IgD expression on B cells is more efficient than IgM but both receptors are functionally equivalent in up-regulation CD80/CD86 co-stimulatory molecules

1980 R. Brink et al. Eur. J. Immunol. 1995.25: 1980-1984

Robert Brink,

Antony Basten

IgD expression on B cells is more efficient than IgM but both receptors are functionally equivalent Christopher C. Goodnow and

in up-regulation CD80/CD86 co-stimulatory Centenary Institute of Cancer Medicine and Cell Biology, molecules Newtown, Australia

The expression and function of IgM and IgD antigen receptors were studied in a series of anti-hen egg lysozyme (HEL) immunoglobulin (1g)-transgenic mice expressing either IgM alone, IgD alone, or both IgM and IgD. B cell surface expression of IgD was found to be more efficient than that of IgM. Thus antigen receptor density on IgD+, IgM- B cells was twofold higher than on IgM+, IgD- B cells despite the presence of sevenfold lower levels of membrane heavy chain mRNA, and coexpression of IgD with IgM led to almost complete inhibition of surface IgM. In addition, less extensive down-regulation of IgD occurred following exposure to antigen in vitro. When regulation of CDSO/CD86 co-stimulatory molecules by surface Ig was examined, up-regulation of the former was initiated at lower antigen concentrations on IgM-, IgD’ compared to IgM’, IgD- B cells. On correcting for antigen receptor density, however, induction of CD80/CD86 by IgM and IgD was comparable. Taken together, these results reinforced the functional similarity of IgM and IgD antigen receptors while at the same time revealing differences in expression which may explain their simultaneous presence on mature B cells.

1 Introduction

Mature B cells coexpress antigen receptors of the IgM and IgD classes [I]. However the functional characteristics of IgM and IgD appear remarkably similar [2-71 with both receptors being non-covalently associated in the B cell membrane with Iga and Igfi polypeptides [8] and eliciting the same intracellular biochemical signals in B cells following receptor cross-linkage [9-121. Not surprisingly therefore, studies of Ig-transgenic mice containing B cells specific for hen-egg lysozyme (HEL) [13-151 have indicated that either IgM or IgD alone can mediate efficient B cell activation in response to HEL [13]. On the other hand, an auxiliary receptor function for IgD is implied by the observation in IgD-“knockout” mice of delayed antibody production and affinity maturation in response to certain T cell-dependent antigens [16] despite a lack of gross differences in the responses of IgD- and wild-type B cells 116, 171.

[I 140861

Present addresses: R. Brink, Whitehead Institute for Biomedical Research, Nine Cambridge Center, Cambridge, MA 02142, USA; C. C:. Goodnow, Howard Hughes Medical Institute and Depart- ment of Microbiology and Immunology, Beckman Center, Stan- ford University, CA 94305, USA Correspondence: Antony Basten, Centenary Institute of Cancer Medicinc and Cell Biology, Locked Bag No. 6, Newtown NSW 2042. Australia (Fax: 61-2-565 6111)

Abbreviations: B 6 C57BL/6 HEL: Hen-egg lysozyme and 6,: p- and S-membrane IgH chains VJO: V region exon of HyHELlO mAb

Key words: B cell / IgM / IgD / CD80 / CD86

To investigate the basis of this phenomenon in more detail, the expression of IgM and IgD antigen receptors per se as well as their abilities to mediate antigen-dependent up- regulation of the CD80 (B7-UBBl) [18, 191 and/or CD86 (B7-2/B70) [ 18, 201 co-stimulatory ligands were examined in anti-HEL Ig-transgenic mice. Surface expression of IgD was found to be more efficient as well as dominant over that of IgM, with the result that IgD’ B cells exhibited a higher antigen receptor density than those expressing IgM alone. Although IgM and IgD did not differ in their intrinsic abilities to up-regulate cell surface CD80/CD86, the increased antigen receptor density associated with IgD expression resulted in more efficient CD80/CD86 induction, indicating a possible basis for the auxiliary receptor function of IgD.

