CD27-mediated activation of murine NK cells

CD27, a member of the TNF receptor superfamily, has been implicated in T cell activation, T cell development, and T cell-dependent Ab production by B cells. In the present study we examined the expression and function of CD27 on murine NK cells. Murine NK cells constitutively expressed CD27 on their surface. Stimulation with immobilized anti-CD27 mAb or murine CD27 ligand (CD70) transfectans solely could induce proliferation and IFN-gamma production of freshly isolated NK cells and enhanced the proliferation and IFN-gamma production of anti-NK1.1-sutimulated NK cells. Although NK cell cytotoxicity was not triggered by anti-CD27 mAb or against CD70 transfectants, prestimulation via CD27 enhanced the cytotoxic activity of NK cells in an IFN-gamma-dependent manner. These results suggest that CD27-mediated activation may be involved in the NK cell-mediated innate immunity against virus-infected or transformed cells expressing CD70.     

TIM-1 signaling in B cells regulates antibody production

Members of the T cell Ig and mucin (TIM) family have recently been implicated in the control of T cell-mediated immune responses. In this study, we found TIM-1 expression on anti-IgM- or anti-CD40-stimulated splenic B cells, which was further up-regulated by the combination of anti-IgM and anti-CD40 Abs. On the other hand, TIM-1 ligand was constitutively expressed on B cells and inducible on anti-CD3⁺ anti-CD28-stimulated CD4⁺ T cells. In vitro stimulation of activated B cells by anti-TIM-1 mAb enhanced proliferation and expression of a plasma cell marker syndecan-1 (CD138). We further examined the effect of TIM-1 signaling on antibody production in vitro and in vivo. Higher levels of IgG2b and IgG3 secretion were detected in the culture supernatants of the anti-TIM-1-stimulated B cells as compared with the control IgG-stimulated B cells. When immunized with T-independent antigen TNP-Ficoll, TNP-specific IgG1, IgG2b, and IgG3 Abs were slightly increased in the anti-TIM-1-treated mice. When immunized with T-dependent antigen OVA, serum levels of OVA-specific IgG2b, IgG3, and IgE Abs were significantly increased in the anti-TIM-1-treated mice as compared with the control IgG-treated mice. These results suggest that TIM-1 signaling in B cells augments antibody production by enhancing B cell proliferation and differentiation.     

Expression of a 33-kDa antigen Tm 1 on lymphocyte surface after modulation of cell surface antigens by IgM monoclonal antibody

The mAb Tm 1 was obtained from a fusion of SP2/O tumor cells with spleen cells from CF1 mouse immunized with T cells modulated by an IgM anti-CD3 mAb.mAb Tm 1 reacted with IgM anti-CD3 modulated T cells (66.6%) but not with unmodulated T cells (4.4%). Tm 1 was not expressed on T cells modulated with either IgG2a or IgG1 anti-CD3 mAb. Immunoprecipitation from 125I-labeled CD3-modulated T cells showed that Tm 1 Ag is a single polypeptide of 33 kDa under reducing and nonreducing conditions. Kinetic studies revealed that Tm 1 was detectable on T cells 10 min after incubation and maximally expressed after 4 h of incubation with IgM anti-CD3 mAb. CD3 expression was markedly modulated by this anti-CD3 mAb after the same period of incubation. Studies with cycloheximide revealed that Tm 1 expression on T cells does not require new protein synthesis. Tm 1 expression persisted long after CD3-reexpression 24 h later. Tm 1 was present on a small fraction of circulating T cells, B cells, and monocytes and absent from granulocytes, platelets, E, and thymocytes. Tm 1 was not expressed on T cells after various activation stimuli but was expressed on B cells upon activation. Additional studies indicate that IgM mAb against other T cell differentiation Ag and IgM mAb against B cell Ag also lead to the expression of Tm 1 on these cells. Thus, modulation of surface Ag by IgM mAb externalizes this cytoplasmic Ag. However, one exception has been noted. Purified mAb Tm 1 was not mitogenic and was unable to block either the T cell proliferation induced by 12-O-tetradecanoyl phorbol-13-acetate plus anti-CD3 mAb and other T cell stimuli, or the B cell proliferation induced by B cell mitogens. The role of Tm 1 on lymphocyte function remains to be determined.     

