Cell surface sialic acid and the regulation of immune cell interactions: the neuraminidase effect reconsidered

E.Ümit Bagriaçik and Kenton S. Miller1

Faculty of Biological Science and The Mervin Bovaird Center for Studies in Molecular Biology and Biotechnology, The University of Tulsa, Tulsa,OK 74104, USA

Received on May 22, 1998; revised on August 10, 1998; accepted onAugust 12, 1998

It has been known for over a decade that sialidase (neuraminidase) treatment could substantially enhance the capacityof resting B cells to stimulate the proliferation of allogeneic and antigen specific, syngeneic T cells. Thus, cell-surface sialic acid was implicated as a potential modulator of immune cell interaction. However, little progress has been made in either identifying explicit roles for sialic acid in this system or in hypothesizing mechanisms to explain the 'neuraminidase effect." Here we show for the first time that cell surface sialic acid on medium incubated B cells blocks access to costimulatory molecules on the B cell surface, and that this is the most likely explanation for the neuraminidase effect. Further, we show that it is likely to be upregulation of ICAM-1 and its subsequent engagement of LFA-1 rather than loss of cell surface sialic acid that in part regulates access to CD86 and other costimulatory molecules. However, we cannot exclude a role for CD86-bound sialic acid on the B cell in modulating binding to T cell CD28. Because sialidase treatment of resting B cells but not resting T cells enables T cell activation, we suggest that sialidase treatment may still be an analogue for an authentic step in B cell activation, and show that for highly activated B cells (activated with polyclonal anti-IgM plus INF-[gamma]) there is specific loss 2,6-linked sialic acid. Potential roles for sialic acid in modulating B cell/T cell collaboration are discussed.

Key words: lymphocyte activation/sialic acid/neuraminidase/costimulation/cellular adhesion/glycocalyx

Introduction

T Cell/B cell collaboration is now a well established prerequisite for the initiation of a humoral immune response to soluble protein antigens (T-dependent antigens). Under these conditions naive lymphocytes (both B and T) require at least two signals for a primary response that results in cellular activation, expansion, and ultimately, differentiation and expression of effector function (Bretscher and Cohn, 1970; Lafferty and Cunningham, 1975). The primary signal is usually delivered via the cell's antigen specific receptor (BCR or TCR) whereas one or more other, distinct cell-surface receptors provide the second costimulatory signal. The ligands required for T cell activation are expressed on professional antigen presenting cells (APCs) such as dendritic cells, macrophages, and activated B cells (for recent reviews, see Foy et al., 1996; Lenschow et al., 1996; Grewal and Flavell, 1998). In contrast, resting B cells are poor APCs (Frohman and Cowing, 1985; Eynon et al., 1992; Fuchs and Matzinger, 1992).

Well over a decade ago, Cowing and co-workers demonstrated that treatment of resting B cells with C.perfringens sialidase significantly enhanced their capacity to present antigen to allogeneic T cells (Cowing and Chapdelaine, 1983; Frohman and Cowing, 1985). Cowing suggested that the basis of this effect was the removal of blocking sialic acid from B cell MHC II molecules. Later, Kearse et al. (1988) suggested that signaling through the B cell receptor led to expression of hyposialylated MHC II which in turn enabled B/T clustering; however, they presented no data documenting B cell membrane desialylation. Shortly thereafter, Grey and coworkers (Krieger et al., 1988) showed that sialylated and desialylated MHC II molecules present antigen with equal efficacy and concluded that MHC bound sialic acid was unlikely to be the cause of the 'neuraminidase effect." Krieger et al. went on to suggest that increased intercellular adhesiveness induced by decreased cell-surface charge was the most likely cause of the neuraminidase effect. These observations implied that changes in the overall cell surface charge might be a regulatory phenomenon associated with B cell activation. Indeed, it is now generally accepted that lymphocyte adhesion is strongly influenced by the sialic acid content of the glycocalyx, which is thought to be developmentally regulated (Springer, 1990) and that B cells rapidly desialylate substantial numbers of cell surface molecules following activation (Tedder et al., 1997). Examination of the literature, however, reveals no well-documented evidence for substantial changes in the sialic acid content of the B cell glycocalyx during any stage of B cell development. Therefore, we have reexamined the question of changes in the sialic acid content of the B cell glycocalyx following different modes of B cell activation.

Krieger et al. also suggested that sialidase-treated B cells might provide some unidentified accessory factor required by T cells for activation. As few monoclonals to T cell costimulatory molecules were available at that time, they were unable test this suggestion. We have evaluated their hypothesis using anti-CD3-triggered T cells to assay for costimulatory signals on syngeneic B cells and found that sialidase-treated B cells do indeed provide costimulatory activity for T cell proliferation. Further, we show that the B cell glycocalyx sialic acid content, although probably not significantly altered with respect to overall surface charge during activation, nonetheless, plays a significant role in modulating T cell access to at least one important B cell costimulatory molecule, CD86.

