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CD72 Ligation Regulates Defective Naive Newborn B Cell Responses
CELLULAR IMMUNOLOGY ARTICLE NO.
175, 179–188 (1997)
CI961033
CD72 Ligation Regulates Defective Naive Newborn B Cell Responses LAURENCE M. HOWARD
AND
DENIS J. REEN
Children’s Research Centre, Our Lady’s Hospital for Sick Children, Crumlin, Dublin 12, Ireland Received June 27, 1996; accepted September 24, 1996
The biological basis for reduced Ig production by naive newborn B cells compared to adult peripheral blood B cells is not fully understood. In a Con A / IL2 T cell-dependent system using ‘‘competent’’ adult T cells, adult B cells produced large amounts of IgM, IgG, and IgA, while cord B cells were restricted to low levels of only IgM production. Cord B cell activation was also diminished. The contribution of specific B–T cell contact-mediated events to the diminished cord B cell response in this system, using mAbs to CD40, CD28, CD80, and CD72, were investigated, as well as regulation of B cell Ig production by cytokines. aCD72 ligation increased cord B cell activation and IgM production, but did not affect adult B cells. Blocking aCD40 mAb inhibited cord B cell Ig production completely, but only partly inhibited adult B cell Ig production even at high concentration, suggesting a greater sensitivity of cord B cells to disruption of the CD40-CD40L interaction. Addition of IL-10 did not increase cord B cell Ig production, while adult B cell Ig production was increased. However, combined addition of IL-10 and aCD72 significantly increased cord B cell Ig production over that in the presence of either aCD72 or IL10 alone, but had no effect on adult B cells over that of IL-10 alone. These data suggest that the diminished T cell-dependent response of cord B cells is due to reduced or absent CD72 ligation. CD72 ligation plays an important role in the induction of primary responses by naive B cells. CD72 modulation of naive B cell sensitivity to IL-10 stimulation may have implications in the induction of class switch, which is deficient in newborn B cells. Since all T cells express CD5 constitutively, these data also suggest the existence of another ligand for CD72. q 1997 Academic Press
INTRODUCTION In humans, neonatal immune defence to bacterial and viral infection is deficient (1–5). In particular, the neonate has an impaired response to bacterial capsular polysaccharide antigens, with poor responses being observed even after repeated exposure (6, 7). In vitro, T cell-independent stimulation of newborn B cells, using
EBV, induces levels of IgM comparable to that of adult B cells (5, 8–10). However, newborn B cells fail to secrete IgG or IgA class-switched isotypes in response to EBV stimulation, while adult B cells secrete large amounts of these isotypes. In contrast, in T cell-dependent systems such as Staphylococcus aureus Cowan I (11, 12), PWM (8, 9, 13, 14), and aCD3 (15, 16), the level of newborn B cell IgM production has been shown invariably to be reduced compared to that of adult B cells, with little or no IgG or IgA production being observed. Generation of newborn Ig-secreting B cells has been shown to be lower than that of adult B cells when stimulated with S. aureus Cowan I or PWM (8, 9, 11, 13, 15). Proliferation of newborn B cells is also reduced compared to adult B cells when stimulated in the aCD3 T cell-dependent system (16). T cells provide help for B cell effector function through both contact interactions and by secreting cytokines that promote B cell development and immunoglobulin secretion (17–19). Contact-mediated help involves many ligand pair interactions on the extracellular membrane surfaces of T and B cells (17–21). These B–T cell interactions are dynamic and involve both B cell stimulation and T cell counterstimulation by B cells (18, 20, 21). Some of the critical B-T cell contact interactions include CD40–CD40L (18, 20–23), CD80/ CD86–CD28/CTLA-4 (24–27) and CD72–CD5 (28–30) ligation. CD40 ligation has been shown to play an important role in activation, proliferation, immunoglobulin production, as well as class-switch induction events in B cells (22, 23, 31). CD40L, the ligand for CD40, is expressed short term on activated T cells (22, 23, 31). CD80 is one of three B cell surface antigens that bind to CD28 and CTLA-4 on T cells (24–27) and is expressed on the surface of activated B cells (24, 27, 32–35). Ligation of CD28 induces T cell activation, proliferation, and secretion of cytokines such as IL-2, IL4, IL-6, and IFN-g (36–42). It is now considered that coligation of CD28 with the TCR is required for effective induction of T cell helper effector function (43, 44). Ligation of CD28 on T cells results in the induction of CD40L expression (38, 39), while ligation of CD40 on B cells results in CD80 expression (45, 46). This has led to the suggestion that a signaling circuit is formed
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by these two ligand pair interactions between the B and T cell (18). Ligation of CD72 on B cells has been shown to induce proliferation (47–50) and to be required for plaque formation in T cell-dependent systems (51). However, the role of CD72 ligation in the T cell-dependent B cell response is less well defined than either CD28 or CD40 ligation. The deficiency of the newborn T cell-dependent B cell response has been associated with diminished function of both B and T cells (15, 16). Newborn B and T cell populations are almost exclusively naive (7, 52–56). This is in contrast to adult cell populations which contain large numbers of differentiated and memory B and T cells, respectively (7, 52, 55, 56). Newborn T cells have been shown to be predominantly suppressor inducers (8, 9, 13, 14) and have a reduced ability to secrete cytokines such as IL-2, IL-4, and IFN-g (12, 57– 60), which promote B cell effector function. Newborn T cells have reduced expression of CD40L upon activation compared to adult T cells, when stimulated with mitogens such as PHA or PMA (61–63). Newborn B cells are less sensitive to CD40 ligation compared to adult B cells (63). It has been suggested that newborn B cells require signals that are not necessary to drive adult B cell effector function (15, 64–66) and that the basis for such signaling requirements are qualitative (64), quantitative (11, 66), or kinetic (66) in origin. However, such signals have not been identified and little is known of the deficiencies of newborn B cells in T cell-dependent systems. The lack of secreted IgG and IgA from class-switched B cells has been associated with the absence of differentiated surface IgD-negative B cells in newborn B cell populations (7, 52–56, 67– 70) which are present in large quantities in adult B cell populations (7, 52, 55, 56). While much attention has been given to the low or absent class-switched response of newborn B cells in T cell-dependent systems, little investigation into the poor primary response of these cells has been carried out. This study investigated the role of T–B cell ligand pair interactions and cytokines in regulating the impaired responses of naive newborn B cells in a Con A plus IL-2, T cell-dependent system that was MHC class II independent (71). This allowed the investigation of naive newborn B cell interactions with competent adult T cells, in the absence of neonatal T cells that are primarily suppressor–inducers (8, 9, 13, 14) and have been shown to be deficient in providing help for B cells (8, 9, 13–16).