Induction of isotype switching and Ig production by CD5+ and CD10+ human fetal B cells.

In the present study the capacity of early fetal B cells to produce Ig was investigated. It is shown that B cells from fetal liver, spleen, and bone marrow (BM) can be induced to produce IgM, IgG, IgG4, and IgE, but not IgA, in response to IL-4 in the presence of anti-CD40 mAb or cloned CD4+ T cells. Even splenic B cells from a human fetus of only 12 wk of gestation produced these Ig isotypes. IFN-alpha, IFN-gamma, and transforming growth factor-beta inhibited IL-4-induced IgE production in fetal B cells, as described for mature B cells. The majority of B cells in fetal spleen expressed CD5 and CD10 and greater than 99% of B cells in fetal BM were CD10+. Highly purified CD10+, CD19+ immature B cells and CD5+, CD19+ B cells could be induced to produce Ig, including IgG4 and IgE, in similar amounts as unseparated CD19+ B cells. Virtually all CD19+ cells still expressed CD10 after 12 days of culture. However, the IgE-producing cells at the end of the culture period were found in the CD19-,CD10- cell population, suggesting differentiation of CD19+,CD10+ B cells into CD19-,CD10- plasma cells. Pre-B cells are characterized by their lack of expression of surface IgM (sIgM). Only 30 to 40% of BM B cells expressed sIgM. However, in contrast to sIgM+,CD10+,CD19+ immature B cells, sorted sIgM-,CD10+,CD19+ pre-B cells failed to differentiate into Ig-secreting cells under the present culture conditions. Addition of IL-6 to these cultures was ineffective. Taken together, these results indicate that fetal CD5+ and CD10+ B cells are mature in their capacity to be induced to Ig isotype switching in vitro as soon as they express sIgM.     

Perturbed homeostasis of peripheral T cells elicits decreased susceptibility to anti-CD3-induced apoptosis in prediabetic nonobese diabetic mice

Activation-induced cell death (AICD) plays a key role in the homeostasis of the immune system. Autoreactive T cells are eliminated through AICD both from the thymus and periphery. In this study, we show that NOD peripheral T cells, especially CD8(+) T cells, display a decreased susceptibility to anti-CD3-induced AICD in vivo compared with T cells from diabetes-resistant B6, nonobese diabetes-resistant, and NOD.B6Idd4 mice. The susceptibility of NOD CD8(+) T cells to AICD varies in an age- and dose-dependent manner upon stimulation in vivo with either a mitogenic or nonmitogenic anti-CD3. NOD T cells preactivated by anti-CD3 in vivo are less susceptible than B6 T cells to TCR-induced AICD. Treatment of NOD mice with a mitogenic anti-CD3 depletes CD4(+)CD25(-)CD62L(+) but not CD4(+)CD25(+)CD62L(+) T cells, thereby resulting in an increase of the latter subset in the spleen. Treatment with a nonmitogenic anti-CD3 mAb delays the onset of T1D in 8.3 TCR transgenic NOD mice. These results demonstrate that the capacity of anti-CD3 to protect NOD mice from T1D correlates with its ability to perturb T cell homeostasis by inducing CD8(+) T cell AICD and increasing the number of CD4(+)CD25(+)CD62L(+) T cells in the periphery.     

