HLA class-II-mediated B-lymphocyte activation: signal transduction and physiologic consequences

(~) INSTITUTPASTEUR/ELSEVIER Paris 1991

Res. Immunol. 1991, 142, 467-474

HLA class-If-mediated B-lymphocyte activation: signal transduction and physiologic consequences D. C h a r r o n , S. B r i c k - G h a n n a m , R. Ramirez and N. Mooney Laboratoire d'Immunog~n~tique mol~culaire, Institut Biomddical des Cordeliers, 15, rue de I'Ecole de Mddecine, 75006 Paris

Introduction Class II antigens of the major histocompatibility complex (MHC) have a well-established role as recognition structures in immune responses. These molecules ensure the genetic restriction of the cellular interactions in the presentation of a processed antigen to T cells (Kaufman et al., 1984; Unanue, 1984). It is worth notin• that these molecules, while expressed on monocytes/macrophages and B lymphocytes, lead to the activation of another cell type, the helper T cell. This is rather unique in the world of cell surface transmembrane proteins, most if not all of which directly trigger the cell on which they are expressed (Williams and Barclay, 1988). The, e is increasing experimental evidence that, besides their peptide-p~esenting function, MHC class II antigens can directly activate B cells and monocytes and induce progression in the cell cycle and/or differentiation (Cambier et al., 1987; Mooney eta/., 1990). These molecules do belong to the immunoglobulin superfamily, other members of which may act either directly or indirectly as activation, adhesion or differentiation antigens. The involvement of class II molecules in the above functions may help to explain a physiological role for the class II molecules which are expressed on non-immune cells and are unlikely to have a major role in peptide presentation. In the physiological situation these include the haematopoietic progenitors, endothelial cells and various epithelial tissues in dutoimmune disorders (Radka et al., 1986). These findings provided a rationale to investigate the direct role of HLA class II molecules in cellular activation. The constitutive expression of these molecules on B lymphocytes facilitated this study. We have reported increased 3H-thymidine uptake by lymphocytes stimulated using immobilized anti-MHC class II mAb (Mooney er al., 1989) and have provided biochemical evidence

for signal transduction via HLA class II antigens on resting B lymphocytes (Mooney et al., 1990). Many second messenger systems involve phospholipase-Cmediated hydrolysis of phosphatidylinositol 4,5-biphosphate and production of inositol 1,4,5-triphosphate which releases Ca 2+ from intracellular stores and l-2-diacylglycerol (DAG), which activates protein kinase C (PKC). Both an increased intracellular calcium flux (Ca~ +) and phosphatidyl inositol biphosphate hydrolysis (Mooney et ai., 1990) were observed in dense B cells in response to anti-HLA class I| antibodies.

DAG accumulation We report here that anti-class-II stimulation of B cells results in an increase in the DAG content of the cells which is both concentration- and timedependent (fig. l). The time cours~ ~f DAG production in B lymphocytes stimulated with anti-DR mAb (Dl. 12) revealed a maximum at 4 min and gradually fell toward the basal level over the ensuing 10 min as shown in figure lB. Although these results were totally consistent with the involvement of a PKC pathway, it was important to consider the possibility that HLA class II B-cell activation may trigger several interacting or independent transduction systems.

cAMP production The adenylate cyclase pathway was tested in the same experimental setting. Neither anti-DR (DI. 12) nor anti-DQ (L2) treatment were shown to increase the production of cAMP above the control level (fig. 2). These results contrast with the observation that in mice, anti-la stimulate cAMP generation in quiescent B cells (Cambier et aL, 1987). This discrepancy

468

D. C H A R R O N E T A L .

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Fig. 1. Concentration dependence and time course of diacyiglycerol accumulation in HLA-class-ll-antibodystimulated B cells.

Fig. 2. Effect of HLA class II antibodies on cAMP production.

A) B cells were stimulated with DI. 12 antibody (directed against HLA DR antigen) for 4 min at the indicated concentrations. B ) B cells were stimulated with Dl.12 at 25 g/ml for various times. These results are representative of ,t different experiments.

