The role of B7-1 and B7-2 costimulation for the generation of CTL responses in vivo

The role of B7-1 and B7-2 costimulatory molecules in the generation of Ag-specific CD8+ CTLs is not well understood. In this paper, we analyze the role of both B7-1 and B7-2 in the generation of CTLs to nonliving, exogenous Ag and to live virus. To analyze the role of B7 costimulation in the induction of CTLs, we blocked B7-1 and/or B7-2 in vivo by injecting C57BL/6 mice with anti-B7-1 and/or anti-B7-2 mAbs; the mice were subsequently immunized with either chicken OVA that had been cross-linked to beads as a model of exogenous Ags or with wild-type and recombinant vaccinia virus expressing different forms of chicken OVA as models of viral Ags. Our results indicate that B7 costimulation is necessary in the generation of CTLs for all of these Ags. Since the B7 molecules could be costimulating CD8+ and/or CD4+ T cells in wild-type animals, we also examined the role of costimulation in the generation of CTLs to exogenous and viral Ag in MHC class II-deficient mice lacking most CD4+ T cells. In these animals, a combination of both mAbs also blocked all CTL responses, indicating that the Th cell-independent activation of CTLs is dependent upon the B7-costimulatory signals supplied to the CD8+ cell. These findings contribute to the understanding of the role of costimulation for the generation of CTLs. We also discuss the implications of these findings on the role of professional APCs in the initiation of CTL responses.     

Complement opsonization is required for presentation of immune complexes by resting peripheral blood B cells

Complement receptor 2 (CD21, CR2) is a B cell receptor for complement degradation products bound to Ag or immune complexes. The role of CD21 in mediating Ag presentation of soluble immune complexes by resting B cells was studied. Complement-coated immune complexes were formed by the incubation of influenza virus with serum from immune donors. These complexes bound to peripheral blood B cells in a complement-dependent manner. The binding required CD21 or, to a lesser extent, complement receptor 1 (CR1, CD35). B cells pulsed with immune complexes containing complement elicited a response from a panel of influenza-specific T cell clones, while those pulsed with immune complexes formed in the absence of complement did not. The expression of the early activation marker CD69 and the costimulatory molecule CD86 were not induced by CD21 ligation alone, suggesting that CD21-mediated Ag presentation occurs independently of B cell activation. Up-regulation of these markers required exposure to T cell factors elicited by the recognition of Ag derived from complement-containing immune complexes. These findings suggest that binding of Ag to CD21 enables Ag-nonspecific B cells to participate in the activation of Ag-specific T cells in a process that occurs independently of well-characterized B cell activation events.     

Expression and function of B7-1 and B7-2 in hapten-induced contact sensitivity

We analyzed the expression and function of the co-stimulatory molecules B7-1 (CD80) and B7-2 (CD86) during contact sensitivity reactions induced by the hapten 2,4-dinitrofluorobenzene (DNFB). In the normal skin, only a few epidermal Langerhans cells or dermal dendritic cells express B7-2. In contrast, following challenge with DNFB, expression of B7-2 is up-regulated in both epidermis and dermis. Importantly, B7-1 is induced later and at lower levels compared to B7-2. Intravenous injections of anti-B7-2 mAb, but not anti-B7-1 mAb partially inhibit the hapten-induced contact sensitivity reaction. Experiments in which mice are injected differentially with anti-B7-2 mAb, either before the afferent or before the efferent phase of the contact sensitivity response, suggest that B7-2 is important for successful antigen priming.     

