has been recently found to display prothymocyte differentiating activity.     

Effects of cholera toxin on human B cells. Cholera toxin induces B cell surface DR expression while it inhibits anti-mu antibody-induced cell proliferation

Experiments were performed to investigate the effect of cholera toxin (CT) on human B cell function. Highly purified (greater than 98% CD20+) human peripheral blood B cells were exposed to CT in the presence or absence of anti-mu antibody. Treatment of highly purified B cells with CT stimulated enhanced expression of surface DR molecules, whereas it did not enhance expression of other B cell surface activation markers including transferrin or IL-2R. Neither the A nor the B subunits of CT by themselves enhanced the expression of surface DR Ag. In addition, 8-bromo-cAMP alone or in combination with the B subunit did not increase the expression of human B cell surface DR Ag. These findings suggest that neither elevation of cAMP nor binding to GM1 ganglioside are sufficient to stimulate this activation parameter in B cells. Associated with CT-mediated enhanced expression of MHC class II molecules we found that CT-treated B cells also served as stronger stimulators, compared with control cells, of both autologous and allogeneic MLR responses in peripheral blood T cells. Although CT stimulated early events in B cell activation, it inhibited anti-mu antibody-induced B cell thymidine incorporation by 55 to 75%. Inhibitory effects of CT were observed even when CT was added to cultures as late as 36 h after the addition of the anti-mu antibody. These results suggest that CT has both a stimulatory and inhibitory effect on human B cells and that the stimulatory effect may be mediated via a cAMP-independent mechanism.     

Leukotriene B4 potentiates the expression and release of Fc epsilon RII/CD23, and proliferation and differentiation of human B lymphocytes induced by IL-4

This study documents the influence of leukotriene (LT) B4 on human B lymphocyte responses. Incubation of freshly isolated B lymphocytes with LTB4, but not LTC4, induced a slight but significant, time- and dose-dependent increase in the surface expression of Fc epsilon RII/CD23 and class II MHC Ag and in the release of soluble CD23. These changes were maximal at 10 nM LTB4 after an incubation period of 48 h. When B lymphocytes were preactivated in vitro with Staphylococcus aureus Cowan strain I (SAC), neither LTB4 nor LTC4 was able to promote proliferation and/or IgG and IgM secretion. In contrast, when resting B lymphocytes were stimulated with a suboptimal concentration (3 U/ml) of IL-4, LTB4, but not LTC4, potentiated both the Fc epsilon RII/CD23 and the class II MHC antigen expression, and the release of soluble CD23 in a dose-dependent manner, without affecting the kinetics of these responses. Furthermore, LTB4, but not LTC4, amplified both the proliferative response and the IgG and IgM secretion induced by addition of a suboptimal dose of IL-4 (3 U/ml) to SAC-preactivated B lymphocytes. Again, LTB4 did not modify the kinetics of the proliferative response promoted by IL-4. Although LTB4 potentiated IL-4-induced IgG and IgM secretion from SAC-activated B lymphocytes, no production of IgE was observed. These data indicate that LTB4 could play a regulatory role in the modulation of IL-4-induced signaling in human B lymphocytes.     

CD40, but not CD154, expression on B cells is necessary for optimal primary B cell responses

CD40 is an important costimulatory molecule for B cells as well as dendritic cells, monocytes, and other APCs. The ligand for CD40, CD154, is expressed on activated T cells, NK cells, mast cells, basophils, and even activated B cells. Although both CD40(-/-) and CD154(-/-) mice have impaired ability to isotype switch, form germinal centers, make memory B cells, and produce Ab, it is not entirely clear whether these defects are intrinsic to B cells, to other APCs, or to T cells. Using bone marrow chimeric mice, we investigated whether CD40 or CD154 must be expressed on B cells for optimal B cell responses in vivo. We demonstrate that CD40 expression on B cells is required for the generation of germinal centers, isotype switching, and sustained Ab production, even when other APCs express CD40. In contrast, the expression of CD154 on B cells is not required for the generation of germinal centers, isotype switching, or sustained Ab production. In fact, B cell responses are completely normal when CD154 expression is limited exclusively to Ag-specific T cells. These results suggest that the interaction of CD154 expressed by activated CD4 T cells with CD40 expressed by B cells is the primary pathway necessary to achieve B cell activation and differentiation and that CD154 expression on B cells does not noticeably facilitate B cell activation and differentiation.     