HUMAN B CELL DIFFERENTIATION: DEPENDENCE ON INTERACTIONS WITH MONOCYTES AND T LYMPHOCYTES VIA CD40, CD80 (B7.1), AND THE CD2-LIGANDS CD48 AND CD58 (LFA-3)

B cell differentiation depends on cellular interactions with T lymphocytes and monocytes via adhesion molecules (AM). In order to characterize AM which are required for B cell differentiation immunoglobulin production using unseparated peripheral blood mononuclear cells (PBMC) was studied. Unstimulated human PBMC were cultured for 9 days with mAb directed at CD2/CD48, /CD58, CD59, CD5/CD72, CD11a—CD18/CD54, CD28/CD80, CD86, CD40/CD40L, or rat CD2 (control). B cell differentiation was quantified measuring IgM and in some cases IgA, IgG, and IgE production. IgM levels were significantly reduced by mAb against CD40, CD48, CD58 and CD80. The reduction was not due to isotype switching to IgA, IgG or IgE. The role of CD40, CD48, CD58 and CD80 was further investigated after depletion of different cell types. Depletion of monocytes and NK cells resulted in no detectable IgM production irrespective of added mAbs. In contrast, IgM production was still present after depletion of T cells and NK cells. Only mAb against CD80 and CD48 significantly reduced IgM production, the reduction of IgM production by anti-CD40 mAb was less than in the presence of T cells. Importantly, anti-CD58 mAb had no effect on IgM production after T cell and NK cell depletion. Taken together, the AM CD40, CD48, CD58, and CD80 are involved in Ig production of unseparated PBMCs. In this model of B cell differentiation only the AM CD58 depend on the presence of T cells while CD48 and CD80 help was found to be T cell independent.     

CD40 stimulation provides an IFN-gamma-independent and IL-4-dependent differentiation signal directly to human B cells for IgE production.

IgE induction from human cells has generally been considered to be T cell dependent and to require at least two signals: IL-4 stimulation and T cell/B cell interaction. In the present study we report a human system of T cell-independent IgE production from highly purified B cells. When human cells were co-stimulated with a mAb directed against CD40 (mAb G28-5), there was induction of IgE secretion from purified blood and tonsil B cells as well as unfractionated lymphocytes. Anti-CD40 alone failed to induce IgE from blood mononuclear cells or purified B cells. The effect of the combination of anti-CD40 and IL-4 on IgE production was very IgE isotype specific as IgG, IgM, and IgA were not increased. Furthermore, anti-CD40 with IL-5 or PWM did not co-stimulate IgG, IgM, or IgA and in fact strongly inhibited PWM-stimulated IgG, IgM and IgA production from blood or tonsil cells. IgE synthesis induced by anti-CD40 plus IL-4 was IFN-gamma independent as is the in vivo production of IgE in humans; the doses of IFN-gamma that profoundly suppressed IgG synthesis induced by IL-4, or IL-4 plus IL-6, had no inhibitory effect on anti-CD40-induced IgE production. Anti-CD23 and anti-IL-6 also could not block anti-CD40 plus IL-4-induced IgE production, but anti-IL-4 totally blocked their effect. IgE production via CD40 was not due to IL-5, IL-6 or nerve growth factor as none of these synergized with IL-4 to induce IgE synthesis by purified B cells. Finally, we observed that CD40 stimulation alone could enhance IgE production from in vivo-driven IgE-producing cells from patients with very high IgE levels; cells that did not increase IgE production in response to IL-4. Taken together, our data suggest that the signals delivered for IgE production by IL-4 and CD40 stimulation may mimic the pathway for IgE production seen in vivo in human allergic disease.     

Molecular interactions mediating T-B lymphocyte collaboration in human lymphoid follicles. Roles of T cell-B-cell-activating molecule (5c8 antigen) and CD40 in contact-dependent help.