Results

Sialidase-treated resting B cells provide effective T cell costimulation in a syngeneic system

In a typical primary mixed lymphocyte reaction, only sialidase-treated allogeneic B cells are stimulatory while sialidase-treated syngeneic B cells are without effect (Figure 1, top). In this case both primary and secondary signals must be provided by the B cells. On the other hand sialidase-treated syngeneic B cells, but not untreated B cells, provide effective costimulation when T cell receptor signaling is triggered by an anti-CD3 directed monoclonal (Figure 1, bottom). This observation supports the suggestion of Krieger et al. that sialic acid on the B cell blocks, in some fashion, access to costimulatory molecules on the B cell surface.


Figure 1. Top: small resting B cells from Balb/c mice and naive T cells from either Balb/c or C57Bl/6 mice were prepared from spleens as described in the Materials and methods section. B Cells were either treated with 0.6 U of sialidase (+) or not (-) and 5 × 105 B cells were mixed with 2.5 × 105 T cells in 0.2 ml of MLC medium in a 96 well flat-bottomed plate. The cells were incubated for 72 h at 37°C, then each well was pulsed with 1 µCi of 3H-thymidine for an additional 18 h, harvested, and the radioactivity of the cells determined by liquid scintillation counting. Bottom: small resting B and T cells prepared from C57Bl/6 spleens were either treated (+) or not (-) with sialidase and then incubated together with 1 µl of culture supernatant of a previously titrated anti-CD3 monoclonal. The cells were incubated and labeled as above.

It is interesting to note that sialidase treatment of the T cells is not effective in promoting costimulation by resting B cells (Figure 1, bottom). This is the first report of such an asymmetry in the effect of sialidase treatment. It should also be noted that the asymmetry exists only with respect to resting B cells as T cell proliferation in response to fixed activated B cells (Krieger et al., 1988, Bagriaçik and Miller, unpublished observations) and nonstimulatory B cell lines (Taira et al., 1986) are restored by sialidase treatment of either the T or the B cell population.

Several known costimulatory/adhesive membrane proteins participate in T cell activation by sialidase-treated resting B cells

To identify the molecules mediating costimulation by sialidase-treated B cells, several monoclonal antibodies to known costimulatory molecules were tested for their ability to block T cell proliferation. A monoclonal specific for an irrelevant MHC was used as a negative reagent control. As seen in Figure 2, the ligand pairs clearly involved in costimulation in this system are CD2/CD48 (blocking by anti-CD48), CD40L/CD40 (blocking by anti-CD40L), and CD28/CD86 (blocking with anti-CD86). In other experiments we have shown that antibodies to CD2, CD54 (ICAM-1), and CD24 (heat stable antigen) are also at least partially blocking, while antibodies to CD40 and CD22 are stimulatory (data not shown). Stimulation by anti-CD40 and anti-CD22 antibodies is consistent with our current understanding of the function of these molecules in the B cell mediated T cell activation (Foy et al., 1996; Tedder et al., 1997). Apparently many of the known costimulatory molecules which may function under different conditions of T cell/B cell collaboration also function during costimulation by sialidase-treated resting B cells. In other experiments we have tested the same panel of monoclonals in assays measuring T cell activation by B cells following treatment of the B cells for 24 h with either with an anti-CD40-directed monoclonal or with LPS. Under these conditions and with the concentrations monoclonals used in Figure 2, no inhibition of T cell activation by the monoclonals was observed (data not shown). This consistent with the observations of others that blocking of CD28-mediated costimulation by anti-CD86 monoclonals is at best incomplete due to the presence of CD80 and other autonomously functioning costimulatory molecules present on the surface of activated APCs (Lenschow et al., 1996).


Figure 2. Small resting B and T cells were isolated as in Fig.1B. B cells were treated with sialidase then incubated with T cells, an anti-CD3 monoclonal, and a second monoclonal as defined. After 42 h (optimal time for anti-CD3 triggered T cell proliferation) the cells were pulsed with tritiated thymidine and counted.

These observations raised the question of whether costimulatory molecules were being induced on the surface of the resting B cells by exposure to anti-CD3 stimulated T cells. In some circumstances engagement of CD40 on B cells by CD40L on T cells (induced by anti-CD3) is known to increase the levels of CD86 and ICAM-1 on B cells, which, subsequently, will induce T cell proliferation (Shinde et al., 1996). It seemed possible that the CD40L expressed on T cells following triggering with anti-CD3 might lead to upregulation of CD86 and ICAM-1 on sialidase-treated B cells versus untreated resting B cells, a possibility which might implicate sialic acid in the regulation of CD40/CD40L interactions. To address this issue we examined expression levels of CD86 and ICAM-1 on sialidase-treated B cells following exposure to T cells which either had or had not been triggered with an anti-CD3 monoclonal. As shown in Figure 3, there is no change in the expression CD86 or ICAM-1 on B cells following in vitro incubation with purified CD4+ T cells which have been triggered with anti-CD3 as compared to the untriggered, resting T cells. When these same cells were assayed for T cell proliferation, it was found that, as before, the T cells in contact with the sialidase-treated but not the untreated B cells proliferated well in response to the anti-CD3 stimulation. Further, analysis of the anti-CD3 triggered population demonstrated that CD40L was indeed being expressed by this population (data not shown).