Monoclonal antibody to CD9 inhibits platelet-induced human endothelial cell proliferation

Platelets are anucleate cytoplasmic fragments derived from bone marrow megakaryocytes, and endothelial cells constitute the barrier between bloodstream and adjacent tissues. Although platelets are thought to regulate the biological functions of endothelial cells, the molecular mechanisms involved are poorly understood. With human umbilical vein endothelial cells and freshly isolated platelets, we established an in vitro model of platelet-induced endothelial cell proliferation. Platelets stimulated endothelial cell proliferation in a dose-dependent manner and transwell experiments with semi-permeable membranes suggested that direct cell-to-cell contacts were required. We developed mAbs against platelets and selected a mAb that blocks their proliferative effect. We purified the antigen by immunoprecipitation and identified it by Q-TOF MS analysis as the tetraspanin CD9. Since both platelets and endothelial cells expressed CD9 strongly on their surfaces we carried out a pre-treatment experiment that showed that CD9 molecules on the endothelial cells participate in the mitogenic effect of the platelets. The inhibitory effect of our mAb was comparable to that of a well-known functional anti-CD9 mAb. These results suggest that the tetraspanin CD9 plays an important role in endothelial regeneration.     

Functional studies on B cell hybridomas with B cell surface antigens. I. Effects of anti-immunoglobulin antibodies on proliferation and differentiation

B cell hybridomas with Ia and IgM molecules on the cell membrane were treated with either purified goat anti-mouse mu antibody (anti-mu) or monoclonal rat anti-mouse IgM antibody (anti-IgM). The spontaneous uptake of [3H] thymidine by these cells was markedly inhibited by both reagents. These hybrid cells could be induced to differentiate into IgM-secreting cells in the presence of these reagents at high frequency. Furthermore, the induction of IgM secretion by B cell hybridomas treated with these antibodies was completely T cell independent, and cell division was not required for the differentiative response to anti-mu. In addition, F(ab')2 fragments of anti-mu showed more effects on proliferation and differentiation of these cells than intact anti-mu. Interestingly, TH2.54, a subline of B cell hybridomas, could generate IgG2a production as well as IgM when incubated with anti-mu. These findings suggest very strongly that the interaction of either goat anti-mu or monoclonal rat anti-IgM with surface IgM molecules on the cell membrane of the B cell hybridomas inhibits in vitro spontaneous proliferation, and results in providing signals for differentiation into Ig-secreting cells without T cell factors.     

Dendritic cell-dependent inhibition of B cell proliferation requires CD22

Recent studies have shown that dendritic cells (DCs) regulate B cell functions. In this study, we report that bone marrow (BM)-derived immature DCs, but not mature DCs, can inhibit BCR-induced proliferation of B cells in a contact-dependent manner. This inhibition is overcome by treatment with BAFF and is dependent on the BCR coreceptor CD22; however, it is not dependent on expression of the CD22 glycan ligand(s) produced by ST6Gal-I sialyltransferase. We found that a second CD22 ligand (CD22L) is expressed on CD11c(+) splenic and BM-derived DCs, which does not contain ST6Gal-I-generated sialic acids and which, unlike the B cell-associated CD22L, is resistant to neuraminidase treatment and sodium metaperiodate oxidation. Examination of splenic and BM B cell subsets in CD22 and ST6Gal-I knockout mice revealed that ST6Gal-I-generated B cell CD22L plays a role in splenic B cell development, whereas the maintenance of long-lived mature BM B cells depends only on CD22 and not on alpha2,6-sialic acids produced by ST6Gal-I. We propose that the two distinct CD22L have different functions. The alpha2,6-sialic acid-containing glycoprotein is important for splenic B cell subset development, whereas the DC-associated ST6Gal-I-independent CD22L may be required for the maintenance of long-lived mature B cells in the BM.     

Effects of cytochalasin B on pancreatic acinar cell structure and secretion

Summary The effects of cytochalasin B (CB) on pancreatic structure and amylase release were studied by use of pancreatic fragments, isolated acini and isolated acinar cells. In pancreatic fragments and isolated acini CB caused the disappearance of microfilaments underlying the apical plasma membrane, loss of apical microvilli and luminal swelling, the last of which was greatly enhanced by addition of protein secretagogues. CB had no effect on basal amylase release but inhibited bethanechol-stimulated amylase in both fragments and acini. Isolated acinar cells, while retaining overall polarity, had lost most of the apical specialization including the microfilament and microvillous complex. Cells were still able to release amylase in response to bethanechol but this release was not affected by CB. The only structural effect of CB on isolated cells was margination of zymogen granules against the plasma membrane. This was, however, not accompanied by increased amylase release. It is concluded that microfilaments are important in maintaining the pancreatic acinar structure. Interference with this structure by CB leads to inhibition of bethanechol-stimulated amylase release. Microfilaments, however, may not play a direct role in secretion.Supported by NIH grant GM-19998 from the United States Public Health Service. — We are indebted to Dr. John Heuser for advice throughout this project and assistance in the preparation of rapidly frozen acini, and to M. Lee for technical assistance     

Separable helper factors support B cell proliferation and maturation to Ig secretion