B7-1 and B7-2 monoclonal antibodies modulate the severity of murine Lyme arthritis

We assessed the role of B7-1 and B7-2 costimulatory molecules on the course of murine Lyme borreliosis because experimental Lyme arthritis is dependent, at least partially, upon the development of the host immune response and these costimulatory molecules have been implicated in CD4+ T-cell differentiation. We demonstrated that Borrelia burgdorferi infection upregulated the surface expression of B7-1 and B7-2 in macrophages and B7-2 expression in B cells. Anti-B7-2 monoclonal antibody (MAb) or both anti-B7-2 and anti-B7-1 MAbs produced a dose-dependent increase in the severity of Lyme arthritis in C3H/HeN mice. In contrast, the administration of an anti-B7-1 MAb reduced the degree of arthritis. These effects occurred independently of significant alteration in B. burgdorferi-specific immune responses, including splenocyte proliferative responses to B. burgdorferi, B. burgdorferi antibody levels and specificity, and mRNA levels of gamma interferon, interleukin-4 (IL-4), IL-10, and IL-12 in the spleen. These results demonstrate that signaling delivered by B7-1 and B7-2 plays a role in determining the severity of acute murine Lyme arthritis.     

The BB1 monoclonal antibody recognizes both cell surface CD74 (MHC class II-associated invariant chain) as well as B7-1 (CD80), resolving the question regarding a third CD28/CTLA-4 counterreceptor

The identification of all CD28/CTLA-4 counterreceptors is critical to our understanding of this pivotal pathway of T cell activation. Clouding our understanding has been the reported discrepancies in expression and function of the B7-1 (CD80) molecule based upon the use of the BB1 vs other anti-B7-1 mAbs. To resolve this issue, we have cloned a BB1-binding molecule from the BB1+B7-1(-) NALM-6 pre-B cell line. Here, we demonstrate that this BB1-binding molecule is identical to the cell surface form of CD74 (MHC class II-associated invariant chain). CD74-transfected cells bound the BB1 mAb but not other anti-CD80 mAbs, CD28-Ig, or CTLA4Ig. Absorption and blocking experiments confirmed the reactivity of BB1 mAb with CD74. A region of weak homology was identified between CD74 and the region of B7-1 encoding the BB1 epitope. Therefore, the BB1 mAb binds to a protein distinct from B7-1, and this epitope is also present on the B7-1 protein. Many of the puzzling observations in the literature concerning the expression of human B7-1 are resolved by an understanding that BB1 staining is the summation of CD74 plus B7-1 expression. This observation requires the field to reconsider studies using BB1 mAb in the analysis of CD80 expression and function.     

Importance of B7-1-expressing host antigen-presenting cells for the eradication of B7-2 transfected P815 tumor cells

We have previously shown that B7-2 (CD86)-transfected P815 tumor cells elicit tumor-eradicating immunity that leads to the regression of the B7-2+ P815 tumor after transient growth in normal DBA/2 mice. Here, we show that both the B7-2 and B7-1 (CD80) molecules contribute to the eradication of B7-2+ P815 tumors as treatment of the mice with both anti-B7-2 and anti-B7-1 mAb was required to prevent B7-2+ P815 tumor regression. The cells that expressed the B7-1 molecule following inoculation of B7-2+ P815 tumor cells into normal mice were not the tumor cells but rather host APCs including MAC-1+ cells present in the draining lymph nodes. Moreover, B7-1-expressing host APCs were found to be important for the rejection of B7-2+ P815 tumors as anti-B7-2 mAb alone, which was ineffective in preventing B7-2+ P815 tumor rejection by normal wild-type mice, was effective in preventing B7-2+ P815 tumor rejection by mice in which the B7-1 gene was disrupted. Finally, consistent with the importance of B7-1-expressing host APCs for the generation of tumor-eradicating immunity against B7-2+ P815 tumor cells, CD4+ T cells (not only CD8+ T cells) were found to participate in tumor-eradicating immunity against B7-2+ P815 tumor cells. Thus, in addition to eliciting tumor-eradicating immunity directly, B7-2+ P815 tumor cells elicit tumor-eradicating immunity indirectly through B7-1-expressing host APCs that present tumor-associated Ags to CD4+ T cells.     