tol–Myers Squibb, Seattle, WA). Purified CD40 mAb 89 was a kind gift from Dr. J. Banchereau (ScheringPlough, Dardilly, France). Purified CD80 mAb BB-1 was a kind gift from Dr. E. A. Clark (University of Seattle, WA). Purified mAb to CD72 (BU40) was obtained from The Binding Site (UK). Human recombinant IL2, IL-6, and IFN-g were obtained from Boehringer Mannheim (Germany). Recombinant human IL-4 and IL-10 were obtained from Genzyme Corporation (Cambridge, MA). Recombinant human IL-5 was obtained from British Biotechnology (UK). Purification of B and T Cells Adult peripheral blood was collected randomly from a large pool of donors in EDTA tubes, from healthy volunteers aged from 19 to 55 years, and cord blood was obtained from full-term newborn infants with a mean gestation age of 41 { 1 weeks (mean { 1 SD). PBMCs and CBMCs were obtained from blood samples by Lymphoprep density gradient centrifugation (Nycomed Pharma AS, Norway). These cells were placed on a Nycoprep 1.063 density gradient (Nycomed Pharma AS) and centrifuged at 350g for 15 min, to remove platelets. The cells obtained were incubated with 10 mM L-leucine methyl ester (Sigma Chemical Co.) to remove monocytes and some NK cells, as has been described (15, 16). The remaining cells were then passed through two rounds of aminoethylisothiouronium bromide (Sigma Chemical Co.)-treated sheep red blood cell (Becton–Dickinson, Mountainview, CA) rosetting, as previously described (72, 73). The rosetted cells from the first round of rosetting were depleted of red cells using 0.17 M ammonium chloride lysis and then incubated on a nylon wool column at 377C for 45 min, giving a purified T cell preparation. The nonrosetted B cells, after the second round of rosetting, were collected and treated with 0.17 M ammonium chloride, to remove contaminating red blood cells. Purities of T and B cell preparations were determined by flow cytometry. Adult peripheral and cord blood B cell preparations contained 81 { 7 and 86 { 6% CD19/ cells, 8 { 6 and 6 { 3% CD3/ cells, 7 { 4 and 6 { 4% CD16/CD56/ NK cells, and 2 { 1 and 2 { 1% CD14/ monocytes, respectively. Adult and cord T cell preparations contained 97 { 2 and 97 { 2% CD3/ cells, 0.9 { 0.5 and 0.8 { 0.5% CD19/ cells, 2 { 1 and 2 { 1% CD16/CD56/ NK cells, and õ0.5 and õ0.5% CD14/ monocytes, respectively. Cell Culture
MATERIALS AND METHODS
Concanavalin A was obtained from Sigma Chemical Company (UK). Monoclonal antibody 9.2, to CD28, in ascites, was a kind gift from Dr. J. A. Ledbetter (Bris-
Cell culture was carried out in RPMI 1640 Dutch modification medium (Gibco BRL, Gaithersberg, NY) containing 20% fetal calf serum (Gibco BRL), 100 U/ ml penecillin, 100 U/ml streptomycin (Imperial Laboratories, UK), with 100 mM L-glutamine (Imperial Laboratories). B Cells (1 1 104) were added to 1 1 105 T cells
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in a 96-well round-bottom tissue culture plate (Nunc, Denmark). Con A at a final concentration of 1 mg/ml and IL-2 at a final concentration of 50 U/ml were added to the recombined cells and the volume in each well was made up to 200 ml, by addition of culture medium. Cultures were carried out in triplicate and incubated at 377C in a 100% humidified atmosphere containing 5% CO2 . For determination of activation, the cells were cultured for 6 days. For measurement of immunoglobulin production, cells were cultured for 12 days. Purified mAbs to CD40, CD72, or CD80 were added at a final concentration of 1 mg/ml, while 9.3 ascites was added at a final dilution of 1/1000. Recombinant human cytokines IL-4, IL-5, IL-6, IL-10, and IFN-g were used at a final concentration of 100 U/ml. Measurement of Immunoglobulin Production After 12 days, the tissue culture plates were centrifuged to pellet the cells and culture supernatants were aspirated from each well. IgM, IgG, and IgA production was determined by ELISA. Briefly, anti-human IgG, IgM, or IgA antisera (Cappel Research Products, Durham, NC) were coated onto 96-well microtiter plates (Corning Inc., U.S.A.) overnight at 47C in 0.1 M carbonate–bicarbonate buffer, pH 9.6. The plates were then washed with PBS containing 0.05% Tween 20. Samples were placed in each well and incubated for 1 hr at room temperature. The wells were washed, and horseradish peroxidase-conjugated anti-human IgG, IgA, or IgM antisera (Tago Inc., Burlingame, CA) were added to each well. The plates were incubated for 1 hr and then washed. Substrate solution, containing 0.08% (w/v) ophenylenediamine and 0.06% (v/v) hydrogen peroxide solution in 0.1 M citrate phosphate buffer, pH 5.0, was then added. The plates were incubated in the dark at room temperature and the absorbance at 405 nm was determined using a Titertek microplate reader. Standard curves and concentration were determined using a smoothed spline semilogarithmic plot (Multicalc, Pharmacia, Sweden).
Statistics Statistical analyses were carried out by either a paired nonparametric Wilcoxon signed rank test or by an unpaired Mann–Whitney test. RESULTS Immunoglobulin Production by Adult Peripheral and Cord Blood B Cells Adult B cells, cocultured with adult T cells, produced large amounts of IgM, IgG, and IgA in response to stimulation with Con A and IL-2 (Fig. 1A). In contrast, immunoglobulin production by cord B cells cocultured with cord T cells was minimal for all Ig isotypes (Fig. 1C). In order to determine the relative contribution of cord B and cord T cells to the diminished Ig production, cord B cells were cocultured with adult T cells (Fig. 1b). In comparison to the response of adult peripheral blood B cells cocultured with adult T cells, cord B cells secreted significantly reduced levels of IgM (P õ 0.01), with no significant IgG or IgA production (n Å 5) (Fig. 1B). No Ig production was observed when adult T cells were cultured alone with Con A and IL-2 in the absence of added B cells, compared to unstimulated adult T cell cultures (n Å 3). Influence of B-T Cell Contact Interactions on Immunoglobulin Production
For measurement of activation of B cells, cells were gently resuspended in the culture wells and transferred to 12 1 75 mm polystyrene tubes (Falcon, Ireland). Phycoerythrin conjugated anti-CD19 mAb (Becton– Dickinson), and FITC-conjugated anti-CD25 or irrelevant control mAbs (Dakopatts AS, Denmark) were added to individual tubes. The contents of the tubes were mixed and incubated in the dark for 15 min. The cells were then washed with PBS containing 20% donor horse serum (Gibco) and resuspended in wash medium, and the cell population was analyzed by flow cytometry (Facscan flow cytometer, Becton–Dickinson). Two thousand CD19/ gated cells were acquired, from which the proportion of CD25/ B cells was determined.
Anti-CD28, CD40, CD72, or CD80 mAbs were added at the start of culture to investigate the contribution of specific contact-mediated signals to the deficient immunoglobulin production by cord B cells. Addition of aCD40 mAb had no significant effect on adult B cell immunoglobulin production, in the presence of adult T cells (Fig. 2a), while it almost completely inhibited IgM production (P õ 0.05) in cord B cell-adult T cell cocultures (Fig. 2b). Ig production in cord B–T cell cocultures was also significantly inhibited (P õ 0.05) by antiCD40 mAb (Fig. 2C). However, in this case, the level of immunoglobulin production was already extremely low. At 10-fold higher concentrations (10 mg/ml) of CD40 mAb, adult B cell Ig secretion was inhibited (data not shown). Addition of aCD28 to adult B–T cell cocultures resulted in significant inhibition (P õ 0.05) of IgM, IgG, and IgA production (Fig. 2A). The addition of aCD28 to cocultures of cord B cells with adult T cells had no significant effect (Fig. 2B), but significantly increased (P õ 0.05) the level of IgM production in cord B–T cell cocultures (Fig. 2C). However, the level of immunoglobulin produced remained low. Anti-CD80 had no significant effect on adult B cell immunoglobulin production (Fig. 2A), although the level of IgM production was slightly raised. Addition of aCD80 to cocultures of cord B cells with adult T cells had no significant effect (Fig.
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FIG. 1. Immunoglobulin production in the Con A / IL-2 system by (A) adult B cells cocultured with autologous adult T cells, (B) cord B cells cocultured with adult T cells, and (C) cord B cells cocultured with autologous cord T cells. Culture supernatants were harvested on Day 12, and Ig levels were determined by ELISA. Data represent means { 1 SEM for five experiments. Statistical analysis was carried out by paired Wilcoxon signed rank test compared to the response in the absence of Con A and IL-2; asterisks denotes P values less than 0.05. Note the different scales between the graphs.