Figure 3. Separately purified populations of small resting B and T cells from C57Bl/6 mice were incubated together either with (thick line) or without (thin line) an anti-CD3 monoclonal at 37°C for 48 h. the cells were then stained with a FITC-labeled B220 monoclonal and either a PE-labeled anti-CD86 monoclonal (left), a PE-labeled anti-ICAM-1 monoclonal (right), or a PE-labeled isotype control monoclonal (stippled area). B cells were identified as the highly FITC-labeled population and the PE signals were compared. Although a subpopulation of activated T cells are known to express B220 (Watanabe and Akaike, 1994), the two cell populations were still clearly distinguishable under our conditions of activation and T cells did not contribute significantly to the PE signal (data not shown).

In light of the above observations we wondered whether incubation of the B cells in medium alone was sufficient to induce CD86 expression beyond that seen on resting cells. Inaba and co-workers (Inaba et al., 1994) had observed upregulation of CD86 on B cells following incubation in vitro. Because this was not the main thrust of their investigation, we decided to explore this issue again. When resting B cells are incubated overnight in complete medium there is a significant upregulation of CD86 (Figure 4, left, dotted line). Surprisingly, there is almost as much CD86 on the surface of a B cell after overnight incubation in complete medium alone as there is on the surface of a cell induced with anti-CD40 (Figure 4, left, thick line), conditions which are known to lead to significant B cell activation and proliferation (see below). However, incubation in medium alone does not induce expression of ICAM-1 (Figure 4, right, dotted line), which is significantly upregulated by treatment of the B cells with anti-CD40 (Figure 4, right, thick line). We have also observed that B cells induced with anti-CD40 exhibit significant homotypic adhesion whereas those incubated in medium alone do not (data not shown). Lymphocyte homotypic adhesion is known to be principally mediated by ICAM-1/LFA-1 interactions (Springer, 1990; Odum et al., 1991); thus, this observation also supports a lack of ICAM-1 on medium incubated cells.


Figure 4. Freshly isolated small resting B cells (thin lines), B cells incubated 24 h in complete medium (dotted lines), or B cells activated for 24 h with anti-CD40 (thick lines) were stained with PE-labeled anti-CD86 (left), anti-ICAM-1 (right), or an isotype control (stippled area).

Sialidase treatment significantly enhances the costimulatory capacity of fixed activated cells but not of fixed resting cells or unfixed activated cells

The above data suggested that medium incubated B cells might not need to be metabolically active to provide effective costimulation following sialidase treatment. To test this hypothesis B cells were incubated either in medium alone or in the presence of an anti-CD40 monoclonal at a concentration sufficient to activate B cells for T cell costimulation (Figure 5A). After 48 h aliquots of cells from each condition were either treated with sialidase or left untreated then fixed with 0.15% paraformaldehyde. As shown in Figure 5B, fixation completely blocks the capacity of CD40-activated B cells to costimulate; however, sialidase treatment significantly restores the capacity of both CD40-activated and medium incubated B cells to provide costimulation, albeit to lower levels than unfixed activated cells. On the other hand, sialidase treatment of unfixed, CD40-activated cells did little to improve their costimulatory capacity (Figure 5C). These experiments have been repeated several times using different modes of B cell activation with no substantive differences in outcome.


Figure 5. The graphs in this figure are derived from a single experiment, but are representative of several experiments conducted at different times.(A) B cells incubated 48 h in complete medium (triangles) or B cells activated for 48 h with anti-CD40 (squares) were used to stimulate anti-CD3 triggered, CD4+ syngeneic T cells in a 48 h proliferation assay. (B) B Cells incubated 48 h in complete medium (triangles) or B cells activated for 48 h with anti-CD40 (squares) were treated with sialidase (open symbols) or not (solid symbols) then fixed with paraformaldehyde and used to stimulate anti-CD3 triggered, CD4+ syngeneic T cells in a 48 h proliferation assay.(C) B Cells activated for 48 h with anti-CD40 were treated with sialidase (open symbols) or not (solid symbols) and used to stimulate anti-CD3 triggered, CD4+ syngeneic T cells in a 48 h proliferation assay. (D) B Cells were activated for 48 h with anti-CD40 (squares) or with anti-IgM/IFN-[gamma] (triangles) were fixed with paraformaldehyde and either treated with sialidase (open symbols) or not (solid symbols) and used to stimulate anti-CD3 triggered, CD4+ syngeneic T cells in a 48 h proliferation assay.(E) B cells activated for 48 h with anti-IgM/IFN-[gamma] were either treated with sialidase (open symbols) or not (solid symbols) and then fixed with paraformaldehyde and used to stimulate anti-CD3 triggered, CD4+ syngeneic T cells in a 48 h proliferation assay. (F) Freshly isolated B cells were either treated with sialidase (open symbols) or not (solid symbols) then fixed with paraformaldehyde and used to stimulate anti-CD3 triggered, CD4+ syngeneic T cells in a 48 h proliferation assay.