2 Materials and methods

2.1 Mice

Production and screening of Ig-transgenic lines has been described [12, 131. MD-3, MD-4 (VH10-p-6 H chain transgene), MM-4, MM-7 (VH10-p), and DD-1, DD-6 (V,10-6) lines were bred on a C57BL/6 (B6) (IgHh) background and expressed transgene encoded IgM” and/or IgDa molecules with the anti-HEL specificity of the HyHEL 10 mAb [21]. MMx DD mice were produced by mating MM and DD parents and identified by the presence of anti-HEL I g M and IgD” in serum.

2.2 Northern blot analysis

Total cellular RNA was prepared as described [22]. To deplete B cells (Ig’), spleen cells (lo7 cells/ml) were incu-

00 14-2980/95/0707- 1 980$10.00 + .25/0 0 VCH Verlagsgesellschaft mbH, D-69451 Weinheim, 1995

Eur. J. Immunol. 1995.25: 1980-1984 Differential expression but functional identity between IgM and IgD 1981

bated with sheep anti-mouse IgG-coated Dynabeads M- 450 (Dynal, Oslo, Norway; 2 x lo7 beads/ml) at 4°C for 30 min in RPMI/2Yo FCS, and bound cells removed by magnetic separation. RNA electrophoresis, blotting, and hybridization to 32P-labeled cDNA probes was as described [23]. Probes used were specific for (i) the rearranged H chain variable region of HyHELlO (V,lO), (ii) membrane exons of y,, (iii) membrane exons of 6,, and (iv) glyceral- dehyde 3-phosphate dehydrogenase (GAPDH; kind gift of Dr. Steve Gerondakis, Walter and Eliza Hall Institute). Membranes originally hybridized with VHIO, y, or 6, probes were rehybridized with the GAPDH probe 10 weeks later after confirming the absence of residual radioactivity. Quantitation of radioactivity was by densitometry using the Gel Scan module of a DU-68 Spectrophotometer

brane H chain mRNA was assessed by comparing hybridization to RNA from B cell-depleted and non-depleted spleens. Relative levels of membrane H chain mRNA per B cell (Fig. 1A) were derived by correcting for (i) non-B cell expression, (ii) amount of mRNA (GAPDH hybridization), and (iii) proportion B220' cells.

I (Beckman, Palo Alto, CA). The B cell specificity of mem-

2.3 Antibodies

The following mAb were used: RS-3.1 (IgM"), AMS-15.1 (IgD"), HyHEL5 (HEL), and RA3-6B2 (B220). CD80/ CD86 was detected using a secreted fusion protein of mouse CTLA-4 and the Fc region of human IgGl (CTLA- 4/1g; kind gift of Dr. Phillip Hodgkin, Australian National University; [24]). PE-labeled RA3-6B2 was from Caltag (South San Francisco, CA) and biotinylated goat anti- human IgG 1 F(ab')2 from Jackson Immunoresearch (West Grove, ME).

2.4 FCM

Spleen cells were stained with fluoresceinated and biotinylated Ab followed by streptavidin-PE (SNPE) (Caltag). HEL-binding Ig was detected with 200 ng/ml HEL (Sigma, St. Louis, MO) followed by HyHELSbiotin and SA/PE. CD80/CD86 molecules wee detected with CTLA- 4/Ig followed by goat anti-human IgGl F(ab'),/biotin and SA/PE. Four or five parameter data were collected on a FACScana (Becton Dickinson & Co., San Jose, CA) and fluorescence profiles derived from cells with lymphoid cell light scatter characteristics. Two-dimensional contour plots were generated with LYSIS II@ software using 5 YO probab- ility contours.

2.5 In vitro cultures

Spleen cells were incubated with RPMI/10 YO FCS at 37 "C in 5 % CO, (2.5 x lo6 cells/ml) in 96-well round-bottom microtiter plates (5 x lo5 celldwell) in the presence or absence of HEL and then washed and stained for cell surface molecules. Staining was analyzed by FCM with data from dead cells excluded on the basis of propidium iodide (PI) incorporation.