Effect of B-cell receptor engagement on CD40-stimulated B cells

Activation of human B cells in vitro either by cross-linking of surface immunoglobulins (sIg) or by triggering CD40 antigen, in the presence of interleukin-10 (IL-10) and interleukin-2 (IL-2), may result in high levels of immunoglobulin secretion in vitro. We studied the combined effects of ligation of the B-cell receptor (BCR) and CD40 [with anti-CD40 monoclonal antibody (mAb)] on B-cell proliferation and production of human immunoglobulin. For this purpose highly purified splenic B cells were cultured with various combinations of anti-CD40 and IL-10/IL-2 or IL-4 in the presence of CD32-transfected L cells. Simultaneous cross-linking of the BCR was achieved by mAb held on CD32-L cells or Staphylococcus aureus (SA). We found that dual BCR and CD40 ligation with IL-10/IL-2 leads to reduced immunoglobulin G (IgG) secretion compared with B cells stimulated with either anti-CD40 and IL-10/IL-2, or compared with B cells stimulated with SA or anti-BCR mAb and IL-10/IL-2. Dual BCR and CD40 ligation with anti-immunoglobulin mAb (anti-kappa + anti-lambda light chains) but not with SA induced a similar reduction in IgM production. The reduced immunoglobulin secretion found during dual ligation is accompanied by increased proliferation. This was independent of cytokine stimulation but SA/CD40-induced proliferation was increased in the presence of IL-10/IL-2, although not with IL-4. The combination anti-kappa and anti-lambda with anti-CD40 showed a long-term suppression of IgG and IgM production (at least 14 days), while anti-kappa or anti-lambda alone, or SA, allowed a moderate recovery of immunoglobulin production by day 14. These results suggest that simultaneous B-cell antigen receptor cross-linking and CD40 engagement via CD40L on T cells induces strong initial proliferation. This may be followed later by antibody production depending on the strength of the BCR signal and the presence of the appropriate cytokines.     

Regulation of C-C chemokine production by murine T cells by CD28/B7 costimulation

C-C chemokines play an important role in recruitment of T lymphocytes to inflammatory sites. T lymphocytes secrete chemokines, but the activation requirements for chemokine production by T cells are uncertain. We studied the regulation of C-C chemokine production by CD28 costimulatory signals by murine T lymphocytes. Splenocytes from BALB/c mice cultured with anti-CD3 mAb expressed macrophage-inflammatory protein (MIP)-1alpha mRNA and secreted MIP-1alpha, which was inhibited by anti-B7-1 plus anti-B7-2 mAbs. MIP-1alpha production by Ag-stimulated T cells from DO.11.10 TCR transgenic mice was augmented by anti-CD28 mAb and increased compared with DO.11.10/CD28(-/-) cells. When T cell costimulation was provided by IL-2, MIP-1alpha was not enhanced. Studies with IL-2, IL-4, STAT4, and STAT6 knock-out mice suggested that chemokine production is controlled by pathways different from those regulating T cell differentiation. Thus, CD28 costimulation may amplify an immune response by stimulating T cell survival, proliferation, and production of chemokines that recruit T cells to inflammatory sites.     

CD72-mediated B cell activation involves recruitment of CD19 and activation of phosphatidylinositol 3-kinase