2B). In cord B–T cell cocultures, the level of IgM production was significantly increased (P õ 0.05), but again the level of IgM produced remained low (Fig. 2C). In adult B–T cell cocultures, addition of aCD72 had no significant effect (P ú 0.05) on adult B cell immunoglobulin production of any isotype (Fig. 2a). In comparison, addition of aCD72 to cocultures of cord B cells with adult T cells significantly increased (P õ 0.05) the level of IgM production (Fig. 2b) and also induced a significant increase in IgA production (P õ 0.05). In cord B– T cell cocultures, the level of IgM and IgG production was also significantly increased (P õ 0.05) by addition of anti-CD72 mAb (Fig. 2c). As was the case with all cord B–T cell cocultures, the immunoglobulin levels remained low. Regulation of Adult and Cord B Cell Activation by mAbs to Contact Associated Antigens The regulation of cord B cell activation through ligation of CD28, CD40, CD72, and CD80 on both B and T cells was investigated, by addition of mAbs to these antigens. Activation was assessed by the expression of CD25 (IL-2Ra) on CD19-positive B cells. As shown in Figs. 3A–3C, cord B cell activation in the presence of either adult or cord T cells, was significantly reduced compared to that of adult B cells cocultured in the presence of adult T cells (P õ 0.01). Addition of anti-CD40 in soluble form to all cultures of adult or cord B cells significantly inhibited (P õ 0.05) activation of B cells (Fig. 3). Addition of anti-CD28 mAb to adult B–T cell cocultures had no effect on adult B cell activation, but significantly increased (P õ 0.05) cord B cell activation when they were cocultured with either adult or cord T cells (Fig. 3). Addition of antiCD80 had no effect on adult or cord B cell activation (Fig. 3). Anti-CD72 addition had no significant effect on adult
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B cell activation, but significantly increased (P õ 0.05) cord B cell activation when they were cocultured with either adult or cord T cells (Fig. 3). However, even in the presence of anti-CD72, the level of cord B cell activation was still significantly reduced (P õ 0.05) compared to that of adult B cells. Combined addition of anti-CD72 and anti-CD28 to cord B–T cell cocultures did not significantly increase the level of cord B cell activation above that of anti-CD72 or anti-CD28 alone (data not shown). Effect of Cytokines on Immunoglobulin Production by Adult and Cord B Cells Addition of IL-4, IL-5, IL-6, and IFN-g had no or minimal effect on Ig production by adult B cells, cocultured with adult T cells (Fig. 4a). Addition of IL-10 to adult B–T cell cocultures significantly increased IgM, IgG, and IgA production, compared to the response in the system alone (P õ 0.05). IL-10 augmented IgM production by adult B cells by approximately fivefold. Addition of any of the cytokines had no, or minimal, effect on immunoglobulin production of any isotype by cord B cells cocultured with adult T cells (Fig. 4B), except for a small but significant increase in IgA production in the presence of IL-10 (P õ 0.05). In cord B– T cell cocultures, addition of IL-5, IL-6, and IFN-g to cocultures had little effect (Fig. 4c). Addition of IL-4 or IL-10 significantly increased cord B cell IgM production (P õ 0.05) in cord B–T cell cocultures, but the response was low. Regulation of Immunoglobulin Production by Combined Addition of Anti-CD72 and IL-10 Since IL-10 caused a significant increase in adult but not cord B cell immunoglobulin production in the
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cocultures with either adult or cord T cells did not affect the level of IgG or IgA production observed in the presence of either aCD72 or IL-10 alone. DISCUSSION In this study we have investigated the deficient T cell-dependent response of cord blood B cells, using a Con A / IL-2 culture system. This system, which is MHC class II-independent (71), allowed coculture of adult T cells and cord B cells thereby excluding any influence that naive newborn T cells might have on the system. T cell-dependent responses require bidirectional contact-mediated interactions between B and T cells (18, 20, 21), as well as cytokine secretion from T
FIG. 2. Regulation of immunoglobulin production by ligation of B-T cell contact antigens using mAbs. (A) Adult B cells cocultured with autologous adult T cells, (B) cord B cells cocultured with adult T cells, and (C) cord B cells cocultured with autologous cord T cells and stimulated with Con A and IL-2. Data represent means { 1 SEM for six experiments. Asterisks denote significant P õ 0.05 in response to mAb addition compared to the system alone, using a paired Wilcoxon signed rank test. Note the different scales of response in each graph.
presence of competent adult T cells, combined stimulation through CD72 ligation in the presence of IL-10 was investigated to determine whether CD72 ligation could restore the lack of responsiveness of cord B cells to added IL-10 (Fig. 5). Adult B cells were unaffected by combined addition of aCD72 and IL-10, compared to added IL-10 alone (Fig. 5A). However, addition of aCD72 in combination with IL-10 synergistically increased cord B cell IgM production (P õ 0.05), in cocultures with adult or cord T cells (Figs. 5B and 5C). The increase in IgM production by cord B cells cocultured with adult T cells was approximately 9-fold (Fig. 5B) and 14-fold when cocultured with cord T cells (Fig. 5B). Combined addition of aCD72 and IL-10 to cord B cell
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FIG. 3. Regulation of B cell activation by ligation of B–T cell contact antigens using mAbs. (A) Adult B cells cocultured with autologous adult T cells, (B) cord B cells cocultured with adult T cells, and (C) cord B cells cocultured with autologous cord T cells and stimulated with Con A and IL-2 for 6 days to induce optimal expression of CD25. Data represent means { 1 SD for five experiments. Asterisks denote significant difference in the activation of CD19/ B cells in the presence of specific mAb compared to in the system alone (P õ 0.05), using a paired Wilcoxon signed rank test.
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cells (17–19). We have shown that both cord B and T cells contribute to the diminished immunoglobulin response, in vitro, of newborn lymphocytes. This supports other studies which have attributed this deficient response to both cord B and T cells (15, 16). However, we have also shown that the deficient response of cord B cells is due in part to specific contact interactive deficiencies between cord B cells and otherwise ‘‘competent’’ adult T cells, and that poor induction of cord B cell activation results in their reduced capacity to respond to cytokine stimulation. It has previously been demonstrated that the induction of effective activation and proliferation of B cells is directly associated with the level of immunoglobulin production in vitro (74, 75). Thus, activation of cord B cells was compared to that of adult B cells, in order to assess its contribution to the deficient immunoglobulin
response of cord B cells. Activation was determined by expression of CD25 (IL-2Ra) on B cells, as it is expressed only during activation and blast stage development of B cells. Since an apparent deficiency in the cross-talk between T cells and cord B cells was observed, this suggests that defective induction of activation and proliferation may affect the overall immunoglobulin response of cord B cells. Furthermore, it has been suggested that newborn B cells are deficient in their capacity to undergo IgA and particularly IgG isotype class switching (15, 16). Cytokines secreted by T cells play an important role in regulating B cell Ig isotype class switching (12, 15, 16, 57–60). In this study, we have shown that naive newborn B cells, stimulated in a T cell-dependent system, have a diminished capacity to respond to cytokine stimulation, compared to adult B cells. This suggests that the capacity of naive newborn B cells to undergo class switch events may be limited due to their insensitivity to cytokine manipulation, in T cell-dependent systems. This paper concentrates therefore on the induction of optimal primary cord B cell IgM responses and investigates the B–T cell interactions that may be responsible for the deficient responses of cord B cells in T cell-dependent systems (15, 16). By using mAbs directed at specific B – T cell interactions, we were able to investigate the contribution of particular ligand pair interactions between T cells and cord B cells to deficient cord B cell responses. mAbs in this study were chosen because of their recognized mode of action. The CD40 mAb, alone, was used to block CD40L – CD40 interactions, in order to determine whether this critical event was present in cord B cell – adult T cell cocultures. CD28 and CD80 mAbs were used to stimulate and block the CD28 signalling pathway, respectively, which is a critical costimulatory event for driving T cell effector function. CD80/CD86 signalling of CD28 in our system is probably minimal, especially when naive B cells are used. CD72 mAb was used in order to determine whether optimal CD72 ligation was occurring in cord B cell cocultures, which is thought to be provided in a T – B cell interaction by CD5. Ligation of CD28 on T cells increased activation of cord, but not adult, B cells in the co-culture system. Ligation of CD72 on B cells augmented cord B cell activation and IgM production, while adult B cell responses were not significantly altered. This leads us to speculate that CD28 and CD72 ligation is diminished in cord B cell cocultures with adult and cord T cells, and that this diminished ligation was due to the diminished expression of their ligands on B and T cells, respectively. This in turn must reflect an inability of cord B cells to interact effectively even with ‘‘competent’’ T cells. Almost all cord B cells are naive (7, 52–56). It is possible that diminished ligation of CD28 on T cells, by cord B cells, may reflect diminished or absent CD80/86 ex-
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FIG. 4. Effect of cytokines on Ig production by (A) adult B cells cocultured with autologous adult T cells, (B) cord B cells cocultured with adult T cells, and (C) cord B cells cocultured with autologous cord T cells and stimulated with Con A and IL-2 for 12 days. All cultures were stimulated with Con A and IL-2. Data represents mean { 1 SEM for six experiments, and data were analyzed using a paired Wilcoxon signed rank test. Asterisks denote significance of P õ 0.05 compared to the response in the absence of cytokines. Note the different scales used for each graph.
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FIG. 5. Effect of addition of aCD72 and IL-10 on immunoglobulin production by (A) adult B cells cocultured with autologous adult T cells, (B) cord B cells cocultured with adult T cells, and (C) cord B cells cocultured with autologous cord T cells and stimulated with Con A / IL-2 for 12 days. Data represent means { 1 SEM for six experiments and were analyzed using a paired Wilcoxon signed rank test. Asterisks denote significant difference compared to Ig production in the system alone (P õ 0.05). Note the different scales of each graph.