3 Results

3.1 Ig-transgenic B cells express higher levels of u. than 6, mRNA

B cells from anti-HEL Ig-transgenic mice were first analyzed for quantitative differences in the expression of IgM and IgD which may account for the auxiliary receptor function of IgD. As a first step, relative levels of membrane forms of p and 6 (y, and 6,) H chain mRNA were measured. B cells from the three types of anti-HEL Ig- transgenic mice express either y,/IgM and 6,/IgD in the normal maturational pattern (MD mice) [13, 141 or express y,/IgM (MM mice) or 6,/IgD (DD mice) alone [ 121. Direct comparison of transgene-encoded membrane H chain mRNA in MM-7 and DD-6 B cells could therefore be achieved using the transgene-specific variable region probe VHIO. Bands detected with this probe in splenic RNA from MM-7 and DD-6 mice were confirmed to represent transgene-encoded 1.1, and 6 , mRNA species respectively (data not shown). Quantitation of these spe-

A

B HEL binding

1 VHIO probe 1 I 6, probe

MM-7 DD-6 DD-6 Non-Tg MD-4

lgMa igDa

I n I

10 10 10

* Fluorescence

Figure 1. Expression of transgene-encoded Ig by anti-HEL Ig- transgenic B cells. (A) Quantitation of membrane H chain mRNA expression. Northern blot analysis and quantitation of the relative levels of membrane H chain species per B cell is described in Sect. 2.2. Species detected with theV,10 probe in MM-7 and DD- 6 B cells were assumed to be the p,,, and 6, mRNA in these cells (dotted lines) thus allowing direct comparison of the levels of p, and 6, mRNA between B cells. (B) IgD' Ig-transgenic B cells express higher levels of antigen receptor than IgD- Ig-transgenic B cells. Spleen cells were stained for FCM either with RA3-6B2/ fluorescein (left) or RA3-6B2/PE (middle and right) to detect B220+ cells and counterstained with HEL + HyHELYbiotin + SA/PE (left panels), RS-3.l/fluorescein (middle panels), or AMS- lS.l/fluorescein (right panels). Staining for HEL-binding, IgMa and IgD" on B220' B cells is indicated. Non-transgenic B6 (IgHh+) B cells served as the negative control for all stains.

1982 R. Brink et al.

cies revealed that the level of 6 , mRNA in DD-6 B cells wa5 sevenfold lower than that of p, mRNA in MM-7 B cells (Fig. IA). Based on this result and on hybridizations with pm- and &,-specific probes, the p, to 6, mRNA ratio was calculated to be 5.9: 1 in MD-4 cells and 4.6: 1 in non- transgenic B cells (Fig. lA), figures consistent with previ- ous estimates of 5 : 1 and 10: 1 for B cells normal spleen [25, 261.

3.2 IgD+ B cells express more sIg than those expressing IgM only

The next step in the quantitative comparison of IgM and IgD expression was to relate the levels of p, and 6, RNA measured in the Ig-transgenic B cells with surface IgM and IgD levels as detected by staining of spleen cells for transgene-encoded H chains (pa and 6") and antigen binding sites (HEL-binding) (Fig. 1B). Upon FCM analysis it was found that expression of IgD as well as IgM by MD-4 mice resulted in increased numbers of antigen receptors (HEL-binding sites) per B cell compared to B cells from MM-4 and MM-7 mice (Fig. 1B). Nevertheless, the number of antigen receptors proved to be highest on B cells from DD-1 and DD-6 mice. Thus, comparison of HEL- binding site densities revealed the presence of twofold or more IgD molecules on DD-1 and DD-6 B cells compared to IgM molecules on MM-4 and MM-7 B cells (Fig. 1B) despite the fact that the levels of 6, mRNA in DD-6 B cells were sevenfold lower than p, mRNA levels in MM-7 B cells (Fig. ZA). Thus the synthesis of 6, and/or its trans- port to or maintenance on the cell surface appeared to be significantly more efficient than that of p,.