A) Cells (2.5 x 105 cells/point) were treated by forskoline 10 -4 M for different lengths of time. B) 2.5 × 105 cells/point were treated for 1 min with anti-HLA class II antibodies (D1.12 and L2) or anti-HLA class I antibody (W6.32).

may well be accounted for by a fine difference in the B-ceU populations studied in human versus the mouse models. Alternatively, the stage o f differentiation o f the B cell could determine the type o f signal transduction pathways which can be triggered~

P K C activity and synthesis

DAG MHC PKC

= diacylglycerol. = major histocompatibility complex. = protein kinase C.

PKC intracellular compartimentalization and activity were analysed in human B cells and B-cell lines. HLA class II ligation induced a definite increase in

PLC PTK

= phospholipase C. = protein tyrosine kinase.

469

H L A C L A S S - I I - M E D I A TED B-L Y M P H O C Y T E A C T I V A T I O N

PKC activity (Brick-Ghannam, 1991). Interestingly, this augmentation involved both the cytosol and membrane fractions. In contrast to PKC responses following phorbol ester treatment, translocation of the cytosolic PKC to the membrane was not observed at any time. Moreover, the peak of PKC activity was observed after 30 to 45 min in contrast with the usual maximum at 5 to 10 min in other systems. This delay led us to assess the synthesis of the enzyme and its transcriptional a n d / o r post-transcriptional regulation. Western blot analysis revealed that anti-class-lI treatment of B cells resulted not only in an increase in the activity of the enzyme but also in an augmentation of the protein using an anti-PKC polyclonal antibody (a gift from F. Huang, NIH) (fig. 3). This increase in PKC was blocked by actinomycin D, and Northern blot analysis confirmed transcriptional regulation (manuscript in preparation). In contrast

1

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3

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5

Fig. 4. Western blot analysis of tyrosine phosphorylation induced via HLA class II molecules in dense B cells. Cells were incubated for I0 min with 10 gg/ml of the following immobilized anti-HLA class II antibodies. Lane 1 : unstimulated; lane 2: anti-lgM (2.5 gg/ml); lane 3: DI.12; lane 4: i.35; lane 5: L2.

CYTOSOL to the mouse, our results demonstrate that the ligation of class II by specific antibodies induced both cytosolic and membrane PKC activity and synthesis. These data reveal that the activation of PKC did not necessarily involve translocation (Brick-Ghannam et al., 1990).

4--82

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Fig. 3. Western blot analysis of PKC in B cells. Cells were treated for 30 min with 12.5 g/ml of different HLA class I1 antibodies or with 20 ng/ml of TPA; 50 g/track of protein are compared. Results are repre.~en tative of three experiments. Lane 1 : control; lane 2: TPA; lane3: L2; lane4; 206; lane 5: 1.35; lane6: B7.21; lane 7: D I. 12.

Tyrosine phosphorylation

Several growth factor receptors are able to signal through intrinsic tyrosine kinase activity. In addition, most of the known tyrosine kinases are either growth factor receptors or protooncogenes involved in growth regulation. Taken together, these findings suggest that tyrosine phosphorylation is intimately linked to growth regulation. We have explored the role of tyrosine kinases in anti-class-l l-induced B-cell activation aild proliferation by Western blotting with specific anti-phosphotyrosine antibodies after stimulation via HLA class II molecules. Both anti-DR (DI. 12) and anti-DQ (L2) mAb were responsible for an increase in a number of tyrosine phosphorylations (fig. 4). It is therefore apparent that ligand binding to class II molecules induces B-cell activation via at least two signal transduct!on pathways involving PKC and a tyrosine kinase. Whether or not these pathways are independent or interactive remains to be determined; protein tyrosine kinase (PTK) dependence of phospholipase C (PLC) activation has recently been reported in B lymphocytes (Carter et al., 1991). Tyrosine phosphorylation is intimately linked to growth regulation, while PKC is often implicated in differentiation pathways. It is conceivable that, depending on the type of HLA class II ligand, the state

470

D. C H A R R O N E T AL.

(glycosylation, maturation)of the class II molecule to which it binds and the cell type (or the stage of differentiation), HLA class I1 signalling activates different pathways, resulting in fine tuning of this complex phenomenon.

Preincubation with anti-lgM for 18 h before sepharose Dl.12 did not significantly alter the proliferative response to anti-lgM alone. However, when B lymphocytes were preincubated with sepharose D1.12 preceding incubation with anti-IgM, the proliferative response was significantly increased compared with the proliferative response to anti-IgM alone (fig. 5b).