pression, which has been shown to be reduced on activated naive B cells (43, 76). The known ligand for CD72, CD5, is expressed constitutively at high levels on both adult and cord T cells (28–30) as well as at low levels on a subpopulation of B cells (28). The deficient CD72 ligation observed on cord B cells in the presence of adult or cord T cells, in this system, suggests, therefore, that an alternative ligand for CD72 may exist, and that this ligand is more effective at inducing signal transduction through CD72 than CD5. It has been suggested that, given the structural similarity to CD23, an antigen which possesses multiple ligands (28), CD72 may also associate with more than one ligand. Furthermore, an alternative ligand for CD5, other than CD72, has also been reported (77), and no functional evidence has yet been presented to show a functional role for CD5–CD72 interactions in the T cell-dependent response. The fact that CD72 ligation on cord B cells was deficient suggests that an alternative CD72 ligand must be induced on activated T cells. CD72 ligation was critical in driving cord B cell activation and IgM production and has been shown to be a potent costimulator of B cell proliferation when used in conjunction with mitogenic stimulation through the B cell receptor (28, 47). When aCD28 and aCD72 were added together no further increase in cord B cell activation was observed over that of addition of either antibody alone, suggesting that the induction of expression of the alternative CD72 ligand on T cells may be under the control of costimulatory events associated with CD28 ligation, and that CD72 ligation may regulate CD80/86 expression on B cells. In support of this argument, costimulation of T cells by CD28 ligation has been shown to induce CD40L expression, and thus play a major role in regulating B-T cell contact activational events (38, 39). aCD80 mAb did not affect cord or adult B cell Ig production, when cocultured with
adult T cells. The CD28/CTLA-4 ligation event is complex and involves two antigens on B cells (CD80 and CD86) and T cells (CD28 and CTLA-4). In cord B–T cell cocultures, aCD80 induced significant IgM production. While the reasons for this are unclear, it may be due to (i) signal transduction through CD80 ligation into the B cell, (ii) differential regulation of CD28/CTLA-4 signal transduction in T cells following ligation by CD80 and CD86, or (iii) induction of a different cytokine secretion profile by T cells due to aCD80 mAb. The CD40-CD40L interaction between B and T cells was investigated using mAb 89, a partial antagonistic antibody that contains an epitope specificity that obstructs CD40L interaction with CD40 on B cells (78). CD40L expression by cord T cells has been shown to be deficient, compared to that of adult T cells (61–63), which express levels of CD40L in excess of that required to drive B cell responses (78). Conversely, cord B cells have been shown to be less sensitive to CD40 ligation than adult B cells (63). Since CD40-CD40L interaction plays an important role in regulating B cell responses (18, 22, 23), it has been suggested that this deficiency in the interaction between cord B and T cells may contribute to the deficient responses of cord B cells (61–63). When cord B cells were cocultured with adult T cells, significant amounts of IgM were produced. Addition of aCD40 mAb, at 1 mg/ml, inhibited this response completely but did not significantly inhibit adult B cell Ig production, suggesting that CD40 ligation was driving cord B cell IgM production, at least in the presence of adult T cells. However higher levels of aCD40 mAb (10 mg/ml), also inhibited IgM production by adult B cells. This suggests that either cord B cells required a higher level of CD40 ligation than adult B cells, for which some evidence exists (63), or that induction of CD40L expression on adult T cells was diminished when these cells were cocultured with cord
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B cells compared to that when cocultured with adult B cells. CD28 ligation, which induces T cell expression of CD40L (38, 39), was deficient in this system when cord B cells were cocultured with adult T cells. Thus both a reduced level of CD40L expression on T cells when cocultured with cord B cells and a requirement for higher levels of CD40 ligation on cord B cells may contribute to the deficient response of cord B cells in this system. With the exception of IL-10, none of the cytokines studied had a positive effect on adult B cell Ig production. In cord B–T cell cocultures, IL-4 increased IgM production. This effect was not observed when cord B cells were cocultured with adult T cells. Cord T cells, being mainly CD45RA cells, do not secrete IL-4 (12, 59), suggesting that addition of IL-4 to cord B–T cell cocultures provided cytokine stimulation that was otherwise absent. In contrast to adult B–T cell cocultures, addition of IL-10 to cocultures of cord B cells with adult T cells did not significantly increase IgM production, while in cord B–T cell cocultures a small increase in IgM production was observed. While adult B cells are highly responsive to IL-10 stimulation, in this system, cord B cells are relatively insensitive to IL-10 stimulation. When aCD72 and IL-10 were added together to cord B–adult T cell cocultures, a large synergistic increase in IgM production was observed. In cord B–T cell cocultures, aCD72 and IL-10 also acted synergistically in increasing IgM production by cord B cells. This was in contrast to adult B cells, in which combined addition of aCD72 and IL-10 did not increase the level of Ig production compared to addition of IL-10 alone. In cord B–adult T cell cocultures, CD72 ligation resulted in induction of responsiveness to IL-10 that was otherwise absent. Since CD72 is already expressed at very high levels on naive cord B cells, it is unlikely that this effect is due to upregulation of CD72 expression on cord B cells, by added IL-10, followed by enhanced signal transduction through mAb ligation of CD72. Thus ligation of CD72 in cord B cell cocultures with either adult or cord T cells must increase their sensitivity to IL-10. The reasons why cord B cells are less sensitive to CD40 ligation than adult B cells has yet to be determined. It has been suggested that driving cord B cell responses requires signals not needed by adult B cells (15, 64–66), and thus their signaling pathways may be somewhat different. Such events may play a role in costimulating and amplifying CD40-dependent signals in naive B cells, and thus in their absence, result in reduced sensitivity to CD40 ligation. It is tempting to speculate that CD72 ligation may play this costimulatory role, supplementing reduced signal transduction in response to CD40 ligation. In support of this argument, CD72 ligation cooperates with CD40/IL-4 signals, augmenting B cell proliferation (47), and, like CD40, rescues apoptotic B cells (79). The induction of
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IL-10 responsiveness of cord B cells is also regulated by CD40 signal transduction (80). Thus it remains to be seen how CD72 and CD40 signaling events are intertwined and whether other CD40 dependent events, such as induction of CD80/86 expression, can be modulated by CD72 ligation. While newborn B cell responsiveness was increased by CD72 ligation, the level of IgM production was still reduced compared to that of adult B cells. B cells can secrete immunoglobulin at high or low rates (67, 81). Thus it is probable that the cord B cell IgM response reflects cells that are capable of only low rates of IgM secretion, while adult B cells are capable of high rates of IgM secretion. B cell development towards high rate Ig secreting cells requires the cell to pass through a number of cell division cycles (67) and is influenced by dendritic cells (81). Development of high-rate Igsecreting plasma cells occurs in the bone marrow and in the mucosa (81). This suggests a further level of immaturity of cord compared to adult B cell populations, that may reflect the primed but inactive status of the newborn immune system compared to an active immune system in adults. The deficient T cell-dependent response of cord blood lymphocytes is due to both T and B cell deficiencies. In this study we have identified deficiencies in specific events in the T cell-dependent response of cord B cells that result in diminished cord B cell differentiation and Ig secretion as well as a reduced ability of cord B cells to costimulate T cells. Ligation of CD72 on B cells significantly influences cord B cell responses, increasing activation, immunoglobulin production, and responsiveness to cytokines such as IL-10. These data suggest that an alternative CD72 ligand, other than CD5, exists, and that this ligand interaction with CD72 plays a critical role in regulating naive B cell responses. ACKNOWLEDGMENTS We thank the nursing staff of the Coombe Women’s Hospital, Dublin, for collecting cord blood samples.