Occupancy of the B cell glycoprotein, CD72 results in syk-independent activation of phospholipase-C gamma and calcium mobilization. The cytoplasmic tail of CD72 does not contain an immunoreceptor tyrosine-based activation motif to directly transduce signals into the B lymphocyte. Hence, we investigated whether other coreceptors such as CD19 and its associated phosphatidylinositol 3-kinase (PI 3-K) were involved in CD72 signaling. Two specific inhibitors of PI 3-K inhibited CD72-stimulated B cell proliferation in a dose-dependent manner. Activation of B lymphocytes via CD72 resulted in recruitment and activation of PI 3-K, which was mediated by CD19. Accordingly, CD72 ligation induced CD19 tyrosine phosphorylation. Thus, lipid products generated as a result of PI 3-K activation may have an important function in CD72-mediated B lymphocyte activation. The kinetics of CD19 tyrosine phosphorylation induced by CD72 ligation were strikingly different from those seen following B cell antigen receptor (BCR) stimulation. A transient increase in the tyrosine phosphorylation of the complement receptors, CD21 and CD35 was observed in BCR- but not CD72-stimulated cells. Co-cross-linking of CD72 and CD19 failed to induce syk tyrosine phosphorylation suggesting that even under these conditions, CD72 signaling was independent of syk activation. A transient and stimulation-dependent physical association between CD19 and CD72 was observed in CD72-ligated cells. These observations suggest a mechanism by which CD72 can recruit CD19 and influence activation of CD19-associated PI 3-K, which appears to be critical for CD72-mediated B cell activation.     

Costimulation through B7-2 (CD86) is required for the induction of a lung mucosal T helper cell 2 (TH2) immune response and altered airway responsiveness

The recruitment of eosinophils into the airways after allergen exposure is dependent on interleukin (IL) 5 secreted from antigen-specific CD4+ T cells of the T helper cell (Th) 2 subset. However, while it is established that costimulation through CD28 is required for TCR-mediated activation and IL-2 production, the importance of this mechanism for the induction of a Th2 immune response is less clear. In the present study, we administered the fusion protein CTLA-4 immunoglobulin (Ig) into the lungs before allergen provocation to determine whether CD28/CTLA-4 ligands are required for allergen-induced eosinophil accumulation and the production of Th2 cytokines. Administration of CTLA-4 Ig inhibited the recruitment of eosinophils into the lungs by 75% and suppressed IgE in the bronchoalveolar lavage fluid. CTLA-4 Ig also inhibited the production of IL-4, IL-5, and IL-10 by 70-80% and enhanced interferon-gamma production from CD3-T cell receptor-activated lung Thy1.2+ cells. Allergen exposure upregulated expression of B7-2, but not B7-1, on B cells from the lung within 24 h. Moreover, airway administration of an anti-B7-2 monoclonal antibody (mAb) inhibited eosinophil infiltration, IgE production, and Th2 cytokine secretion comparable in magnitude to that observed with CTLA-4 Ig. Treatment with an anti-B7-1 mAb had a small, but significant effect on eosinophil accumulation, although was less effective in inhibiting Th2 cytokine production. The anti-B7-2, but not anti-B7-1, mAb also inhibited antigen-induced airway hyperresponsiveness in vivo. In all of the parameters assessed, the combination of both the anti-B7-1 and anti-B7-2 mAb was no more effective than anti-B7-2 mAb treatment alone. We propose that strategies aimed at inhibition of CD28 interactions with B7-2 molecules may represent a novel therapeutic target for the treatment of lung mucosal allergic inflammation.     

Monoclonal antibodies recognising differentiation antigens on porcine B cells

Nineteen monoclonal antibodies (mAbs) submitted to the porcine CD Workshop were analysed on porcine B cells from different lymphoid tissues by flow cytometry and immunohistochemistry. Six mAbs only bound to pig B cells and three of these were assigned cluster numbers, 76-7-4 (No. 001):CD1, K231-3B2 (No. 027):CD25 and CC51 (No. 100):CD21. Of the three remaining B cell positive mAbs, one was assigned to a swine cluster, CC55 (No. 101):SWC7, a second, LIG4 (No. 123), recognised cell surface IgM and the third, MUC106A (No. 072), remained unassigned.     