Since anti-IgM(Fab)2 plus IFN-[gamma] activated B cells are potent antigen presenting cells expressing high levels of CD86 (Morokata et al. 1995), we decided to investigate the capacity of sialidase to enhance costimulation by these cells following fixation. If loss of surface charge due to desialylation were a normal and significant feature of B cell activation, then treatment of highly activated, fixed B cells with sialidase should only minimally enhance their capacity to costimulate, as seen with unfixed cells. To test this hypothesis, we activated resting B cells with these reagents and then fixed the cells with paraformaldehyde as above. Unlike the anti-CD40 activated cells (Figure 5C), these cells were effective costimulators following fixation and were as active as the fixed anti-CD40 activated cells which had been sialidase treated (Figure 5D). On the other hand, treatment of the fixed anti-IgM/IFN-[gamma]-activated cells with sialidase enhanced their capacity to costimulate to very high levels, i.e., beyond that seen even with unfixed, CD40-activated cells (compare Figure 5A and 5E). Note that the non-sialidase-treated sample is the same in Figure 5D and 5E. The apparent difference is due to the high level of T cell stimulation seen with the anti-IgM/IFN-[gamma]-activated, sialidase-treated cells. Thus, following fixation the costimulatory capacity of even highly activated cells expressing high levels of CD86 and other costimulatory and adhesive molecules is still significantly enhanced by sialidase treatment. This observation suggested that loss cell surface charge might not be a major event during B cell activation.

Krieger et al. had observed that fixation of resting cells eliminated their capacity to stimulate allogeneic T cell proliferation and that sialidase treatment could not restore the costimulatory activity. As shown in Figure 5F, we have confirmed their observation and attribute the inability of fixed, sialidase-treated resting cells to costimulate to the lack of CD86 on the resting cell surface (Figure 4, left, thin line), thus supporting the hypothesis of Krieger et al. that resting B cells must upregulate a costimulatory ligand before sialidase treatment can be effective, ostensibly CD86.

Highly activated B cells do not globally alter their N-linked sialic acid content

During germinal center formation, B lymphocytes show significant increases in the binding of peanut agglutinin (PNA), a lectin sensitive to the sialylation state of terminal Gal[beta]1,3GalNAc residues carried on O-linked glycans. It has been shown that T cell allo-blasts (Forman and Puré, 1991) and supernatants from anti-CD3 stimulated CD4+ T cells (Lahvis and Cerny, 1997) can cause the conversion of splenic B cells from a PNA- to a PNA+ phenotype. Further, Baum and co-workers have shown, at least for T cells, that the PNA binding phenotype is tightly controlled by the regulated expression of various sialyltransferases (Gillespie et al., 1993; Baum et al., 1996). In related experiments we have found that IFN-[gamma]is able to substitute for T cell supernatant for the induction of PNA receptors on the B cell surface and the development of a partial germinal center phenotype (Bagriaçik and Miller, unpublished observations).

B Cells that had been activated either by incubation in medium alone, with a monoclonal anti-CD40 antibody, or with polyclonal anti-IgM (Fab)2 plus IFN-[gamma] were evaluated for the binding of PNA and Jacalin (Gal[alpha]1,3GalNAc), ECA (Gal[beta]1,4GlcNAc), SNA (NeuAc[alpha]2,6Gal), MAL II (NeuAc[alpha]2,3Gal[beta]1,4GlcNAc), and WGA (NeuAc and GlcNAc) (see Cummings, 1994, for lectin specificity). As shown in Figure 6, both PNA and Jackalin binding is increased following B cell activation with anti-IgM plus IFN-[gamma], but not following medium incubation or activation with anti-CD40. This suggests that anti-IgM plus IFN-[gamma] induces the de novo appearance of Gal[beta]1,3GalNAc residues. It is likely that these new residues are associated with the B cell CD45 molecule (Cook et al., 1987).


Figure 6. B cells were incubated 48 h in complete medium alone (dotted line), with anti-CD40 (thin line), or with anti-IgM/IFN-[gamma] (thick line), then stained with biotinylated jacalin (JAC), PNA, SNA, ECA, MAA II, or WGA, then counterstained with PE-streptavidin and analyzed by flow cytometry. Stippled area shows binding of PE-streptavidin in the absence of lectin.

There is little change, however, in the mean fluorescence intensity for either MAL II, ECA, or WGA. ECA is a particularly sensitive marker for sialidase activity on N-linked carbohydrates, and we use it routinely to assess the effectiveness of our sialidase treatments (data not shown). There is a modest but reproducible reduction in the number of cells binding SNA (~10%). Interestingly, those cells affected seem to go from being highly SNA positive to being completely SNA negative. The lack of an intermediate level of binding suggests a limited diversity and/or a high turn over rate of SNA targets on the B cell surface. The existence of two distinct murine B cell populations under all three conditions, i.e., medium incubated, anti-CD-40 stimulated, and anti-IgM/IFN-[gamma] stimulated, which are distinguishable by SNA binding is quite reproducible but is currently unexplained. Because of the large number of cells found in the negative peak (10-20%) versus the purity of our B cells preparations (95-98%), it is unlikely that these cells are contaminates.