Before discussing the putative ligands of the HLA class II molecules, it is of interest to consider the physie~ogical consequences of HLA class II ligation. Anti-class respt~nses

II influences

anti-it-induced

The effect of anti-class II antibodies (20-40 Fg/ml) on (Ca~ +) was examined, since the in" (Ca z+ . classically . c r ease m i ) is described as one of the first intracellular events following surface Ig crosslinking. Figure 6 illustrates isometric displays of calcium mobilization by high-density B lymphocytes. Figure 6a shows the typical increase in intracellular free calcium after cross-linking of mIgM by a monoclonal anti-IgM (BPA2.I-CGC). Figure6b shows that DI.12 ( 4 0 F g / m l ) elevated (CaZi+) after cross-linking with a rabbit anti-mouse F(ab')2 resulted in elevated (Ca 2+) comparable to the increase induced by anti-IgM. The increased (Ca~ +) suggests that class II antigen signalling is part of the same pathway as anti- F. However 2-D PAGE patterns of the cellular proteins synthesized after anti- F , PMA or anti-class II treatment shows that different stimuli do not result in precisely the same state of activation of the cell. This is also the case for the induction of protein phosphorylation. Differences in the kinetics of activation of the different signalling pathways may also explain these data.

B-cell

E-lymphocyte activation in vitro is generally obtai.r:.~d after cross-linking of surface immunoglobulin ,~ith an anti-it antibody. Anti-it mAb are variable in ¢~eir ability to induce proliferative responses. ImmG~lization of the antibody may result in a proliferative response which is not observed with the same antibody in soluble form. A sepharose-conjugated anti-MHC-class-II DRantibody induced proliferation of a resting Blymphocyte population (fig. 5a). This effect was not epitope-restricted, as a sepharose-conjugated antiMHC class II antibody also induced proliferation (data not shown). Costimulation of resting B cells with a suboptimal concentration of mitogenic antiIgM (2.5 Itg/ml) and sepharose Dl.12 (6 Itg/ml) resulted in a greater stimulation than with either reagent alone.

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S.I.

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AntiDR

Anticlass I

AntiDP

b

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AntiIgM

Anti- D1.12 IgM +D1.12

AntiIgM 18 hrs + D1.12

D1.12 18 hrs + AntiIgM

Fig. 5. a) Proliferation of high-density B lymphocytes in the presence of sepharose-conjugated D I. 12 (4 ~.g/ml). The stimulation index is shown (control cpm were less than 3,500; n =4 mean+SE). b) Proliferation of high-density B lymphocytes in response to either anti-lgM (2.5 ~.g/ml) or sepharose DI. 12 (6 ~g/ml) added either singly or after an 18-h preincubation with anti-IgM or sepharose DI. 12, respectively.

H L A C L A S S - I L M E D I A TED B-L Y M P H O C Y T E A C T I V A T I O N

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Fig. 6. Isometric displays of (Cai2÷) mobilization by high-density B cells. Antibodies were added after baseline measuremer~t for 2 rain; (Ca~ +) was recorded for 2 min before addition of a rabbit antimouse cross-linking F(ab') 2 (50 i.tg/ml) and monotoring for a further 6 rain. a) (Ca~+) after addition of a monoclonal anti-lgM; b) (Ca 2+) after addition of Dl.12 followed by RAM.

Furthermore, the influence of class II signalling on signalling via slgM was revealed by the demonstration that anti-class-ll treatment was able to induce proliferation in B Go cells treated with an otherwise non-mitogenic anti%t. Similarly to thymus-dependent antigens, a nonmitogenic anti-slg antibody binds to slg without inducing a proliferative response. We have used a nonmitogenic anti-slg as a model in order to investigate the potential contribution of HLA-class-II-moleculemediated signalling in T-cell-dependent antigen responses. The presence of an anti-HLA-DR antibody (Dl.12) preceding or at the same time as a nonmitogenic anti-slg resulted in a significant proliferative response which was not observed with the antisIg alone. This stimulation was more marked when the D 1.12 was present for a short time before the addition of anti-sIg (up to 8 h; fig. 7a,b) and was not significant after 24 h of pre-incubation with D l.12 (fig. 7a,b)o The presence of an anti-sIg followed by addition of DI.12 also resulted in a proliferative response which was still induced following 24-h preincubation with anti-sIg. On the contrary, preincubation with DI. 12 did not change the proliferative response to a mitogenic anti-slg (fig. 7c,d), nor was the proliferative response to a mitogenic antisIg increaseJ by later addition of DI. 12. An antibody directed against the MHC class I antigens (W6.32) was used as an isotype control, and pre-incubation of B cells with this antibody did not significantly induce increased proliferation (fig. 8d).