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40. Seder, R. A., Germain, R. N., Linsley, P. S., and Paul, W. E., J. Exp. Med. 179, 299, 1994. 41. Sommer, F., Rollinghoff, M., and Lohoff, M., Eur. J. Immunol. 23, 2498, 1993. 42. McArthur, J. G., and Raulet, D. H., J. Exp. Med. 178, 1645, 1993. 43. Lenschow, D. J., Sperling, A. I., Cooke, M. P., Freemon, G., Rhee, L., Decker, D. C., Gray, G., Nadler, L. M., Goodnow, C. C., and Bluestone, J. A., J. Immunol. 153, 1990, 1994. 44. William, Y. H., Cooke, M. P., Goodnow, C. C., and Davis, M. M., J. Exp. Med. 179, 1539, 1994. 45. Ranheim, E. A., and Kipps, T. J., J. Exp. Med. 177, 925, 1993. 46. Yellin, M. J., Sinning, J., Covey, L. R., Sherman, W., Lee, J. J., Glickman-Nir, E., Sippel, K. C., Rogers, J., Cleary, A. M., Parker, M., Chess, L., and Lederman, S., J. Immunol. 153, 666, 1994. 47. Kamal, M., Katira, A., and Gordon, J., Eur. J. Immunol. 21, 1419, 1991. 48. Pezzutto, A., Dorken, B., Valentine, M. A., Shu, G. L., and Clark, E. A., in ‘‘Leukocyte Typing IV, White Cell Differentiation Antigens’’ (Knapp, W., Dorken, B., Gilks, W. R., Rieber, E. P., Schmidt, R. E., Stein, H., and Kr. von dem Borne, A. E. G., Eds.), p. 178, Oxford Univ. Press, London 1989. 49. Subbarao, B., Morris, J., and Balayut, A. R., Cell. Immunol. 112, 329, 1988. 50. Subbarao, B., and Melchers, F., Curr. Topics Microbiol. Immunol. 113, 72, 1984. 51. Yakura, H., Shu, F. W., Boyse, E. A., and Tang, L. J., J. Exp. Med. 153, 129, 1981. 52. Gathings, W. E., Lawton, A. R., and Cooper, M. D., Eur. J. Immunol. 7, 804, 1977. 53. Heldrup, J., Kalm, O., and Prellner, K., Acta Paediatr. 81, 125, 1992. 54. Durandy, A., Thuillier, L., Forveille, M., and Fischer, A., J. Immunol. 144, 60, 1990. 55. Lai, R., Visser, L., and Poppema, S., Br. J. Haematol. 87, 251, 1994. 56. Rainaut, M., Pagniez, M., Hercend, T., Daffos, F., and Forestier, F., Hum. Immunol. 18, 331, 1987. 57. Von Freeden, U., Zessack, N., van Valen, F., and Burdach, S., Pediatr. Res. 30, 270, 1991. 58. Kruse, A., Neustock, P., Reuter, M., and Kirchner, H., J. Interferon Res. 13, 221, 1993. 59. Lewis, D. B., Prickett, K. S., Larsen, A., Grabstein, K., Weaver, M., and Wilson, D. B., Proc. Natl. Acad. Sci. USA 85, 3, 1988. 60. Kruse, A., Rink, L., Rutenfranz, I., Kolanczyk, B., and Kirchner, H., J. Interferon Res. 12, 13, 1992. 61. Brugnoni, D., Airo, P., Graf, D., Marconi, M., Lebowitz, M., Plebani, A., Giliani, S., Malacarne, F., Cattaneo, R., Ugazio, A. G., Albertini, A., Kroczek, R. A., and Notarangelo, L. D., Eur. J. Immunol. 24, 919, 1994. 62. Fuleihan, R., Ahern, D., and Geha, R. S., Eur. J. Immunol. 24, 925, 1994. 63. Durandy, A., De Saint Basile, G., Lisowska-Grosspierre, B., Gauchat, J. F., Forveille, M., Kroczek, R. A., Bonnefoy, J. Y., and Fischer, A., J. Immunol. 154, 560, 1995. 64. Rijkers, G. T., Dollekamp, I., and Zegers, B. J., J. Immunol. 141, 388, 1988. 65. Lawton, A. R., Int. J. Technol. Asses. Health Care 10, 54, 1994. 66. Orson, F. M., De Seau, V., Pike, S., and Blaese, M., J. Immunol. 133, 8, 1984. 67. Jelinek, D. F., and Lipsky, P. E., Adv. Immunol. 40, 1, 1987.
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CI961033
CD72 Ligation Regulates Defective Naive Newborn B Cell Responses LAURENCE M. HOWARD
AND
DENIS J. REEN
Children’s Research Centre, Our Lady’s Hospital for Sick Children, Crumlin, Dublin 12, Ireland Received June 27, 1996; accepted September 24, 1996
The biological basis for reduced Ig production by naive newborn B cells compared to adult peripheral blood B cells is not fully understood. In a Con A / IL2 T cell-dependent system using ‘‘competent’’ adult T cells, adult B cells produced large amounts of IgM, IgG, and IgA, while cord B cells were restricted to low levels of only IgM production. Cord B cell activation was also diminished. The contribution of specific B–T cell contact-mediated events to the diminished cord B cell response in this system, using mAbs to CD40, CD28, CD80, and CD72, were investigated, as well as regulation of B cell Ig production by cytokines. aCD72 ligation increased cord B cell activation and IgM production, but did not affect adult B cells. Blocking aCD40 mAb inhibited cord B cell Ig production completely, but only partly inhibited adult B cell Ig production even at high concentration, suggesting a greater sensitivity of cord B cells to disruption of the CD40-CD40L interaction. Addition of IL-10 did not increase cord B cell Ig production, while adult B cell Ig production was increased. However, combined addition of IL-10 and aCD72 significantly increased cord B cell Ig production over that in the presence of either aCD72 or IL10 alone, but had no effect on adult B cells over that of IL-10 alone. These data suggest that the diminished T cell-dependent response of cord B cells is due to reduced or absent CD72 ligation. CD72 ligation plays an important role in the induction of primary responses by naive B cells. CD72 modulation of naive B cell sensitivity to IL-10 stimulation may have implications in the induction of class switch, which is deficient in newborn B cells. Since all T cells express CD5 constitutively, these data also suggest the existence of another ligand for CD72. q 1997 Academic Press
INTRODUCTION In humans, neonatal immune defence to bacterial and viral infection is deficient (1–5). In particular, the neonate has an impaired response to bacterial capsular polysaccharide antigens, with poor responses being observed even after repeated exposure (6, 7). In vitro, T cell-independent stimulation of newborn B cells, using
EBV, induces levels of IgM comparable to that of adult B cells (5, 8–10). However, newborn B cells fail to secrete IgG or IgA class-switched isotypes in response to EBV stimulation, while adult B cells secrete large amounts of these isotypes. In contrast, in T cell-dependent systems such as Staphylococcus aureus Cowan I (11, 12), PWM (8, 9, 13, 14), and aCD3 (15, 16), the level of newborn B cell IgM production has been shown invariably to be reduced compared to that of adult B cells, with little or no IgG or IgA production being observed. Generation of newborn Ig-secreting B cells has been shown to be lower than that of adult B cells when stimulated with S. aureus Cowan I or PWM (8, 9, 11, 13, 15). Proliferation of newborn B cells is also reduced compared to adult B cells when stimulated in the aCD3 T cell-dependent system (16). T cells provide help for B cell effector function through both contact interactions and by secreting cytokines that promote B cell development and immunoglobulin secretion (17–19). Contact-mediated help involves many ligand pair interactions on the extracellular membrane surfaces of T and B cells (17–21). These B–T cell interactions are dynamic and involve both B cell stimulation and T cell counterstimulation by B cells (18, 20, 21). Some of the critical B-T cell contact interactions include CD40–CD40L (18, 20–23), CD80/ CD86–CD28/CTLA-4 (24–27) and CD72–CD5 (28–30) ligation. CD40 ligation has been shown to play an important role in activation, proliferation, immunoglobulin production, as well as class-switch induction events in B cells (22, 23, 31). CD40L, the ligand for CD40, is expressed short term on activated T cells (22, 23, 31). CD80 is one of three B cell surface antigens that bind to CD28 and CTLA-4 on T cells (24–27) and is expressed on the surface of activated B cells (24, 27, 32–35). Ligation of CD28 induces T cell activation, proliferation, and secretion of cytokines such as IL-2, IL4, IL-6, and IFN-g (36–42). It is now considered that coligation of CD28 with the TCR is required for effective induction of T cell helper effector function (43, 44). Ligation of CD28 on T cells results in the induction of CD40L expression (38, 39), while ligation of CD40 on B cells results in CD80 expression (45, 46). This has led to the suggestion that a signaling circuit is formed
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by these two ligand pair interactions between the B and T cell (18). Ligation of CD72 on B cells has been shown to induce proliferation (47–50) and to be required for plaque formation in T cell-dependent systems (51). However, the role of CD72 ligation in the T cell-dependent B cell response is less well defined than either CD28 or CD40 ligation. The deficiency of the newborn T cell-dependent B cell response has been associated with diminished function of both B and T cells (15, 16). Newborn B and T cell populations are almost exclusively naive (7, 52–56). This is in contrast to adult cell populations which contain large numbers of differentiated and memory B and T cells, respectively (7, 52, 55, 56). Newborn T cells have been shown to be predominantly suppressor inducers (8, 9, 13, 14) and have a reduced ability to secrete cytokines such as IL-2, IL-4, and IFN-g (12, 57– 60), which promote B cell effector function. Newborn T cells have reduced expression of CD40L upon activation compared to adult T cells, when stimulated with mitogens such as PHA or PMA (61–63). Newborn B cells are less sensitive to CD40 ligation compared to adult B cells (63). It has been suggested that newborn B cells require signals that are not necessary to drive adult B cell effector function (15, 64–66) and that the basis for such signaling requirements are qualitative (64), quantitative (11, 66), or kinetic (66) in origin. However, such signals have not been identified and little is known of the deficiencies of newborn B cells in T cell-dependent systems. The lack of secreted IgG and IgA from class-switched B cells has been associated with the absence of differentiated surface IgD-negative B cells in newborn B cell populations (7, 52–56, 67– 70) which are present in large quantities in adult B cell populations (7, 52, 55, 56). While much attention has been given to the low or absent class-switched response of newborn B cells in T cell-dependent systems, little investigation into the poor primary response of these cells has been carried out. This study investigated the role of T–B cell ligand pair interactions and cytokines in regulating the impaired responses of naive newborn B cells in a Con A plus IL-2, T cell-dependent system that was MHC class II independent (71). This allowed the investigation of naive newborn B cell interactions with competent adult T cells, in the absence of neonatal T cells that are primarily suppressor–inducers (8, 9, 13, 14) and have been shown to be deficient in providing help for B cells (8, 9, 13–16).