Neisserial porins induce B lymphocytes to express costimulatory B7-2 molecules and to proliferate

The neisserial porins are the major protein components of the outer membrane of the pathogenic Neisseria (N. meningitidis and N. gonorrhoeae). They have been shown to be able to enhance the immune response to poorly immunogenic substances (e.g., polysaccharides, peptides, glycolipids, etc.). To explore the basis of their potent adjuvant activity, the effect of the neisserial porins on T-B cell interactions and T cell costimulation was examined. Neisserial porins increased the surface expression of the costimulatory ligand B7-2 (CD86) but did not affect the expression of B7-1 (CD80). In addition, incubation with the neisserial porins increased the T lymphocyte costimulatory ability of B lymphocytes, which was inhibited by anti-B7-2 but not anti-B7-1 monoclonal antibodies. Upregulation of B7-2 on the surface of B lymphocytes may be the mechanism behind the immunopotentiating activity of neisserial porins.     

The role of B7-1 and LFA-3 in costimulation of CD8+ T cells

This study compares the ability of LFA-3 (CD58) and B7-1 (CD80) ligands to provide costimulatory signals for superantigen (SAg)-stimulated CD8+ and CD4+ T cells. We show that B7-1 and LFA-3 costimulation activate CD8+ T cells to proliferation, cytokine production (IL-2, TNF, and IFN-gamma), and cytotoxicity. A long-lasting proliferative response was observed after combined DR/B7-1/LFA-3 costimulation. Detailed analysis of SEA-activated CD8+ T cells revealed that maximal production of IFN-gamma was seen in LFA-3-costimulated cells, while production of IL-2 was mainly induced after B7-1 costimulation. A fivefold increase in the IFN-gamma production was observed when activated CD8+ T cells were costimulated with Chinese hamster ovary (CHO)-DR/LFA-3 cells compared with the secretion induced by CHO-DR/B7-1. In contrast, SEA-treated CD4+ T cells costimulated with B7-1 or LFA-3 gave rise to a similar production of IFN-gamma, suggesting a preferential function for the CD2/LFA-3 pathway in the regulation of IFN-gamma in CD8+ T cells. Moreover, the generation of CTL was supported similarly by B7-1 and LFA-3 costimulation, but not by CHO-DR cells. We conclude that ligation of the CD28 and CD2 receptors mediate distinct effect on CD8+ and CD4+ T cell effector functions.     

Ligation of CD28 receptor by B7 induces formation of D-3 phosphoinositides in T lymphocytes independently of T cell receptor/CD3 activation

The co-stimulatory role of B7/CD28 interactions is important in promoting T cell activation. Very little is known about the intracellular events that follow CD28 engagement although recent evidence has implicated coupling of CD28 to a protein tyrosine kinase signal transduction pathway. In this study we have investigated the putative role of D-3 phosphoinositides as mediators of CD28 receptor signaling, since phosphoinositide (PI) 3-kinase, the enzyme responsible for D-3 phosphoinositide formation, is a known substrate for protein tyrosine kinases associated with certain T cell surface receptors such as CD4 and interleukin-2 receptor. The lipid products of PI 3-kinase activity have been suggested to play a role in mitogenic signaling and growth regulation in other cells. Chinese hamster ovary cells (CHO) previously transfected with B7 cDNA, induced time-dependent elevation above basal levels of phosphatidylinositol(3,4)-bisphosphate (PtdIns(3,4)P2) and PtdIns(3,4,5)P3, while parental CHO cells that did not express B7 had no effect on these lipids. Moreover, the elevation of these same lipids by CD3 ligation was potentiated in an additive manner by CHO-B7+ but not by CHO-B7- cells. CHO-B7+ and CHO-B7- cells did not activate phospholipase C as evidenced by their inability to modulate basal or CD3-induced changes in the levels of phosphatidic acid or D-4 and D-5 phosphoinositides. These data imply that PI 3-kinase but not phospholipase C, may be an important signal transduction molecule with respect to CD28-mediated co-stimulation and T cell activation following ligation by B7.     