I

lgMa l o 10 10 10 10 10

Fzgure 2. sIgD expression is dominant over IgM expression on B cells from MM x D D Ig-transgenic mice. Spleen cells from MM x DD, MM and D D mice were stained for IgM" (RS-3.U biotin + SA/PE) and IgD" (AMS-lS.l/fluorescein). Profiles of the parental MM and D D lines are arranged along the top and left sides of the diagram respectively and those corresponding to their MM x D D progeny positioned in grid fashion relative to the parental profiles. Non-transgenic B6 (IgHb+) (top left) as well as MD-3 Ig-transgenic and BALB/c (IgH"+) non-transgenic spleen cells (bottom right) are also included.

Eur. J. Immunol. 1995.25: 1980-1084

3.3 Preferential expression of membrane IgD over IgM

The molecular basis of the inverse relationship between membrane H chain mRNA levels and sIg expression on MM and DD B cells was investigated by mating MM and DD mice to yield hybrid Ig-transgenic mice carrying both vH1o-p and VH10-6 H chain transgenes (MM x DD Ig- transgenic mice). In this way it was possible to test whether the more efficient expression of sIg on B cells from DD versus MM mice was due to a higher rate of membrane H chain synthesis. If true, the ratio of 1gD:IgM on MM x DD B cells should be similar to that on DD compared to MM B cells (i.e. 2: 1). However, surface levels of IgD" on B cells from three independent MM x DD lines (MM-4 x DD-6, MM-7 x DD-6, MM-7 x DD-1) did not vary from those in the DD parental lines, whereas IgM" levels were 20 to 50-fold lower than in parental MM mice (Fig. 2). In other words, MM X DD B cells expressed 40 to 100 fold more IgD than IgM. Dominant expression of transgene-encoded IgD over IgM in MM x DD mice was specific for membrane Ig since production of secreted anti- HEL IgM" by MM X DD B cells both spontaneously and in response to HEL-SRBC was not reduced compared to MM B cells (data not shown).

3.4 CD80/CD86 up-regulation occurs equally efficiently via IgM and LgD

Although the more efficient surface expression of IgD compared to IgM may contribute to the auxiliary receptor function of IgD (discussed below), it was also possible that differences in IgM and IgD function may explain the preferential recruitment of IgD' B cells into T cell-dependent responses [16]. We therefore tested whether IgD may be better than IgM at signaling antigen-dependent upregula- tion of CD80/CD86 co-stimulatory molecules. To do this, spleen cells from MM-4 (IgM', IgD-) and DD-6 (IgM-, IgD') mice were mixed and cultured with HEL. Induction of CD80/CD86 (CTLA-4/Ig binding) was measured after counter-staining with antibodies directed against IgM" and IgDa to distinguish MM-4 and DD-6 B cells. At all concentrations of HEL, induction of CD80/CD86 was greater on DD-6 B cells (Fig. 3A). Moreover, MM-4 B cells required a higher concentration of HEL to initiate up- regulation (Fig. 3A). However, before these data could be used in a direct comparison of up-regulation of CD80/ CD86 by IgM and IgD, it was necessary to correct for the greater antigen receptor density on DD-6 B cells (Fig. 1B). Thus, the relative number of antigen-binding sites per B cell was determined by measuring HEL-binding to uncultured MM-4 and DD-6 B cells (Fig. 3C) and plotted against post-culture levels of CD80/CD86 (Fig. 3A). As shown in Fig. 3D, induction of CD80/CD86 on MM-4 and DD-6 B cells required the initial occupation of identical numbers of antigen-binding sites (HEL-binding = 300 units). Moreover, the relationship between increased expression of CD80/CD86 and the initial amount of HEL bound by the B cells was the same for MM-4 and DD-6 B cells up to the point of receptor saturation on the MM-4 cells (HEL-binding = 2200 units) (Fig. 3D). Thus, on a molecule-for-molecule basis, IgM and IgD were equally efficient in signaling CD80/CD86 up-regulation.