The 24-hr culture supernatant of Con A-activated spleen cells (SN) contains helper factors that enable maturation to high-rate polyclonal Ig secretion and enhance proliferation in cultures of mouse B cells activated with the F(ab')2 fragment of class-specific rabbit antimouse IgM antibody (anti-Ig). When interleukin 2 (IL 2), also called T cell growth factor, is removed from SN by absorption with an IL 2-dependent cell line at either 4 degrees C or 37 degrees C, all the helper activity for anti-Ig-activated B cells is also removed. Partial removal of IL 2 results in partial removal of helper activity for B cells. However, the IL 2-depleted SN appears to contain another helper factor, TRF, that enables anti-Ig-activated B cell cultures to mature to high-rate Ig secretion. This TRF activity is revealed by adding purified human IL 2 or an IL 2-containing supernatant of a cloned, lectin-activated T cell hybridoma line (FS6-14.13) to Il 2-depleted SN, which restores the polyclonal antibody response to anti-Ig. The hybridoma supernatant by itself supports proliferation of anti-Ig-activated B cell cultures, as measured by an increase in cell number, but not maturation to Ig secretion. This proliferative response is likewise IL 2 dependent, although purified IL 2 with anti-Ig is not sufficient. These experiments define separable combinations of factors acting on anti-Ig-activated B cell cultures, one of which (SN) results in both proliferation and maturation to high-rate Ig secretion, whereas the other (hybridoma supernatant) results in proliferation only. IL 2 appears to be an essential component of both combinations, although the target cell for IL 2 action in this system remains to be determined.     

LPS-mediated cell surface expression of CD74 promotes the proliferation of B cells in response to MIF

Macrophage migration inhibitory factor (MIF) is a chemokine-like inflammatory cytokine, which plays a pivotal role in the pathogenesis of inflammatory and cardiovascular diseases as well as cancer. We previously identified MIF as a novel B cell chemokine that promotes B cell migration through non-cognate interaction with the CXC chemokine receptor CXCR4 and CD74, the surface form of MHC class II invariant chain. In this study, we have analyzed the regulation of the MIF receptors under inflammatory conditions by investigating the impact of lipopolysaccharide (LPS), tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) on CD74 and CXCR4 expression in B lymphocytes. We found that both LPS and TNF-α stimulation of primary B cells and the human B myeloma cell line RPMI-8226 enhanced protein expression as well as mRNA levels of CD74 in a time- and dose-dependent manner. By contrast, no effect on CXCR4 expression was observed. Selective inhibition of IκBα phosphorylation significantly attenuated LPS-induced expression of CD74, suggesting the contribution of NF-κB signaling pathways to the regulation of CD74 expression. Importantly, individual or simultaneous blockade of MIF or CD74 using specific neutralizing antibodies markedly affected B cell proliferation after LPS exposure. Taken together, our findings unveil a connection between the pro-proliferative activity of MIF/CD74 signaling in B cells and inflammation, offering novel target mechanisms in inflammatory cardiovascular or autoimmune pathogenesis.     

Human peripheral blood T helper cell-induced B cell activation results in B cell surface expression of the CD23 (BLAST-2) antigen

We have developed an in vitro system to assess the early stages of B cell activation induced by peripheral blood T helper cells. Peripheral blood mononuclear cells are cultured for 16 hr with anti-CD3 monoclonal antibody (mAb), T lymphocytes are then removed by sheep red blood cell rosette depletion, and expression of the B cell surface activation antigen CD23 (BLAST-2) is assessed by indirect immunofluorescence. Anti-CD3 mAb, but not a control anti-CD5 mAb, stimulates the expression of CD23 on 20-50% of peripheral blood B cells cultured with autologous T cells. T cell subset depletion studies show that the CD4+ T cell subset is responsible for anti-CD3-mediated induction of CD23 on autologous B cells. Anti-CD3-induced, T helper cell-dependent CD23 expression is not MHC-restricted, as allogeneic combinations of T and non-T cells, cultured in the presence of anti-CD3 antibody, also result in the expression of B cell CD23. Individuals whose monocyte Fc receptors bind murine IgG1 mAb poorly fail to trigger T cell proliferation in response to murine IgG1 anti-CD3 mAb and also fail to express B cell CD23 following culture of PBMC with IgG1 anti-CD3 mAb, while the usual expression of CD23 is seen after culture with IgG2a anti-CD3 mAb. The mechanism of anti-CD3-induced B cell activation was addressed in experiments using a two-chamber culture system. While little IL-4 activity was detected in anti-CD3-stimulated culture supernatants, optimal induction of CD23 was observed when T and B cells were cultured together in a single chamber. This suggests that under physiologic conditions, in which quantities of lymphokine may be limiting, close physical contact between the anti-CD3-activated Th cell and B cell may be required for CD23 expression. The anti-CD3-induced BLAST-2 assay will facilitate the analysis of Th cell-mediated B cell activation in any individual and should permit us to separately evaluate the roles of Th cells and B cells in the impaired immunoregulation characteristic of autoimmune disorders.