Gross and colleagues (Gross et al. 1996) have developed a fluorescent substrate, FITC-conjugated CMP-neuraminic acid (CMP-NANA-Fl) useful for assaying sialyltransferase activity. They used this reagent to demonstrate the existence of an ecto-sialyltransferase activity on human B cells and B cell lines which is capable of reconstituting known carbohydrate B cell differentiation markers (CDw75 and HB-6) following sialidase treatment. We have used a similar approach to evaluate the sialylation state of N-linked terminal galactose residues on medium-incubated versus CD40-activated B cells. In our hands the combination of CMP-NANA-Fl and rat liver 2,6 sialyltransferase (EC2.4.99.1) efficiently labels sialidase treated cells compared with untreated cells (Figure 7, top). When this enzyme-substrate combination is used on medium incubated cells, there is modest incorporation in the absence of exogenously added enzyme; however, there is significant incorporation when enzyme is added to the reaction. This is consistent with the significant level of ECA binding by these cells and supports the existence of numerous terminal Gal[beta]1,4GlcNAc residues, the favored substrate for the rat liver enzyme. Interestingly, when the same experiment is performed with CD40 activated cells, there is significant incorporation of substrate without exogenously added enzyme (Figure 7, bottom). This is consistent with the activation of an endogenous ecto-sialyltransferase in murine B cells as observed for human cells by Gross et al. Further, addition of exogenous enzyme does not lead to further incorporation indicating these two enzymes may compete for the same targets on the B cell surface. Most importantly though, there is no significant difference in total incorporation between medium incubated cells and CD40 activated cells, again suggesting that there are no large quantitative differences in the level of N-linked sialic acid between these two populations.


Figure 7. Top: small resting B cells were incubated in sialyltransferase buffer as described in Materials and methods, either alone (stippled area), with CMP-NANA-Fl (thin line), or with CMP-NANA-Fl plus rat liver2,6 sialyltransferase (thick line), either with (Sialidase) or without (Untreated) prior sialidase treatment. The cells were then washed and analyzed by flow cytometry. Bottom: B cells incubated 24 h in complete medium alone (Medium), or with anti-CD40 (Anti-CD40) were incubated in sialyltransferase buffer as described in Materials and methods, either alone (stippled area), with CMP-NANA-Fl (thin line), or with CMP-NANA-Fl plus rat liver 2,6 sialyltransferase (thick line). The cells were then washed and analyzed by flow cytometry.

From these results we conclude that while there is some specific loss of 2,6-linked sialic acid and de novo expression of Gal[beta]1,3GalNAc residues following B cell activation with anti-IgM plus IFN-[gamma], there are no significant quantitative differences in sialic acid content between CD40-activated and medium-incubated cells which could contribute significantly to the enhanced costimulatory capacity of the CD40 activated cells.

Discussion

It has been known for over a decade that sialidase treatment could substantially enhance the capacity of B cells to stimulate the proliferation of allogeneic and antigen specific syngeneic T cells (Cowing and Chapdelaine, 1983; Frohman and Cowing, 1985). Although these observations implicated cell-surface sialic acid as a potential modulator of immune cell interaction, little progress had been made in either identifying explicit roles for sialic acid in this system or in hypothesizing mechanisms to explain the 'neuraminidase effect." Although Cowing and coworkers suggested that hypersialylation of MHC II on resting B cells might block MHC/TCR engagement, Krieger et al. (1988), using purified IAd in liposomes bound to glass beads, found that MHC II from small resting B cells presents antigen as effectively as that purified from activated cells, and that sialidase treatment was without effect. Additionally, Nag et al. (1992) found that both in vitro deglycosylated I-As and untreated I-As could induce T cell energy when exposed to T cells in soluble form. It is now clear that resting B cells express MHC II molecules capable of antigen binding and TCR engagement (Zhong et al., 1997), an event which leads to partial activation of the T cell. However, they fail to effectively display the costimulatory ligands required for full T cell activation (Ho et al., 1994; Croft et al., 1997; Jaishwal and Croft, 1997). Kreiger et al. postulated three possibilities for the presentation defect in resting B cells: (1) these cells present too little MHC II; (2) these cells fail to express a necessary accessory factor; (3) these cells lack the necessary 'cell interaction" molecules. They were able to discount option 1, but were unable to decide between 2 and 3. In this report we show that both suppositions 2 and 3 are correct.