tol–Myers Squibb, Seattle, WA). Purified CD40 mAb 89 was a kind gift from Dr. J. Banchereau (ScheringPlough, Dardilly, France). Purified CD80 mAb BB-1 was a kind gift from Dr. E. A. Clark (University of Seattle, WA). Purified mAb to CD72 (BU40) was obtained from The Binding Site (UK). Human recombinant IL2, IL-6, and IFN-g were obtained from Boehringer Mannheim (Germany). Recombinant human IL-4 and IL-10 were obtained from Genzyme Corporation (Cambridge, MA). Recombinant human IL-5 was obtained from British Biotechnology (UK). Purification of B and T Cells Adult peripheral blood was collected randomly from a large pool of donors in EDTA tubes, from healthy volunteers aged from 19 to 55 years, and cord blood was obtained from full-term newborn infants with a mean gestation age of 41 { 1 weeks (mean { 1 SD). PBMCs and CBMCs were obtained from blood samples by Lymphoprep density gradient centrifugation (Nycomed Pharma AS, Norway). These cells were placed on a Nycoprep 1.063 density gradient (Nycomed Pharma AS) and centrifuged at 350g for 15 min, to remove platelets. The cells obtained were incubated with 10 mM L-leucine methyl ester (Sigma Chemical Co.) to remove monocytes and some NK cells, as has been described (15, 16). The remaining cells were then passed through two rounds of aminoethylisothiouronium bromide (Sigma Chemical Co.)-treated sheep red blood cell (Becton–Dickinson, Mountainview, CA) rosetting, as previously described (72, 73). The rosetted cells from the first round of rosetting were depleted of red cells using 0.17 M ammonium chloride lysis and then incubated on a nylon wool column at 377C for 45 min, giving a purified T cell preparation. The nonrosetted B cells, after the second round of rosetting, were collected and treated with 0.17 M ammonium chloride, to remove contaminating red blood cells. Purities of T and B cell preparations were determined by flow cytometry. Adult peripheral and cord blood B cell preparations contained 81 { 7 and 86 { 6% CD19/ cells, 8 { 6 and 6 { 3% CD3/ cells, 7 { 4 and 6 { 4% CD16/CD56/ NK cells, and 2 { 1 and 2 { 1% CD14/ monocytes, respectively. Adult and cord T cell preparations contained 97 { 2 and 97 { 2% CD3/ cells, 0.9 { 0.5 and 0.8 { 0.5% CD19/ cells, 2 { 1 and 2 { 1% CD16/CD56/ NK cells, and õ0.5 and õ0.5% CD14/ monocytes, respectively. Cell Culture
MATERIALS AND METHODS
Concanavalin A was obtained from Sigma Chemical Company (UK). Monoclonal antibody 9.2, to CD28, in ascites, was a kind gift from Dr. J. A. Ledbetter (Bris-
Cell culture was carried out in RPMI 1640 Dutch modification medium (Gibco BRL, Gaithersberg, NY) containing 20% fetal calf serum (Gibco BRL), 100 U/ ml penecillin, 100 U/ml streptomycin (Imperial Laboratories, UK), with 100 mM L-glutamine (Imperial Laboratories). B Cells (1 1 104) were added to 1 1 105 T cells
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in a 96-well round-bottom tissue culture plate (Nunc, Denmark). Con A at a final concentration of 1 mg/ml and IL-2 at a final concentration of 50 U/ml were added to the recombined cells and the volume in each well was made up to 200 ml, by addition of culture medium. Cultures were carried out in triplicate and incubated at 377C in a 100% humidified atmosphere containing 5% CO2 . For determination of activation, the cells were cultured for 6 days. For measurement of immunoglobulin production, cells were cultured for 12 days. Purified mAbs to CD40, CD72, or CD80 were added at a final concentration of 1 mg/ml, while 9.3 ascites was added at a final dilution of 1/1000. Recombinant human cytokines IL-4, IL-5, IL-6, IL-10, and IFN-g were used at a final concentration of 100 U/ml. Measurement of Immunoglobulin Production After 12 days, the tissue culture plates were centrifuged to pellet the cells and culture supernatants were aspirated from each well. IgM, IgG, and IgA production was determined by ELISA. Briefly, anti-human IgG, IgM, or IgA antisera (Cappel Research Products, Durham, NC) were coated onto 96-well microtiter plates (Corning Inc., U.S.A.) overnight at 47C in 0.1 M carbonate–bicarbonate buffer, pH 9.6. The plates were then washed with PBS containing 0.05% Tween 20. Samples were placed in each well and incubated for 1 hr at room temperature. The wells were washed, and horseradish peroxidase-conjugated anti-human IgG, IgA, or IgM antisera (Tago Inc., Burlingame, CA) were added to each well. The plates were incubated for 1 hr and then washed. Substrate solution, containing 0.08% (w/v) ophenylenediamine and 0.06% (v/v) hydrogen peroxide solution in 0.1 M citrate phosphate buffer, pH 5.0, was then added. The plates were incubated in the dark at room temperature and the absorbance at 405 nm was determined using a Titertek microplate reader. Standard curves and concentration were determined using a smoothed spline semilogarithmic plot (Multicalc, Pharmacia, Sweden).
Statistics Statistical analyses were carried out by either a paired nonparametric Wilcoxon signed rank test or by an unpaired Mann–Whitney test. RESULTS Immunoglobulin Production by Adult Peripheral and Cord Blood B Cells Adult B cells, cocultured with adult T cells, produced large amounts of IgM, IgG, and IgA in response to stimulation with Con A and IL-2 (Fig. 1A). In contrast, immunoglobulin production by cord B cells cocultured with cord T cells was minimal for all Ig isotypes (Fig. 1C). In order to determine the relative contribution of cord B and cord T cells to the diminished Ig production, cord B cells were cocultured with adult T cells (Fig. 1b). In comparison to the response of adult peripheral blood B cells cocultured with adult T cells, cord B cells secreted significantly reduced levels of IgM (P õ 0.01), with no significant IgG or IgA production (n Å 5) (Fig. 1B). No Ig production was observed when adult T cells were cultured alone with Con A and IL-2 in the absence of added B cells, compared to unstimulated adult T cell cultures (n Å 3). Influence of B-T Cell Contact Interactions on Immunoglobulin Production
For measurement of activation of B cells, cells were gently resuspended in the culture wells and transferred to 12 1 75 mm polystyrene tubes (Falcon, Ireland). Phycoerythrin conjugated anti-CD19 mAb (Becton– Dickinson), and FITC-conjugated anti-CD25 or irrelevant control mAbs (Dakopatts AS, Denmark) were added to individual tubes. The contents of the tubes were mixed and incubated in the dark for 15 min. The cells were then washed with PBS containing 20% donor horse serum (Gibco) and resuspended in wash medium, and the cell population was analyzed by flow cytometry (Facscan flow cytometer, Becton–Dickinson). Two thousand CD19/ gated cells were acquired, from which the proportion of CD25/ B cells was determined.
Anti-CD28, CD40, CD72, or CD80 mAbs were added at the start of culture to investigate the contribution of specific contact-mediated signals to the deficient immunoglobulin production by cord B cells. Addition of aCD40 mAb had no significant effect on adult B cell immunoglobulin production, in the presence of adult T cells (Fig. 2a), while it almost completely inhibited IgM production (P õ 0.05) in cord B cell-adult T cell cocultures (Fig. 2b). Ig production in cord B–T cell cocultures was also significantly inhibited (P õ 0.05) by antiCD40 mAb (Fig. 2C). However, in this case, the level of immunoglobulin production was already extremely low. At 10-fold higher concentrations (10 mg/ml) of CD40 mAb, adult B cell Ig secretion was inhibited (data not shown). Addition of aCD28 to adult B–T cell cocultures resulted in significant inhibition (P õ 0.05) of IgM, IgG, and IgA production (Fig. 2A). The addition of aCD28 to cocultures of cord B cells with adult T cells had no significant effect (Fig. 2B), but significantly increased (P õ 0.05) the level of IgM production in cord B–T cell cocultures (Fig. 2C). However, the level of immunoglobulin produced remained low. Anti-CD80 had no significant effect on adult B cell immunoglobulin production (Fig. 2A), although the level of IgM production was slightly raised. Addition of aCD80 to cocultures of cord B cells with adult T cells had no significant effect (Fig.
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FIG. 1. Immunoglobulin production in the Con A / IL-2 system by (A) adult B cells cocultured with autologous adult T cells, (B) cord B cells cocultured with adult T cells, and (C) cord B cells cocultured with autologous cord T cells. Culture supernatants were harvested on Day 12, and Ig levels were determined by ELISA. Data represent means { 1 SEM for five experiments. Statistical analysis was carried out by paired Wilcoxon signed rank test compared to the response in the absence of Con A and IL-2; asterisks denotes P values less than 0.05. Note the different scales between the graphs.