An anti-HLA class II DQ antibody (L2) was tested in the same system, and similarly to D 1.12, addition of L2 either at the same time or preceding anti-lg resulted in a proliferative response (fig. 8b). Prestimulation with an anti-HLA class II antibody directed against DR, DP and DQ (1.35) did not, however, result in significant proliferation compared to either anti-lg or 1.35 alone (fig. 8a). The results of this study demonstrate that signal transduction via HLA class II antigens can have an enhancing effect on the proliferation of resting human B lymphocytes in response to signalling via slg, the putative antigen receptor. Furthermore, this study demonstrates that class II signalling is influential at an early stage in B-lymphocyte activation. If the signalling via a non-mitogenic anti-slg can be compared to that of a T-cell-dependent antigen, this result suggests that it is the lack of the HLA class II signalling which results in T-cell dependence. In this physiological situation, the combination of both signals may auginent the T-cell dependent signal, while the requirement for both signals may prevent aberrant activation by a putative physiological ligand in the absence of antigen. The use of a non-mitogenic anti-slg a a model for a T-cell-dependent antigen reveals two important features of HLA-class-II-mediated signalling (Mooney, 1991, in press). Firstly, HLA class I1 signalling can provide the first signal in the process of B-lymphocyte proliferation; secondly, this signal may be important for B-lymphocyte responses to T-cell-dependent antigens.

472

D. C H A R R O N E T A L . 3H-thymldln8 (cpm) :5 . . . . . . . . . . . .

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Fig. 7. 3H-thymidine uptake was determined after stimulation with either soluble anti-HLA class II DR (DI. 12, 10 g.g/ml), anti-lgM (non-mitogenic 10 F,g/ml) or mitogenic anti-IgM (2,5 ~,g/ml). Solid bars represent proliferation when DI. 12 was constantly present and anti-lgM added following the indicated number of hours.

CD4 class II interactions in B-lymphocyte activation As anti-g, is used experimentally to mimic antigen binding to surface Ig, anti-class II mAb is intended to reproduce the effect of a physiological binder of the HLA class It molecule. CD4 has been described as a ligand of the HLA class II molecule (Doyle and Strominger, 1987). An HLA class II CD4 interaction was shown to operate in T/B-cell cognate interaction as suggested, by the synergistic inhibitory effect of anti-CD4 and of anti-DR mAb (Fischer et al., 1986). CD4 class II binding was directly demonstrated by B-cell conjugate formation with CD4-transfected L ceils (Doyle and Strominger, 1987). The exact nature of the CD4 class II molecular contacts involved remains controversial. Adhesionlike sequences have been suggested, as well as other

epitopes (Mazerolles et al., 1988; Clayton et al., 1989). These data led us to investigate the role of CD4 class II binding in B-cell signal transduction. Evidence of such involvement was obtained from the following observation: soluble CD4 induces phosphorylation of proteins documented by 2-D PAGE, similar to patterns generated by anti-class II; soluble CD4 increases IP2-IP3 generation in resting and in activated B cells; soluble CD4 inhibits the induction by anticlass II of a proliferative response of B cells in response to non-mitogenic anti ~t. Thus, in three independent assays, soluble CD4 generates responses in B ceils which are highly simi-

H L A CLASS-II-MEDIA TED B-L Y M P H O C Y T E A C T I V A T I O N 3H-thymlOIne (cpm) ................. 101" .......................................................................................................................................................

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Fig. 8. 3H-thymidine uptake was determined after stimulation with either anti-HLA mAb or nonmitogenic anti-lgM. Solid bars represent proliferation when DI. 12 was constantly present and antiIgM added following the indicated number of hours.

lar to those induced with anti-class II antibodies. These data raise the possibility that anti-class II mAb used experimentally may, in fact, mimic the CD4 molecule in physiological T/B-cell contacts.