Rescue of thymocytes from glucocorticoid-induced cell death mediated by CD28/CTLA-4 costimulatory interactions with B7-1/B7-2

During the differentiation of thymocytes to mature T cells the processes of positive and negative selection result in signals that either protect thymocytes from cell death, or delete, through apoptosis, thymocytes with self-reactive T cell receptors (TCR). Glucocorticoids have been shown to induce thymocyte apoptosis and are produced within the thymic microenvironment. Furthermore, steroid-induced apoptosis of thymocytes has been suggested as a potential mechanism for removal of nonselected thymocytes. In this report, we demonstrate that thymocytes can be rescued from glucocorticoid-induced apoptosis by incubation with cells that express high levels of B7-1 or B7-2. In addition, the ability to be rescued by B7-1 and/or B7-2 can precede expression of the TCR. We demonstrate that CD3(+)-depleted or CD3+/ TCR-beta(+)-doubly depleted thymocytes can be rescued from glucocorticoid-induced apoptosis through the interaction of CD28 or CTLA-4 on thymocytes with cells bearing high levels of B7-1 or B7-2. Furthermore, these transfected cells are major histocompatibility complex (MHC) class II negative and, while they may express MHC class I, there is no preferential rescue of CD8+ thymocytes in the presence of glucocorticoids. Together, these data suggest that the rescue of thymocytes from glucocorticoids can be independent of the TCR. We also demonstrate that, in addition to CD28, CTLA-4 is expressed on thymocytes, suggesting that rescue from glucocorticoid-induced cell death can be mediated by both CD28 and CTLA-4. A CTLA-4Ig fusion protein which binds to both B7-1 and B7-2 was shown to completely block the rescue of thymocytes from glucocorticoid-induced cell death. Therefore, we conclude that interactions between B7-1/B7-2 and CD28/CTLA-4 are sufficient and necessary for rescue of thymocytes from glucocorticoid-induced cell death.     

Expression of costimulatory molecules, B7-1 and B7-2 on human gastric carcinoma

Costimulation of T cells via B7-1 and B7-2 molecules on a tumor has been shown to be important for eliciting cell-mediated antitumor immunity. We studied the surface expression of B7-1 and B7-2 in 24 cases of gastric carcinoma from the primary locus, 20 cases of metastatic carcinoma from malignant ascites, 20 cases of benign gastric mucosa and 7 gastric carcinoma cell lines by two-color flow cytometry with mAb CD80 and CD86. The B7-1 and B7-2 molecules were expressed by 6 cell lines, and 1 cell line showed the predominant expression of B7-2 but not B7-1. Almost all patients with primary gastric carcinoma and benign gastric mucosa showed high levels of expression of the B7-1 and B7-2, revealing approximately 40%-60% positive cells. However, the percentage of B7-1-positive cells of poorly differentiated primary carcinomas was significantly lower than that of well-differentiated carcinoma and normal mucosa (P < 0.01). Furthermore, all of the metastatic carcinoma cells revealed consistently very low or undetectable levels of expression of the B7-1 molecule, only 8% (mean) of cells being positive, despite showing higher levels of B7-2 expression. Thus, it seems likely that decreased or deleted expression of B7-1 correlates with the grade of tumor differentiation, tumor progression and metastasis. These results suggest that the B7-1 molecule on the gastric carcinoma bearing CD80+CD86+ is abrogated during tumor invasion and/or metastasis, and the tumor finally acquires the CD80-CD86+ phenotype. Consequently, inadequate B7-1 costimulation may contribute to the escape of tumors from destruction by the host's immune system.     