2B). In cord B–T cell cocultures, the level of IgM production was significantly increased (P õ 0.05), but again the level of IgM produced remained low (Fig. 2C). In adult B–T cell cocultures, addition of aCD72 had no significant effect (P ú 0.05) on adult B cell immunoglobulin production of any isotype (Fig. 2a). In comparison, addition of aCD72 to cocultures of cord B cells with adult T cells significantly increased (P õ 0.05) the level of IgM production (Fig. 2b) and also induced a significant increase in IgA production (P õ 0.05). In cord B– T cell cocultures, the level of IgM and IgG production was also significantly increased (P õ 0.05) by addition of anti-CD72 mAb (Fig. 2c). As was the case with all cord B–T cell cocultures, the immunoglobulin levels remained low. Regulation of Adult and Cord B Cell Activation by mAbs to Contact Associated Antigens The regulation of cord B cell activation through ligation of CD28, CD40, CD72, and CD80 on both B and T cells was investigated, by addition of mAbs to these antigens. Activation was assessed by the expression of CD25 (IL-2Ra) on CD19-positive B cells. As shown in Figs. 3A–3C, cord B cell activation in the presence of either adult or cord T cells, was significantly reduced compared to that of adult B cells cocultured in the presence of adult T cells (P õ 0.01). Addition of anti-CD40 in soluble form to all cultures of adult or cord B cells significantly inhibited (P õ 0.05) activation of B cells (Fig. 3). Addition of anti-CD28 mAb to adult B–T cell cocultures had no effect on adult B cell activation, but significantly increased (P õ 0.05) cord B cell activation when they were cocultured with either adult or cord T cells (Fig. 3). Addition of antiCD80 had no effect on adult or cord B cell activation (Fig. 3). Anti-CD72 addition had no significant effect on adult
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B cell activation, but significantly increased (P õ 0.05) cord B cell activation when they were cocultured with either adult or cord T cells (Fig. 3). However, even in the presence of anti-CD72, the level of cord B cell activation was still significantly reduced (P õ 0.05) compared to that of adult B cells. Combined addition of anti-CD72 and anti-CD28 to cord B–T cell cocultures did not significantly increase the level of cord B cell activation above that of anti-CD72 or anti-CD28 alone (data not shown). Effect of Cytokines on Immunoglobulin Production by Adult and Cord B Cells Addition of IL-4, IL-5, IL-6, and IFN-g had no or minimal effect on Ig production by adult B cells, cocultured with adult T cells (Fig. 4a). Addition of IL-10 to adult B–T cell cocultures significantly increased IgM, IgG, and IgA production, compared to the response in the system alone (P õ 0.05). IL-10 augmented IgM production by adult B cells by approximately fivefold. Addition of any of the cytokines had no, or minimal, effect on immunoglobulin production of any isotype by cord B cells cocultured with adult T cells (Fig. 4B), except for a small but significant increase in IgA production in the presence of IL-10 (P õ 0.05). In cord B– T cell cocultures, addition of IL-5, IL-6, and IFN-g to cocultures had little effect (Fig. 4c). Addition of IL-4 or IL-10 significantly increased cord B cell IgM production (P õ 0.05) in cord B–T cell cocultures, but the response was low. Regulation of Immunoglobulin Production by Combined Addition of Anti-CD72 and IL-10 Since IL-10 caused a significant increase in adult but not cord B cell immunoglobulin production in the
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cocultures with either adult or cord T cells did not affect the level of IgG or IgA production observed in the presence of either aCD72 or IL-10 alone. DISCUSSION In this study we have investigated the deficient T cell-dependent response of cord blood B cells, using a Con A / IL-2 culture system. This system, which is MHC class II-independent (71), allowed coculture of adult T cells and cord B cells thereby excluding any influence that naive newborn T cells might have on the system. T cell-dependent responses require bidirectional contact-mediated interactions between B and T cells (18, 20, 21), as well as cytokine secretion from T
FIG. 2. Regulation of immunoglobulin production by ligation of B-T cell contact antigens using mAbs. (A) Adult B cells cocultured with autologous adult T cells, (B) cord B cells cocultured with adult T cells, and (C) cord B cells cocultured with autologous cord T cells and stimulated with Con A and IL-2. Data represent means { 1 SEM for six experiments. Asterisks denote significant P õ 0.05 in response to mAb addition compared to the system alone, using a paired Wilcoxon signed rank test. Note the different scales of response in each graph.
presence of competent adult T cells, combined stimulation through CD72 ligation in the presence of IL-10 was investigated to determine whether CD72 ligation could restore the lack of responsiveness of cord B cells to added IL-10 (Fig. 5). Adult B cells were unaffected by combined addition of aCD72 and IL-10, compared to added IL-10 alone (Fig. 5A). However, addition of aCD72 in combination with IL-10 synergistically increased cord B cell IgM production (P õ 0.05), in cocultures with adult or cord T cells (Figs. 5B and 5C). The increase in IgM production by cord B cells cocultured with adult T cells was approximately 9-fold (Fig. 5B) and 14-fold when cocultured with cord T cells (Fig. 5B). Combined addition of aCD72 and IL-10 to cord B cell
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FIG. 3. Regulation of B cell activation by ligation of B–T cell contact antigens using mAbs. (A) Adult B cells cocultured with autologous adult T cells, (B) cord B cells cocultured with adult T cells, and (C) cord B cells cocultured with autologous cord T cells and stimulated with Con A and IL-2 for 6 days to induce optimal expression of CD25. Data represent means { 1 SD for five experiments. Asterisks denote significant difference in the activation of CD19/ B cells in the presence of specific mAb compared to in the system alone (P õ 0.05), using a paired Wilcoxon signed rank test.
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cells (17–19). We have shown that both cord B and T cells contribute to the diminished immunoglobulin response, in vitro, of newborn lymphocytes. This supports other studies which have attributed this deficient response to both cord B and T cells (15, 16). However, we have also shown that the deficient response of cord B cells is due in part to specific contact interactive deficiencies between cord B cells and otherwise ‘‘competent’’ adult T cells, and that poor induction of cord B cell activation results in their reduced capacity to respond to cytokine stimulation. It has previously been demonstrated that the induction of effective activation and proliferation of B cells is directly associated with the level of immunoglobulin production in vitro (74, 75). Thus, activation of cord B cells was compared to that of adult B cells, in order to assess its contribution to the deficient immunoglobulin
response of cord B cells. Activation was determined by expression of CD25 (IL-2Ra) on B cells, as it is expressed only during activation and blast stage development of B cells. Since an apparent deficiency in the cross-talk between T cells and cord B cells was observed, this suggests that defective induction of activation and proliferation may affect the overall immunoglobulin response of cord B cells. Furthermore, it has been suggested that newborn B cells are deficient in their capacity to undergo IgA and particularly IgG isotype class switching (15, 16). Cytokines secreted by T cells play an important role in regulating B cell Ig isotype class switching (12, 15, 16, 57–60). In this study, we have shown that naive newborn B cells, stimulated in a T cell-dependent system, have a diminished capacity to respond to cytokine stimulation, compared to adult B cells. This suggests that the capacity of naive newborn B cells to undergo class switch events may be limited due to their insensitivity to cytokine manipulation, in T cell-dependent systems. This paper concentrates therefore on the induction of optimal primary cord B cell IgM responses and investigates the B–T cell interactions that may be responsible for the deficient responses of cord B cells in T cell-dependent systems (15, 16). By using mAbs directed at specific B – T cell interactions, we were able to investigate the contribution of particular ligand pair interactions between T cells and cord B cells to deficient cord B cell responses. mAbs in this study were chosen because of their recognized mode of action. The CD40 mAb, alone, was used to block CD40L – CD40 interactions, in order to determine whether this critical event was present in cord B cell – adult T cell cocultures. CD28 and CD80 mAbs were used to stimulate and block the CD28 signalling pathway, respectively, which is a critical costimulatory event for driving T cell effector function. CD80/CD86 signalling of CD28 in our system is probably minimal, especially when naive B cells are used. CD72 mAb was used in order to determine whether optimal CD72 ligation was occurring in cord B cell cocultures, which is thought to be provided in a T – B cell interaction by CD5. Ligation of CD28 on T cells increased activation of cord, but not adult, B cells in the co-culture system. Ligation of CD72 on B cells augmented cord B cell activation and IgM production, while adult B cell responses were not significantly altered. This leads us to speculate that CD28 and CD72 ligation is diminished in cord B cell cocultures with adult and cord T cells, and that this diminished ligation was due to the diminished expression of their ligands on B and T cells, respectively. This in turn must reflect an inability of cord B cells to interact effectively even with ‘‘competent’’ T cells. Almost all cord B cells are naive (7, 52–56). It is possible that diminished ligation of CD28 on T cells, by cord B cells, may reflect diminished or absent CD80/86 ex-
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FIG. 4. Effect of cytokines on Ig production by (A) adult B cells cocultured with autologous adult T cells, (B) cord B cells cocultured with adult T cells, and (C) cord B cells cocultured with autologous cord T cells and stimulated with Con A and IL-2 for 12 days. All cultures were stimulated with Con A and IL-2. Data represents mean { 1 SEM for six experiments, and data were analyzed using a paired Wilcoxon signed rank test. Asterisks denote significance of P õ 0.05 compared to the response in the absence of cytokines. Note the different scales used for each graph.