A role for li

Several questions remain concerning the state of the class II molecule which is engaged in signal transduction. Ii is a 3 l-kDa glycoprotein associated with H L A class II molecules (Charron and McDevitt, 1980). The majority of the class II ([3, ~) complexes exposed at the cell surface are devoid of the Ii chain. This is particularly the case for activated B cells, in which only a small contingent (5 %) of the cell surface class II molecules are associated with li. In resting B cells which are not engaged in cell processing

and presentation, the percentage of class II molecules associated with Ii appears to be higher. In a preliminary study, we have found that an anti-li antibody (LN2) is able to generate IP2-1P3. This was observed in class II + B cells as well as in class II- B cells which are, however, cell-surface-li +. Whether Ii is required for signal transduction remains to be investigated. The Ii chain may act as a transducer intermediate of the class II molecule. Alternatively, li may transduce a signal independently of its physi.cal association with a class II molecule. In this case, the pathway implicated may be identical or different. Transfection experiments are in progress to investigate this question. Key-words: HLA, MHC, B lymphocyte, Transduction; Class II antigens.

474

D. C H A R R O N

References

Brick-Ghannam, C., Mooney, N. & Charran, D. (1991), Signal transduction in B lymphocytes. Human. Irnrnunol., 30, 202-207. Cambier, J.C., Newell, M.K., Justement, L.B., McGuire, J.C., Leach, K.L. &Chen, Z.Z. (i987), Ia binding ligands and cAMP stimulate nuclear translocation of PKC in B lymphocyte. Nature (Lond.), 327,629-631. Carter, R.H., Park, D.J., Rhee, S.G. & Fearon, D.T. (1991), Tyrosine phosphorylation of phospholipase C induced by membrane immunoglobulin in B lymphocytes. Proc. nat. Acad. Sci. (Wash.), 88, 2745-2749. Charron, D.J. & McDevitt, H.O. (1980), Characterization of HLA-DR region antigens by two-dimensional gel electrophoresis molecular genotyping. J. exp. Med., 152, 18s. Clayton, L.K., Sieh, M., Pious, D.A. & Reinherz, E.L. (1989), Identification of human CD4 residues affecting class II MHC versus HIV lgpl20 binding. Nature (Lond.), 339, 548. Doyle, C. & Strominger, J.L. (1987), Interactions between CD4 and class II MHC molecules mediates cell adhesions. Nature (Lond.), 330, 256-259. Fischer, A., Sterkers, G., Charron, D.J. & Durandy, A. (1986), Possible T4-HLA class II interaction as an essential event in antigen-specific helper T-lymphocytedependent B cell activation. Europ. J. ImrnunoL, 16, llll-lll6. Kaufman, J.F., Auffray, C., Korman, A.J., Shackelford,

ET AL.

D.A. & Strominger, J. (1984), The class II molecules of the human and murine major histocompatibility complex. Cell, 36, 1. Mazerolles, F., Durandy, A., Piatier-Tonneau, D., Charron, D., Montagnier, L., Auffray, C. & Fischer, A. (1988), Immunosuppressive properties of synthetic peptides derived from CD4 and HLA-DR antigens. Cells, 55, 497-504. Mooney, N., Grillot-Courvalin, C., Hivroz, C. & Charron, D. (1989), A role for MHC class II antigens in B cell activation. J. Autoirnmun., 2, 215-223. Mooney, N., Hivroz, C., Talebian-Ziai, S., GrillotCourvalin, C. & Charron, D. (1989), Signal transduction via MHC class II antigens on B lymphocytes. J. lmmunogenet., 16~ 273-281. Mooney, N., Grillot-Courvalin, C., Hivroz, C., Ju~ L.Y. & Charron, D. (1990), Early biochemical events following MHC class II mediated signaling on human B lymphocytes. J. IrnrnunoL, 145, 2070-2076. Mooney, N., Van Alewik, D., Brick-Ghannam, C. & Charron, D. (1991), HLA class II antigen-mediated induction of a proliferative response to anti-IgM in human B lymphocytes. Int. J. Cancer (in press). Radka, S., Charron, D.J. & Brodsky, F. (1986), Class II molecules of the human major histocompatibility complex considered as differentiation markers. Hum. Irnrnunol., 16, 390-400. Unanue, E.R. (1984), Antigen-presenting function of the macrophage. Ann. Rev. Irnrnunol., 2, 395-428. Williams, A.F. & Barclay, A.N. (1988), Domains for cell surface recognition. Ann. Rev. Irnrnunol., 6, 381-406.