In lymphoid follicles, CD4+ T lymphocytes provide contact-dependent stimuli to B cells that are critical for the generation of specific antibody responses in a process termed Th function. The CD4+ T cell-restricted surface activation protein, 5c8 Ag (T-BAM), has recently been shown to be a component of the contact-dependent helper signal to B cells. To further dissect this process, we utilized a Jurkat T cell lymphoma clone, termed D1.1, that constitutively expresses T-BAM and activates peripheral B cells to express surface CD23 in a contact-dependent mechanism that is inhibited by mAb anti-T-BAM (5c8). Similar to its effect on peripheral B cells, Jurkat D1.1 activates B cells from lymphoid organs, as well as a B cell lymphoma clone, RAMOS 266,4CN 3F10 (RAMOS 266), to up-regulate surface CD23. Interestingly, mAb to the B cell surface molecule, CD40 (mAb G28-5 and B-B20), inhibit D1.1 induced activation of RAMOS 266 and peripheral and lymphoid B cells. In contrast, mAb to CR2 or the adhesion molecules, LFA1, LFA3, or ICAM-1, have little effect. The inhibitory effect of anti-CD40 mAb on B cell activation induced by D1.1 is specific because anti-CD40 potentiates, rather than inhibits, the up-regulation of CD23 on B cells induced by rIL-4. Moreover, cross-linking CD40 molecules by anti-CD40 mAb bound to Fc gamma RII+ (CD32) L cells induces B cell CD23 expression. In vivo, T-BAM-expressing cells are CD4+ T cells that are restricted to lymphoid organs and are localized in the mantle and centrocytic zones of lymphoid follicles and the spleen periarteriolar lymphoid sheath in association with CD40+ B cells. Taken together, these data demonstrate that T-BAM on T cells and CD40 on B cells are involved in contact-dependent T-B help interactions that occur in lymphoid follicles.     

Inhibitory coreceptors activated by antigens but not by anti-Ig heavy chain antibodies install requirement of costimulation through CD40 for survival and proliferation of B cells

Ag-induced B cell proliferation in vivo requires a costimulatory signal through CD40, whereas B cell Ag receptor (BCR) ligation by anti-Ig H chain Abs, such as anti-Ig micro H chain Ab and anti-Ig delta H chain Ab, alone induces proliferation of B cells in vitro, even in the absence of CD40 ligation. In this study, we demonstrate that CD40 signaling is required for survival and proliferation of B cells stimulated by protein Ags in vitro as well as in vivo. This indicates that the in vitro system represents B cell activation in vivo, and that protein Ags generate BCR signaling distinct from that by anti-Ig H chain Abs. Indeed, BCR ligation by Ags, but not by anti-Ig H chain Abs, efficiently phosphorylates the inhibitory coreceptors CD22 and CD72. When these coreceptors are activated, anti-Ig H chain Ab-stimulated B cells can survive and proliferate only in the presence of CD40 signaling. Conversely, treatment of Ag-stimulated B cells with anti-CD72 mAb blocks CD72 phosphorylation and induces proliferation, even in the absence of CD40 signaling. These results strongly suggest that activation of B cells by anti-Ig H chain Abs involves their ability to silence the inhibitory coreceptors, and that the inhibitory coreceptors install requirement of CD40 signaling for survival and proliferation of Ag-stimulated B cells.     

Treatment of human B cell lymphoma xenografts with a CD3 x CD19 diabody and T cells

The use of anti-CD3 x antitumor bispecific Abs is an attractive and highly specific approach in cancer therapy. Recombinant Ab technology now provides powerful tools to enhance the potency of such immunotherapeutic constructs. We designed a heterodimeric diabody specific for human CD19 on B cells and CD3epsilon chain of the TCR complex. After production in Escherichia coli and purification, we analyzed its affinity, stability, and pharmacokinetics, and tested its capacity to stimulate T cell proliferation and mediate in vitro lysis of CD19+ tumor cells. The effect of the diabody on tumor growth was investigated in an in vivo model using immunodeficient mice bearing a human B cell lymphoma. The CD3 x CD19 diabody specifically interacted with both CD3- and CD19-positive cells, was able to stimulate T cell proliferation in the presence of tumor cells, and induced the lysis of CD19+ cells in the presence of activated human PBL. The lytic potential of the diabody was enhanced in the presence of an anti-CD28 mAb. In vivo experiments indicated a higher stability and longer blood retention of diabodies compared with single chain Fv fragments. Treatment of immunodeficient mice bearing B lymphoma xenografts with the diabody and preactivated human PBL efficiently inhibited tumor growth. The survival time was further prolonged by including the anti-CD28 mAb. The CD3 x CD19 diabody is a powerful tool that should facilitate the immunotherapy of minimal residual disease in patients with B cell leukemias and malignant lymphomas.     