Eur. J. Immunol. 1995.25: 1980-1984 Differential expression but functional identity between IgM and IgD 1983

A 2 5 0 , I

01 1 1 10 100

HEL (nurnl)

I

HEL (ngirnl)

250 - D ::!m 50 0 1000 2000 3000 4000

HEL (nglrnl)

Figure 3. IgM and IgD mediate up-regulation of CD80/CD86 with equal efficiency but IgM undergoes more extensive down- regulation. (A, B) MM-4 and DD-6 spleen cells were mixed, incubated with HEL at 37°C for 12 h, stained for IgMa (RS-3.U fluorescein) or IgD" (AMS-l5.l/fluorescein) and counterstained for CD80/CD86 (CTLA-4/Ig + anti-human IgGl/biotin + SA/ PE). Mcan PE fluorescence on live (PI-) I@'' DD-6 (0) and IgM'" MM-4 (0) B cells indicated relative expression of CD80/ CD86. Mean fluorescein fluorescence indicated the level of surface IgD" (DD-6. 0 ) or IgMa (MM-4, 0) and was recorded as a proportion of the levels present on cells cultured in the absence of HEL. (C) The amounts of HEL bound by MM-4 and DD-6 B cells at the start of the cultures were estimated by first staining MM-4 and DD-6 spleen cells with RA3-6B2/fluorescein and then with HEL at 4°C in the presence of 0.1 % NaN3. Bound HEL was detected with HyHELShotin + SA/PE and quantitated as the mean PE fluorescence on HEL-binding B220' cells for MM-4 (0) and DD-6 (0) B cells. (D) For each HEL concentration, HEL bound at the outset of the culture was plotted against post-culture levels of CD80/CD86 on MM-4 (0) and DD-6 (0) B cells. (E) For each HEL concentration, HEL bound at the outset of the culture was plotted versus post-culture antigen receptor levels on MM-4 (IgM, 0) and DD-6 (IgD", 0) B cells.

3.5 Antigen-dependent down-regulation is greater for IgM than IgD

In addition to up-regulating co-stimulatory molecules such as CD80/CD86, exposure of B cells to antigen leads to down-regulation of both membrane IgM and IgD molecules [27]. In the current experiments, down-regulation of antigen receptors was observed on both MM-4 and DD-6 B cells following culture with HEL (Fig. 3B). Although the concentration of HEL required to initiate IgD down- regulation on DD-6 B cells was lower than that needed to initiate down-regulation of IgM on MM-4 B cells

0 10 20

Time (hours)

Figure 4. Greater down-regulation of IgM compared to IgD increases the antigen receptor ratio on IgD' compared to 1gD- Ig- transgenic B cells. Spleen cells were incubated with 200 ndml HEL for 0, 12 or 24 h. (A) MD-4 spleen cells were stained with RA3-6B2PE and either RS-3.l/fluorescein or AMS-lS.l/fluorescein following incubation with HEL and the levels of surface IgMd (0) and IgD" (0) on live B cells determined as for Fig. 3B. BALB/c (IgH"') non-transgenic B cells showed no reduction in surface IgM" or IgD" following incubation with HEL (data not shown). (B) Cultured spleen cells from MD-4 (O) , MM-4 (0), MM-4 x DD-6 (a) mice and non-transgenic B6 mice (0) were stained with RA3-6B2/fluorescein and with HEL + HyHELS/biotin + SA/PE to detect transgene-encoded antigen receptors. Rela- tive HEL-binding was quantitated as the mean PE fluorescence of HEL-binding B220' cells.

(Fig. 3B), correction for antigen receptor levels revealed that down-regulation in each case depended on an initial occupancy of identical numbers of HEL-binding sites per B cell (Fig. 3E). Nevertheless, the extent of antigen receptor down-regulation was consistently less on DD-6 compared to MM-4 B cells (Fig. 3E). Consequently, IgM and IgD were similar in their capacities to signal antigen- dependent antigen receptor down-regulation, whereas the extent to which they were themselves down-regulated was greater for IgM than for IgD. Preferential down-regulation by HEL of IgM versus IgD was shown also to occur when IgM and IgD were coexpressed on MD-4 B cells (Fig. 4A), confirming that IgM antigen receptors are intrinsically more susceptible to this form of modulation.