Here we show for the first time that sialidase treatment restores the costimulatory capacity of fixed B cells which have previously been either medium incubated or CD40 activated. Under conditions of mild activation, i.e., using a nonimmobilized anti-CD40 monoclonal, the stimulatory capacities of fixed medium incubated cells and fixed activated cells are virtually identical, both before and after sialidase treatment (Figure 5B). This is an important observation, because the stimulatory capacity of the two unfixed cell populations differs substantially (Figure 5A). This observation demonstrates for the first time that expressing high levels of CD86 alone is insufficient for costimulation. In light of the fact that both populations express almost equal amounts of CD86 on their surfaces (Figure 4), while only the CD40 activated cells express high levels of ICAM-1 (Figure 4), it seems reasonable to postulate that the upregulation of ICAM-1 (or other equivalent adhesion enhancing molecules) is a necessary prerequisite for successful engagement of T cell CD28 by CD86. The alternative explanation, that ICAM-1 alone is responsible for T cell costimulation in the CD40 activated cell, is belied by the legion of reports implicating CD86 as the primary T cell costimulatory molecule in this and other systems (for reviews, see Foy et al., 1996; Lenschow et al., 1996; Grewal and Flavell, 1998). Thus it seems very likely that ICAM-1 and CD86 collaborate on the CD40 activated cell to provide effective T cell costimulation. Indeed, it has been noted that T cells from LFA-1, and CD28 knock-out mice exhibit profoundly reduced proliferation in response to Con A stimulation, as well as to alloantigen (Shahinian et al., 1993; Shier et al., 1996). That both systems exhibit similar defects suggests that LFA-1/ICAM-1 and CD86/CD28 make distinct contributions to T cell activation, each of which is necessary but not sufficient for full proliferation. Restoration of fixation blocked costimulation to both medium and anti-CD40 activated B cells by neuraminidase treatment suggests that fixation may be blocking LFA-1/ICAM-1 function and that in this system LFA-1/ICAM-1 function is primarily one of bridging the glycocalyx and increasing adhesion. It also seems clear that on the resting cell, a number of costimulatory ligands besides CD86 are exposed by sialidase treatment, as evidenced by the monoclonal blocking experiments (Figure 2). It seems likely that none of these costimulatory signals is individually sufficient to activate a resting T cell. However, in combination and when exposed by sialidase treatment of the B cell, they are sufficient to push at least a portion of the anti-CD3 triggered T cells into DNA synthesis.

Although sialidase treated B cells do not upregulate ICAM-1 in response to anti-CD3 triggered T cells, it is unlikely that T cell CD40L fails to engage CD40 on the sialidase-treated B cell because anti-CD40L antibody is blocking for T cell proliferation (Figure 2). It is more likely CD40L does engage CD40 but that CD40 signaling is insufficient to cause significant upregulation of ICAM-1. It has been reported that CD40Llow T cells (generated by triggering the T cell with low amounts anti-CD3, as in our experiments) are unable to induce proliferation of small resting B cells unless the B cells are simultaneously triggered with anti-sIg (Poudrier and Owens, 1994). Further, in that report the T dependent proliferation of the anti-sIg-triggered B cells was blocked by antibody to ICAM-1. In any event, it is seems clear that costimulation of anti-CD3 triggered T cells by sialidase-treated resting B cells does not require upregulation of ICAM-1.

It is interesting to note, however, that sialidase treatment of T cells, which also increases adhesion, is not sufficient to allow resting B cells to costimulate (Figure 1). This suggests to us that removal of sialic acid from the surface of the resting cell plays some additional role in preparing the cell to function in costimulation. We have noticed the enhanced binding of two anti-CD86 monoclonals (PO3 and GL-1) to B cells following sialidase treatment, while the binding of CTLA-4Ig was unperturbed (Bagriaçik and Miller, unpublished observations). However, it must be keep in mind that the binding sites for CTLA-4 and CD28 on CD86, while overlapping, are not identical and CD28 has a much reduced affinity for CD86 compared with CTLA-4 (Kariv et al., 1996). As we have not tested the binding of soluble CD28 to sialidase-treated cells, it remains a possibility that this asymmetry arises as a result of enhanced CD28 interactions with asialo-CD86 and thus warrants further investigation. On the other hand, B cell surface sialic acid is known to negatively modulate the interaction of CD22 with T-cell CD45 (Sgroi et al., 1993; Braesch-Andersen and Stamenkovic, 1994), and there are other highly sialylated costimulatory molecules on the surface of the resting B cell, e.g., HSA (CD24), which may also respond to sialidase treatment with enhanced T cell ligand binding; thus, sialidase treatment may yet be an analog for an authentic step in B cell activation.

Morokata et al. (1992) attributed the enhanced stimulatory capacity of anti-IgM plus IFN-[gamma] treated B cells compared with B cells activated by treatment with anti-IgM alone to enhanced expression of CD86. However, CD86 expression was only enhanced 2-fold by the addition of IFN-[gamma] whereas CD86 expression was enhanced 15-fold on anti-IgM triggered cells compared with untreated resting population. Further, in our hands paraformaldehyde-fixed, anti-IgM plus IFN-[gamma] activated cells are costimulatory whereas fixed CD40 activated cells are not, although both express high levels of CD86 and ICAM-1 compared with resting cells. The major difference that we have observed between the two populations resides in the sialylation state of specific molecules, i.e., CD45 - the PNA receptor (Cook et al., 1987) and those molecules affected by the loss of 2,6-linked sialic acid as detected by SNA binding (Figure 6). We suggest that these changes may account for the ability of this cell population to costimulate without sialidase treatment following fixation. The development of methods for the comparative analysis of the glycosylation state of nanogram amounts of purified glycoproteins should aid in sorting out this question (Goodarze and Turner, 1997).