CD28-independent costimulation of T cells by OX40 ligand and CD70 on activated B cells.

OX40 and its ligand (OX40L) have been implicated in T cell-dependent humoral immune responses. To further characterize the role of OX40/OX40L in T-B cell interaction, we newly generated an anti-mouse OX40L mAb (RM134L) that can inhibit the costimulatory activity of OX40L transfectants for anti-CD3-stimulated T cell proliferation. Flow cytometric analyses using RM134L and an anti-mouse OX40 mAb indicated that OX40 was inducible on splenic T cells by stimulation with immobilized anti-CD3 mAb in a CD28-independent manner, while OX40L was not expressed on resting or activated T cells. OX40L was inducible on splenic B cells by stimulation with anti-IgM Ab plus anti-CD40 mAb, but not by either alone. These activated B cells exhibited a potent costimulatory activity for anti-CD3-stimulated T cell proliferation and IL-2 production. Anti-CD80 and anti-CD86 mAbs partially inhibited the costimulatory activity, and further inhibition was obtained by their combination with RM134L and/or anti-CD70 mAb. We also found the anti-IgM Ab- plus anti-CD40 mAb-stimulated B cells exhibited a potent costimulatory activity for proliferation of and IL-2 production by anti-CD3-stimulated CD28- T cells from CD28-deficient mice, which was substantially inhibited by RM134L and/or anti-CD70 mAb. These results indicated that OX40L and CD70 expressed on surface Ig- and CD40-stimulated B cells can provide CD28-independent costimulatory signals to T cells.     

In vivo regulation of the murine syngeneic mixed lymphocyte reaction

Previous work from this laboratory has suggested that a CD8+ T suppressor (Ts) cell network regulated the murine syngeneic mixed lymphocyte reaction (SMLR). We have attempted to disrupt this network by the inoculation of anti-CD8 monoclonal antibodies (mAb) in vivo. Intraperitoneal inoculation of three mAbs resulted in a marked increase in the proliferation of CD4+, self-Ia-reactive splenic T cells in vitro to syngeneic, but not to allogeneic, spleen cells. Suppression was not limited to a specific mouse strain as the enhanced SMLR was reproducible following anti-CD8 treatment of three strains of mice. In vivo depletion of CD8+ T cells was not a prerequisite for enhancement of the SMLR as several mAb to CD8 augmented the SMLR independent of their capacity to cause CD8 T cell depletion. Moreover, enhancement of the SMLR could be mimicked in vitro by inclusion of anti-CD8 mAb in in vitro cultures of responder T cells and irradiated Ia+ syngeneic stimulators. Since the in vitro SMLR was enhanced following mAb treatment, it was expected that the in vivo SMLR would also be increased. However, no evidence of increased in vivo autoreactivity could be detected following in vivo treatment with anti-CD8 mAb, indicating that other mechanisms in addition to CD8+ regulatory T cells acted to regulate the in vivo activity of autoreactive T cells.     