B70 antigen is a second ligand for CTLA-4 and CD28

The membrane antigen B7/BB1 (refs 1, 2) is expressed on activated B cells, macrophages and dendritic cells, and binds to a counter-receptor, CD28, expressed on T lymphocytes and thymocytes. Interaction between CD28 and B7 results in potent costimulation of T-cell activation initiated through the CD3/T-cell receptor complex. Discrepancies between results with anti-CD28 and anti-B7 antibodies have suggested the existence of a second ligand for CD28 and CTLA-4 (refs 3, 6-8). We have generated a monoclonal antibody, IT2, that reacts with a 70K glycoprotein (B70). B70 complementary DNA was cloned from a B-lymphoblastoid cell line library and encodes a new protein of the immunoglobulin superfamily with limited homology to B7. B70 is expressed on resting monocytes and dendritic cells and on activated, but not resting, T, NK and B lymphocytes. IT2 substantially inhibited the binding of a CTLA4-immunoglobulin fusion protein to human B-lymphoblastoid cell lines and, together with anti-B7 antibody, completely blocked CTLA-4 binding. Further IT2 efficiently inhibited primary allogeneic mixed lymphocyte responses. These findings indicate that B70 is a second ligand for CD28 and CTLA-4 and may play an important role for costimulation of T cells in a primary immune response.     

Evidence for an important interaction between a complement-derived CD21 ligand on follicular dendritic cells and CD21 on B cells in the initiation of IgG responses

The addition of Ags to mononuclear leukocyte cultures typically elicits modest Ab responses, implying that cosignals beyond those provided by T cells and macrophages may be needed. Recently, we reported that Ab responses could be dramatically enhanced (10-1000-fold) by the addition of follicular dendritic cells (FDC), suggesting that FDC may provide an important costimulatory signal. This result prompted a study of molecules involved in FDC-mediated enhancement of Ab responses stimulated by specific Ag with memory T and B cells or nonspecifically by the addition of LPS. In this study, we report evidence supporting the concept that FDC bear a ligand that engages complement receptor II (CR2 or CD21) on B cells and provides a critical cosignal for both Ag-specific and polyclonal responses. A blockade of the CR2 ligand on FDC by the use of soluble CR2 or a blockade of CR2 on B cells by use of CR2 knockout mice (or B cells with CR2 blocked) reduced Ab responses from the microg/ml to the ng/ml range (10-1000-fold reductions). FDC from C3 knockout mice, which cannot generate the CR2-binding fragments (iC3b, C3d, and C3dg), were unable to provide costimulatory activity, suggesting the CR2 ligand on FDC consists of C3 fragments. FDC trap complement-activating Ag-Ab complexes, and it appears that FDC present B cells with both specific Ag to engage B cell receptors and a CR2 ligand to engage B cell-CR2. In short, optimal induction of specific Ab responses appears to require the combination of specific Ag and costimulatory molecules from both T cells and FDC.     

CD1 expression defines subsets of follicular and marginal zone B cells in the spleen: beta 2-microglobulin-dependent and independent forms

We have used multicolor FACS analysis, immunohistology, and functional assays to study the expression of CD1 on B cell subsets from normal and beta 2m-/- mice. Two B cell subpopulations were identified that express high levels of CD1 in normal mice: splenic marginal zone B cells (IgMhigh IgDlow CD21high CD24intermediate CD23- CD43-) and a newly identified subpopulation of follicular B cells. The latter cells are unusual, because they are IgDhigh CD23+, like follicular B cells, but express high levels of CD21 and IgM, an expression pattern that is associated with marginal zone B cells. Therefore, the high-level expression of CD1 and CD21 was found to be closely associated on splenic B cells. Immunohistology confirmed the expression of CD1 on marginal zone B cells and on clusters of B cells in splenic follicles. Both the high-level CD1 expression by these cells and the low-level CD1 expression by subpopulations of B cells in the spleen, lymph node, peritoneal cavity, and bone marrow were markedly reduced in beta 2m-/- mice. Despite this, a CD1-restricted T cell clone proliferated vigorously in response to LPS-activated spleen cells that had been obtained from both beta 2m-/- and wild-type mice. This response was inhibited by the 3C11 anti-CD1 mAb. These results show the heterogeneity of B cell subsets in their expression of the beta 2m-dependent form of CD1. They further suggest that a beta 2m-independent form of CD1 is expressed on B cells that can stimulate T cells; however, this form is not easily visualized with the anti-CD1 mAb used here.