We have shown that incubation of resting B cells with anti-IgM plus IFN-[gamma] induces specific changes in the carbohydrate composition of the B cell glycocalyx (Figure 6). That such changes do not occur following anti-CD40 stimulation is indicative of the differential signaling pathways which must control expression of the various glycosyltransferases involved in production of the B cell surface glycoconjugates. It will be important to dissect the various levels of controls which influence the production of each phenotype. While there are several reports of developmental control on such enzymes in T cells, this is the first report of an apparent down regulation of a 2,6-sialyltransferase in B cells. Why only a portion of the B cells express SNA binding glycans is currently not known but is under active investigation in our laboratory. Whether SNA binding defines distinct and functionally important B cell compartments remains to be seen.

Materials and methods

Mice

Six 8 week old, male and female Balb/c (H-2d) and C57Bl/6 (H-2b) mice were purchased from Jackson Laboratory, Bar Harbor, ME, and maintained in the vivarium at University of Tulsa.

Isolation of small resting B cells

Resting B cells were isolated as described by Croft and Swain (1995) with minor modifications. Briefly, spleens were collected aseptically from 5- to 8-week-old male or female mice and mashed through a steel screen into 10 ml of Hank's balanced salt solution (HBSS). Cells were incubated in complete medium (RPMI-1640 supplemented with glutamine, 2-mercaptoethanol, penicillin, streptomycin, and 10% fetal calf serum) on plastic at 37°C to remove adherent cells, and then red blood cells were lysed by incubation at room temperature with 3 ml of Tris-ammonium chloride solution, pH 7.2. Nonadherent cells were separated on a four-layer (50, 60, 70, and 80%) discontinuous gradient of Percoll (Pharmacia, Uppsala-Sweden) by 20 min centrifugation at 600 × g and cells from 60-70% interface were recovered. These cells were treated with a cocktail of anti-Thy1.2, anti-CD8, anti-CD4, and anti-Mac1[alpha] monoclonal antibodies at 4°C for 30 min and were depleted by two rounds of complement-mediated lysis using Low-Tox guinea pig complement (Accurate Chemical, Westbury, NY). Remaining cells were again separated on Percoll (as above) and cells at the 60%-70% interface were recovered. These cells were estimated to be 95-98% pure B cells by CD19 staining and flow cytometry.

B Cell activation

B Cells were activated by incubation in 24 well tissue culture plates in complete medium (106/ml) containing either anti-CD40 (10 µg/ml) or anti-IgM (30 µg/ml) plus IFN-[gamma] (30 U/ml). After 24-48 h, depending on the experiment, cells were harvested and used for T cell activation or lectin staining.

Isolation of small resting CD4+ T cells

CD4+ T cells were prepared according to the protocol of Croft et al. (1992) with only minor modifications. Spleen cells were incubated in 100 × 20 mm polystyrene tissue culture dishes (Corning, Park Ridge, IL) for 1 h at 37°C, 5% CO2 to remove adherent cells. Nonadherent cells were incubated with a cocktail of anti-Mac-1[alpha], anti-HSA (J11d), anti-CD8 (3.155) monoclonal antibodies on ice for 30 min. Cells having bound monoclonals were lysed in HBSS containing 10% Low-Tox guinea pig complement at 37°C in a water bath for 45 min. The depletion process was repeated twice. The depleted cells were then passed over a nylon wool column.

The B and T cell preparations described above were routinely stained with monoclonal antibodies specific for known B (CD19) and T (Thy-1) markers and analyzed by flow cytometry. The resting B and CD4+ T cell preparations used in this study were found to be >98% and >94% pure, respectively.

Sialidase and mitomycin-C treatment of resting B cells

For sialidase treatment (Powell et al., 1987), resting B cells were incubated in HBSS containing 0.6 U Clostridium perfringens sialidase (EC 3.2.1.18) (Sigma, St. Louis, MO) per 5 × 107 cells for 30 min at 37°C. Cells were then washed twice by centrifugation. B cells were incubated in HBSS with 100 cells for 30 min at 37°C. Cells were then washed twice by centrifugation. B cells were incubated in HBSS with 100 with medium by centrifugation.

Proliferation assays

For the allogeneic proliferation system (Frohman and Cowing, 1985), B cells (5.0 × 105 cells) from Balb/c mice were incubated with 2.5 × 105 T cells from C57Bl/6 mice, in triplicate wells of 96-well, flat-bottom microtiter cell culture plate (Falcon, Lincoln Park, NJ) in 200 T cells from C57Bl/6 mice, in triplicate wells of 96-well, flat-bottom microtiter cell culture plate (Falcon, Lincoln Park, NJ) in 200 µl of RPMI-1640 medium (Sigma, St. Louis, MO) containing 10% fetal bovine serum, 2 mM l-glutamine, 25 mM HEPES, 5 U/ml penicillin, and 5 µg/ml streptomycin (Sigma, St. Louis, MO) 37°C, 5% CO2 for 80 h. The cell cultures were pulsed with 1 µCi/well of 3H-thymidine (ICN Radiochemicals, Irvine, CA) 24 h before harvesting on a microanalytical cell harvester (M.A. Bioproducts, Walkersville, MD). The amount of 3H-thymidine incorporation was measured by liquid scintillation counting (LS-3801-Beckman, Irvine, CA).