The preferential down-regulation of IgM over IgD leads to the prediction that the higher levels of antigen receptor expressed on IgD' compared to IgD- B cells before antigen exposure should increase further in relative terms after incubation with antigen. This was confirmed by monitoring total antigen receptor levels (HEL-binding) following HEL-mediated downregulation in vitro (Fig. 4B) which demonstrated that the ratio of antigen receptors on IgD' (MD and MM X DD) B cells versus IgD- (MM) B cells did indeed increase under these conditions (Fig. 4B).

4 Discussion

Two findings reported here support the conclusion that 6, H chains are expressed more efficiently as surface Ig than

1984 R. Brink et al. Eur. J. Immunol. 1995.25: 1980-1984

Medical Foundation, University of Syndney and an Australian Postgraduate Research Award.

Received February 9,1995; in revised form May 8,1995; accepted May 9, 1995.

p,, H chains. First, IgD levels on DD B cells were 2-fold higher than those of IgM on MM B cells (Fig. 1B) despite the 7-fold lower levels of 6 , mRNA in DD-6 B cells compared to p, mRNAin MM-7 B cells (Fig. 1A). Secondly, B cells from MM x DD mice expressed 20 to 50-fold lower levels of surface IgM but identical levels of surface IgD when compared with B cells from the original MM and DD parental lines (Fig. 2). Not only do these findings indi- cate 6, is expressed more efficiently than p,,, on the B cell membrane, but also that the two molecules compete directly for expression as membrane Ig.

Competition between p,,, and 6, for expression on B cells points to the existence of limiting cellular component(s) necessary for surface Ig expression. A possible candidate is the IgalIgB heterodimer [8]. Although surface IgD can be expressed as a glycosylphosphatidyl inositol-linked protein [28], it is expressed in association with Iga/Igp on both MD and DD B cells ([29], unpublished data). If Iga/Igp is a crucial limiting component for sIg expression, then the dominant expression of IgD over IgM on MM x DD B cells could be explained by a preferential association of IgalIgB with 6,, over p, [30].

Exposure of Ig-transgenic B cells to HEL for 12 h in vitvo led to more pronounced down-regulation of sIgM than sIgD (Figs. 3E and 4A). Since HEL-induced down- regulation of Ig on Ig-transgenic B cells is mediated predominantly by endocytosis (unpublished data), this phenomenon may be due to more efficient re-expression of sIgD. Alternatively, IgD may be more resistant to endocytosis, consistent with the relatively slow tunover rate of slgD compared to sIgM [31].

While differences in IgM and IgD expression were identified in this study, the catalog of functional similarities between IgM and IgD was extended by demonstrating equivalent mediation of antigen-dependent CD80/CD86 up-regulation (Fig. 3D). This apparent dichotomy between IgM and IgD receptor function and expression raises the question as to what mechanisms underlie the auxiliary receptor function of IgD identified in IgD- deficient mice [16]. In particular, how is it that IgD can enhance T cell-dependent B cell responses when IgM is efficient as IgD in mediating two of the key events in this process, namely processing and presentation of bound antigens [3. 41 and induction of co-stimulatory molecules (Fig. 3D)? A possible answer comes from the observation here of differential antigen-dependent up-regulation of CD80/CD86 on DD-6 versus MM-4 B cells. Thus, although IgD was not intrinsically better than IgM at signaling CD80/CD86 up-regulation (Fig. 3D), the higher receptor levels on DD-6 B cells (Fig. 3C) allowed them to respond more efficiently to HEL than MM-4 B cells, espe- cially at low antigen concentrations (Fig. 3A). The higher sIg levels associated with IgD expression may therefore lead to more efficient induction of co-stimulatory molecules, thereby allowing increased recruitment of IgD+ B cells into antigen-driven, T cell-dependent responses [16]. Fur- thermore, maintenance of high levels of antigen receptor on IgD' B cells after antigen exposure (Fig. 4B) may also enhance the responsiveness of IgD+ B cells.

Work supported by Q Program Grant from the National Health and Medical Research Council of Australia. R. B. was supported by the

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IgD expression on B cells is more efficient than IgM but both receptors are functionally equivalent in up-regulation CD80/CD86 co-stimulatory molecules