Phenotypic and functional analysis of murine CD3+,CD4-,CD8- TCR-gamma delta-expressing peripheral T cells

Murine CD3+,CD4-,CD8- peripheral T cells, which express various forms of the TCR-gamma delta on their cell surface, have been characterized in terms of their cell-surface phenotype, proliferative and lytic potential, and lymphokine-producing capabilities. Three-color flow cytofluorometric analysis demonstrated that freshly isolated CD3+,CD4-, CD8- TCR-gamma delta lymph node cells were predominantly Thy-1+,CD5dull,IL-2R-,HSA-,B220-, and approximately 70% Ly-6C+ and 70% Pgp-1+. After CD3+,CD4-,CD8-splenocytes were expanded for 7 days in vitro with anti-CD3-epsilon mAb (145-2C11) and IL-2, the majority of the TCR-gamma delta cells expressed B220 and IL-2R, and 10 to 20% were CD8+. In comparison to CD8+ TCR-alpha beta T cells, the population of CD8+ TCR-gamma delta-bearing T cells exhibited reduced levels of CD8, and about 70% of the CD8+ TCR-gamma delta cells did not express Lyt-3 on the cell surface. Functional studies demonstrated that splenic TCR-gamma delta cells proliferated when stimulated with mAb directed against CD3-epsilon, Thy-1, and Ly-6C, but not when incubated with an anti-TCR V beta 8 mAb, consistent with the lack of TCR-alpha beta expression. In addition, activated CD3+,CD4-,CD8- peripheral murine TCR-gamma delta cells were capable of lysing syngeneic FcR-bearing targets in the presence of anti-CD3-epsilon mAb and the NK-sensitive cell line, YAC-1, in the absence of anti-CD3-epsilon mAb. Finally, activated CD3+, CD4-,CD8-,TCR-gamma delta+ splenocytes were also capable of producing IL-2, IL-3, IFN-gamma, and TNF when stimulated in vitro with anti-CD3-epsilon mAb.     

CTLA-4 engagement and regulatory CD4+CD25+ T cells independently control CD8+-mediated responses under costimulation blockade

Blockade of costimulatory signals is a promising therapeutic target to prevent allograft rejection. In this study, we sought to characterize to what extent CTLA-4 engagement contributes to the development of transplantation tolerance under the cover of CD40/CD40L and CD28/CD86 blockade. In vitro, we found that inhibition of the primary alloresponse and induction of alloantigen hyporesponsiveness by costimulation blockade was abrogated by anti-CTLA-4 mAb. In addition, regulatory CD4(+)CD25(+) T cells (T(REG)) were confirmed to play a critical role in the induction of hyporesponsiveness by anti-CD40L and anti-CD86 mAb. Our data indicated that CTLA-4 engagement is not required for activation or suppressor function of T(REG). Instead, in the absence of either CTLA-4 signaling or T(REG), CD8(+) T cell division was enhanced, whereas the inhibition of CD4(+) T cell division by costimulation blockade remained largely unaffected. In vivo, the administration of additional anti-CTLA-4 mAb abrogated anti-CD40L- and anti-CD86 mAb-induced cardiac allograft survival. Correspondingly, rejection was accompanied by enhanced allograft infiltration of CD8(+) cells. We conclude that CTLA-4 signaling and T(REG) independently cooperate in the inhibition of CD8(+) T cell expansion under costimulation blockade.     

Cell surface comodulation of CD4 and T cell receptor by anti-CD4 monoclonal antibody

In our study we have used anti-CD4 mAb to investigate the cell surface association between CD4 and the Ag-specific TCR complex on mature peripheral T cells. Anti-CD4 mAb was administered in vivo and in vitro and its effects on CD4 and CD3 cell surface expression were determined. In vivo, anti-CD4 mAb reduced cell surface expression of its ligand, CD4, and secondarily also reduced cell surface expression of CD3/TCR on CD4+ splenic T cells. In vitro, multivalent cross-linking of CD4 by anti-CD4 mAb and either FcR+ cells or anti-Ig mAb also resulted in decreased surface expression of CD4 and specific comodulation of CD3/TCR. The secondary reduction in cell surface CD3/TCR expression induced by CD4 cross-linking could be pharmacologically disrupted by high doses of PMA, indicating that the comodulation of CD3 with CD4 was dependent upon intracellular mediators, possibly including protein kinase C. These results demonstrate that, in the presence of anti-CD4 mAb, CD4 is functionally associated with the CD3/TCR complex, and that this association is dependent upon the activity of intracellular mediators. Such intracellular mediators might induce the coordinate down-modulation of physically unassociated CD4 and CD3/TCR molecules, or, alternatively, might promote a physical interaction between CD4 and CD3/TCR molecules.     