For the proliferation of syngeneic cells, 2 × 105 B cells were incubated with 1 × 104 T cells in 200 µl of MLC medium containing 10 µl of 1:80 dilution of an anti-CD3 monoclonal antibody culture supernatant (monoclonal R3/7.159, a gift of Dr. J. Klein, University of Tulsa). Cells were incubated for 42 h in round-bottomed, 96-well tissue culture plates at 37°C, 5% CO2; pulsed with 3H-thymidine for 6 h; and then harvested. Thymidine incorporation was measured by liquid scintillation counting.

Cell staining with lectins

Cell staining with lectins was performed as previously described (Bagriaçik et al., 1996). All cells were stained with biotinylated lectins from Vector Labs., Inc., Burlingame, CA; 5 × 105 cells were stained in 100 µl final volume of HBSS containing 5-20 µg/ml of lectin for 30 min in the dark at 4°C. The cells were then counterstained with phycoerythrin (PE)-conjugated streptavidin (PharMingen, San Diego, CA) and fixed with 200 µl of 3.7% formaldehyde prepared in HBSS.

Antibodies and immunofluorescence analysis

Hybridoma lines, GL-1 (anti-B7.2), M1/70 (anti-mac1[alpha]), GK1.5 (anti-CD4), AF6-120 (anti-MHC class II), J11d (anti-HSA), J1j.10 (anti-Thy1.2), 3.155 (anti-CD8) were obtained from the ATCC. Monoclonal antibodies, 1D3 (anti-CD19), HM40-3 (anti-CD40), HM48-1 (anti-CD48), RM2-5 (anti-CD2), MR1 (anti-CD40L), RM4-5 (anti-CD4), 30-H12 (anti-Thy1.2), 53-6.7 (anti-CD8), 23G2 (anti-CD45RB), 2.4G2 (anti-CD16/CD32), and 3E2 (anti-ICAM-1) were purchased from PharMingen (San Diego, CA).

Cells (106) were first incubated with an Fc receptor blocker (anti-CD16/CD32) according to the manufacturer recommendations and then titrated concentrations of monoclonal antibodies conjugated either with phycoerythrin (PE) or biotin in 100 µl of HBSS containing 0.01% sodium azide for 30 min at 4°C. The cells were then washed and fixed in 2% formaldehyde prepared in HBSS. For biotin labeled antibody staining, PE-conjugated streptavidin (PharMingen, San Diego, CA) was used as a secondary reagent.

Cells stained with lectins or antibodies were analyzed using an EPICS 751 flow cytometer interfaced with a Cicero data acquisition unit running Cyclops software (Cytomation Inc. Fort Collins, CO). List mode data was analyzed with the WinMDI 2.6 program written by J. Trotter of The Scripps Research Institute. All flow diagrams were smoothed 10 times using the WinMDI smoothing routine to increase clarity.

Labeling cell surface Gal[beta]1,4GalNAc with CMP-NANA-Fl

Carbohydrate termini were labeled using rat liver [alpha]2-6 sialyltransferase (Boehringer Mannheim Corp., Indianapolis, IN) according to the protocol of Whitehart et al. (1990). Briefly, after purification of the enzyme on CDP-hexanolamine-Sepharose and Sephadex G-50, 2 × 107 cells will be labeled in 50 µl of sialyltransferase buffer containing 6.25 nmol of cytidine-5[prime]-monophospho-9-(3-fluoresceinyl-thioureido)-9-deoxy-N-acetyl-neuraminic acid (CMP-NANA-Fl) and terminated by the addition of 1 ml of ice-cold HEPES-buffered saline. Cells were washed in HBSS and analyzed by flow cytometry as above.

Acknowledgments

We thank Dr. John Klein for critically reviewing the manuscript. This work was supported by grants to K.S.M. from The Mervin Bovaird Center for Studies in Molecular Biology and Biotechnology and The National Institute of Allergy and Infectious Diseases.

Abbreviations

APC, antigen presenting cell; B220, leukocyte common antigen (CD45R);BCR, B cell receptor (IgM or IgD); HSA, heat stable antigen (CD24); CD40L, CD40 ligand (CD154); CTLA-4, (CD152); ECA, Erythrina cristagalli agglutinin; FITC, fluorescein; ICAM-1, intracellular adhesion molecule 1 (CD54); IgM, immunoglobulin class M; Jacalin, Artocarpus integrifolia agglutinin; LFA-1, lymphocyte function associated antigen 1 (CD11a/CD18); LPS, lipopolysaccharide from E.coli; MAA II, Maackia amurensis agglutinin II; MHC II, major histocompatibility locus, class II; PE, phycoerythrin; PNA, Arachis hypogaea agglutinin; sIg, surface immunoglobin (IgM or IgD); SNA, Sambucus nigra agglutinin; TCR, T cell receptor; WGA, Tritcum vulgaris agglutinin.

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1To whom correspondence should be addressed at: Faculty of Biological Science, 600 South College Avenue, The University of Tulsa, Tulsa, OK 74104-3189


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