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FIG. 5. Effect of addition of aCD72 and IL-10 on immunoglobulin production by (A) adult B cells cocultured with autologous adult T cells, (B) cord B cells cocultured with adult T cells, and (C) cord B cells cocultured with autologous cord T cells and stimulated with Con A / IL-2 for 12 days. Data represent means { 1 SEM for six experiments and were analyzed using a paired Wilcoxon signed rank test. Asterisks denote significant difference compared to Ig production in the system alone (P õ 0.05). Note the different scales of each graph.
pression, which has been shown to be reduced on activated naive B cells (43, 76). The known ligand for CD72, CD5, is expressed constitutively at high levels on both adult and cord T cells (28–30) as well as at low levels on a subpopulation of B cells (28). The deficient CD72 ligation observed on cord B cells in the presence of adult or cord T cells, in this system, suggests, therefore, that an alternative ligand for CD72 may exist, and that this ligand is more effective at inducing signal transduction through CD72 than CD5. It has been suggested that, given the structural similarity to CD23, an antigen which possesses multiple ligands (28), CD72 may also associate with more than one ligand. Furthermore, an alternative ligand for CD5, other than CD72, has also been reported (77), and no functional evidence has yet been presented to show a functional role for CD5–CD72 interactions in the T cell-dependent response. The fact that CD72 ligation on cord B cells was deficient suggests that an alternative CD72 ligand must be induced on activated T cells. CD72 ligation was critical in driving cord B cell activation and IgM production and has been shown to be a potent costimulator of B cell proliferation when used in conjunction with mitogenic stimulation through the B cell receptor (28, 47). When aCD28 and aCD72 were added together no further increase in cord B cell activation was observed over that of addition of either antibody alone, suggesting that the induction of expression of the alternative CD72 ligand on T cells may be under the control of costimulatory events associated with CD28 ligation, and that CD72 ligation may regulate CD80/86 expression on B cells. In support of this argument, costimulation of T cells by CD28 ligation has been shown to induce CD40L expression, and thus play a major role in regulating B-T cell contact activational events (38, 39). aCD80 mAb did not affect cord or adult B cell Ig production, when cocultured with
adult T cells. The CD28/CTLA-4 ligation event is complex and involves two antigens on B cells (CD80 and CD86) and T cells (CD28 and CTLA-4). In cord B–T cell cocultures, aCD80 induced significant IgM production. While the reasons for this are unclear, it may be due to (i) signal transduction through CD80 ligation into the B cell, (ii) differential regulation of CD28/CTLA-4 signal transduction in T cells following ligation by CD80 and CD86, or (iii) induction of a different cytokine secretion profile by T cells due to aCD80 mAb. The CD40-CD40L interaction between B and T cells was investigated using mAb 89, a partial antagonistic antibody that contains an epitope specificity that obstructs CD40L interaction with CD40 on B cells (78). CD40L expression by cord T cells has been shown to be deficient, compared to that of adult T cells (61–63), which express levels of CD40L in excess of that required to drive B cell responses (78). Conversely, cord B cells have been shown to be less sensitive to CD40 ligation than adult B cells (63). Since CD40-CD40L interaction plays an important role in regulating B cell responses (18, 22, 23), it has been suggested that this deficiency in the interaction between cord B and T cells may contribute to the deficient responses of cord B cells (61–63). When cord B cells were cocultured with adult T cells, significant amounts of IgM were produced. Addition of aCD40 mAb, at 1 mg/ml, inhibited this response completely but did not significantly inhibit adult B cell Ig production, suggesting that CD40 ligation was driving cord B cell IgM production, at least in the presence of adult T cells. However higher levels of aCD40 mAb (10 mg/ml), also inhibited IgM production by adult B cells. This suggests that either cord B cells required a higher level of CD40 ligation than adult B cells, for which some evidence exists (63), or that induction of CD40L expression on adult T cells was diminished when these cells were cocultured with cord
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B cells compared to that when cocultured with adult B cells. CD28 ligation, which induces T cell expression of CD40L (38, 39), was deficient in this system when cord B cells were cocultured with adult T cells. Thus both a reduced level of CD40L expression on T cells when cocultured with cord B cells and a requirement for higher levels of CD40 ligation on cord B cells may contribute to the deficient response of cord B cells in this system. With the exception of IL-10, none of the cytokines studied had a positive effect on adult B cell Ig production. In cord B–T cell cocultures, IL-4 increased IgM production. This effect was not observed when cord B cells were cocultured with adult T cells. Cord T cells, being mainly CD45RA cells, do not secrete IL-4 (12, 59), suggesting that addition of IL-4 to cord B–T cell cocultures provided cytokine stimulation that was otherwise absent. In contrast to adult B–T cell cocultures, addition of IL-10 to cocultures of cord B cells with adult T cells did not significantly increase IgM production, while in cord B–T cell cocultures a small increase in IgM production was observed. While adult B cells are highly responsive to IL-10 stimulation, in this system, cord B cells are relatively insensitive to IL-10 stimulation. When aCD72 and IL-10 were added together to cord B–adult T cell cocultures, a large synergistic increase in IgM production was observed. In cord B–T cell cocultures, aCD72 and IL-10 also acted synergistically in increasing IgM production by cord B cells. This was in contrast to adult B cells, in which combined addition of aCD72 and IL-10 did not increase the level of Ig production compared to addition of IL-10 alone. In cord B–adult T cell cocultures, CD72 ligation resulted in induction of responsiveness to IL-10 that was otherwise absent. Since CD72 is already expressed at very high levels on naive cord B cells, it is unlikely that this effect is due to upregulation of CD72 expression on cord B cells, by added IL-10, followed by enhanced signal transduction through mAb ligation of CD72. Thus ligation of CD72 in cord B cell cocultures with either adult or cord T cells must increase their sensitivity to IL-10. The reasons why cord B cells are less sensitive to CD40 ligation than adult B cells has yet to be determined. It has been suggested that driving cord B cell responses requires signals not needed by adult B cells (15, 64–66), and thus their signaling pathways may be somewhat different. Such events may play a role in costimulating and amplifying CD40-dependent signals in naive B cells, and thus in their absence, result in reduced sensitivity to CD40 ligation. It is tempting to speculate that CD72 ligation may play this costimulatory role, supplementing reduced signal transduction in response to CD40 ligation. In support of this argument, CD72 ligation cooperates with CD40/IL-4 signals, augmenting B cell proliferation (47), and, like CD40, rescues apoptotic B cells (79). The induction of
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IL-10 responsiveness of cord B cells is also regulated by CD40 signal transduction (80). Thus it remains to be seen how CD72 and CD40 signaling events are intertwined and whether other CD40 dependent events, such as induction of CD80/86 expression, can be modulated by CD72 ligation. While newborn B cell responsiveness was increased by CD72 ligation, the level of IgM production was still reduced compared to that of adult B cells. B cells can secrete immunoglobulin at high or low rates (67, 81). Thus it is probable that the cord B cell IgM response reflects cells that are capable of only low rates of IgM secretion, while adult B cells are capable of high rates of IgM secretion. B cell development towards high rate Ig secreting cells requires the cell to pass through a number of cell division cycles (67) and is influenced by dendritic cells (81). Development of high-rate Igsecreting plasma cells occurs in the bone marrow and in the mucosa (81). This suggests a further level of immaturity of cord compared to adult B cell populations, that may reflect the primed but inactive status of the newborn immune system compared to an active immune system in adults. The deficient T cell-dependent response of cord blood lymphocytes is due to both T and B cell deficiencies. In this study we have identified deficiencies in specific events in the T cell-dependent response of cord B cells that result in diminished cord B cell differentiation and Ig secretion as well as a reduced ability of cord B cells to costimulate T cells. Ligation of CD72 on B cells significantly influences cord B cell responses, increasing activation, immunoglobulin production, and responsiveness to cytokines such as IL-10. These data suggest that an alternative CD72 ligand, other than CD5, exists, and that this ligand interaction with CD72 plays a critical role in regulating naive B cell responses. ACKNOWLEDGMENTS We thank the nursing staff of the Coombe Women’s Hospital, Dublin, for collecting cord blood samples.
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