CD40 ligation activates murine macrophages via an IFN-gamma-dependent mechanism resulting in tumor cell destruction in vitro

We have shown previously that agonistic anti-CD40 mAb induced T cell-independent antitumor effects in vivo. In this study, we investigated mechanisms of macrophage activation with anti-CD40 mAb treatment, assessed by the antitumor action of macrophages in vitro. Intraperitoneal injection of anti-CD40 mAb into C57BL/6 mice resulted in activation of peritoneal macrophages capable of suppressing B16 melanoma cell proliferation in vitro, an effect that was greatly enhanced by LPS and observed against several murine and human tumor cell lines. Anti-CD40 mAb also primed macrophages in vitro to mediate cytostatic effects in the presence of LPS. The tumoristatic effect of CD40 ligation-activated macrophages was associated with apoptosis and killing of tumor cells. Activation of macrophages by anti-CD40 mAb required endogenous IFN-gamma because priming of macrophages by anti-CD40 mAb was abrogated in the presence of anti-IFN-gamma mAb, as well as in IFN-gamma-knockout mice. Macrophages obtained either from C57BL/6 mice depleted of T and NK cells by Ab treatment, or from scid/beige mice, were still activated by anti-CD40 mAb to mediate cytostatic activity. These results argued against the role of NK and T cells as the sole source of exogenous IFN-gamma for macrophage activation and suggested that anti-CD40 mAb-activated macrophages could produce IFN-gamma. We confirmed this hypothesis by detecting intracytoplasmic IFN-gamma in macrophages activated with anti-CD40 mAb in vivo or in vitro. IFN-gamma production by macrophages was dependent on IL-12. Taken together, the results show that murine macrophages are activated directly by anti-CD40 mAb to secrete IFN-gamma and mediate tumor cell destruction.     

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.     

Resolution of three nonproliferative immature splenic B cell subsets reveals multiple selection points during peripheral B cell maturation.

Although immature/transitional peripheral B cells may remain susceptible to selection pressures before full maturation, the nature and timing of these selection events remain unclear. We show that correlated expression of surface (s) IgM (sIgM), CD23, and AA4 defines three nonproliferative subpopulations of immature/transitional peripheral B cells. We designate these populations transitional (T) 1 (AA4(+)CD23(-)sIgM(high)), T2 (AA4(+)CD23(+)sIgM(high)), and T3 (AA4(+)CD23(+)sIgM(low)). Cells within all three subsets are functionally immature as judged by their failure to proliferate following sIgM cross-linking in vitro, and their rapid rate of turnover in vivo as assessed by 5-bromo-2'-deoxyuridine labeling. These labeling studies also reveal measurable cell loss at both the T1-T2 and T2-T3 transitions, suggesting the existence of multiple selection points within the peripheral immature B cell pool. Furthermore, we find that Btk-deficient (xid) mice exhibit an incomplete developmental block at the T2-T3 transition within the immature B cell pool. This contrasts markedly with lyn(-/-) mice, which exhibit depressed numbers but normal ratios of each immature peripheral B cell subset and severely reduced numbers of mature B cells. Together, these data provide evidence for multiple selection points among immature peripheral B cells, suggesting that the B cell repertoire is shaped by multiple unique selection events that occur within the immature/transitional peripheral B cell pool.