Ig-specific T-B cell interaction

Eur. J. Immunol. 1990.20: 833-839

Svetlana M. Mazel, Alexander Y. Rudensky and VitaGj L. Yurin Laboratory of Immunology, All-Union Research Institute for Genetics and Selection of Industrial Microorganisms, Moscow

833

Immunoglobulin-specific T-B cell interaction 111. B cell activation by immunoglobulinrecognizing T cell clones Immunoglobulin (1g)-specificT-B cell interactions were studied in the model of Tcell recognition of Ig x chain Igx-lb allotype in Igx-l-congenic rats. Using Igx-lb-recognizingmajor histocompatibility complex (MHC)-restricted T helper clones from August rats we have shown that Igx-lb+ B cells from congenic August.lb rats presented Igx-lb epitope of the processed self-synthesized Ig to Tclones. This interaction was found to be a bidirectional regulatory event inducing specific MHC-dependent proliferation of both interacting T cell and B cell as well as Ig(1gx-lb) synthesis. Small Igx-lb+ B cells were capable of inducing T clone proliferation and becoming activated in response to the same T clone. Limiting dilution analysis suggested that every tenth cell in Igx-lb+ B cell population is involved in this interaction. The bystander activation of Igx-la+ B cells byT clones in the presence of irradiated Igx-lb+ spleen cells, if observed,was less than the level of specific Igx-lb+ B cell proliferation. In contrast to a 20-fold increase of Ig(1gx-lb) levels upon stimulation of Igx-lb+lla+B cell population from heterozygous (August x August.1b)FI rats by T clones, a “nonspecific” increase of Ig(1gx-la) was not observed.This result demonstrates the requirement for direct T-B contact for B cell activation to occur. The data suggest a great functional potency of T-B interactions mediated by T cell recognition of Ig-derived peptide/MHC class I1 complexes on the B cell surface.The implication of the data for idiotypic regulation enables us to propose the existence of putative idiopeptidic network T-B cell interactions.

1 Introduction

Ig-specificT-B cell interaction, are of primary importance. There is much experimental data demonstrating the “connectivity” of T and B cell antigen recognition repertoires, for example, involvement of T cells in Id network interactions with B cells [6,7] and influences of IgR expression on the antigen receptor repertoire of T lymphocyte subsets [8-lo]. Recently, A. Coutinho’s group obtained experimental evidence for co-selection of bothTand B cell clones to express a particular Id [ l l , 121.

The “antigen bridge” concept of T-B cell cooperation suggests the requirement of a specificTand B cell interaction mediated by Ag to mount B cell clone activation and differentiation resulting in antibody production [11. At present, the modified version of this concept taking into consideration the data of recent studies on antigenmediated MHC-restricted T-B cell interaction [2-51 could be described as follows. Ag-specific B cell binds native protein Ag molecules via its IgR. Ag molecules bound by Previously,we developed a new model system of Ig-specific sIg undergo endocytosis and subsequent intracellular pro- T-B cell interaction mediated by T cell recognition of Igx cessing (partial proteolysis and denaturation).The peptidic L chain allotype (Igx-lb) in inbred rat strains [13, 141. It fragments of the processed antigen are then expressed in was shown that normal Igx-lb+ B cells are able to present association with MHC class I1 molecules on the B cell Igx-lb determinants of self-synthesized IgR to Igx-lbsurface and can be recognized by the MHC-restricted hpmune T cells [14]. To analyze the B cell presentation of Ag-specificTh cells. MHC-restricted B cell presentation of Igx-lb determinant we have established MHC-restricted an antigen to immune T cell appears to be a cellular T cell clones from August rats (RT-lC;Igx-la) recognizing mechanism of clonal antigen-driven activation of B cells. Igx-lb determinant of both serum Ig molecules and membrane Ig of B cells from congenic August.lb rats It should be emphasized that both the recognition reper- (RT-lC; Igx-lb) [15]. It has been shown that the toires of antigen-specific Th cells and B cells should be Igx-lb-recognizingTcell clones were specific for the BrCN selected prior to antigen priming. Thus, the potential peptide 176-214 [pLk-lb(176-214),approx. M, 60001 from mechanisms of T and B cell clonal selection and regulatory the Igx-lb x chain C-terminus. Sequences 176-214of rat x interactions in the absence of antigen, in particular, chains of Igx-la and Igx-lb allotypes carry only three allelic amino acid substitutions at positions 184,185 and 188 [16]. This peptide was found to be completely devoid of Igx-lb serological activity and did not require additional process[I 78191 ing to be recognized by T cell clones. These results were Correspondence: Vitalij L. Yurin, Laboratory of Immunology, later confirmed in the experiments using synthetic peptides Institute for Genetics and Selection of Industrial Microorganisms, corresponding to different regions of C,(Igx-lb) domain (manuscript in preparation). Thus, these data suggest that Dorozhnij pr. 1, Moscow 113545, USSR the MHC-restricted T cell recognition of the processed form of B cells’own IgR could be considered as a basis of the Abbreviations: RT-1: Rat MHC SC: Spleen cell(s) 0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1990

0014-2980/90/0404-0833$02.Sol0

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Eur. J. Immunol. 1990.20: 833-839

S. M. Mazel, A.Y. Rudensky und V L.Yurin

direct Ig-specific T-B cell interaction in our experimental system. In this report we describe specific activation of Igx-lb+ B cells by the Igx-lb-recognizing T cell clones, which resulted in B cell proliferation and Ig synthesis. Based on recent experiments we suggest the MHC-restricted T cell recognition of the processed self IgR determinants could serve as a possible mechanism of selective B cell activation in the absence of exogenous antigen.

2 Materials and methods 2.1 Animals August (RT-lC, Igx-la) rats were purchased from the animal facility “Stolbovaya” of the Academy of Medical Sciences USSR. Congenic August. l b (RT-lC,Igx-lb) rats as well as (August x August.1b)Fl hybrids were obtained from our animal facility. Female rats were aged 2-3 months. The priming of August rats to Igx-lb and PPD was described previously [141. 2.2 Antibodies Affinity-purified anti-Igx-lb antibodies (AB) were prepared as described earlier [13]. Affinity-purified rabbit anti-Ig rat antibodies were isolated from hyperimmune antisera on Sepharose 4B (Pharmacia, Uppsala, Sweden) conjugated with Ig from August rats. mAb specific for RT-lBC molecule (YR5/24) was obtained from Serotec (Bicester, GB). The rat anti-Igx-lb mAb C3B9 (IgG1) was obtained from the fusion of mouse myeloma cells X63Ag8.653 with spleen cells (SC) from an August rat immune to IgG (Igx-lb) [17]. The mouse anti-Igx-la mAb L2C5 (IgG1) and mouse anti-rat C, mAb (IgG1) [18] were kindly provided by Dr.A.V. Chervonsky (USSR Cancer Research Center). Antibodies were labeled with 1251by the chloramine-T method [19].

2.5 B cell proliferative response B cells (l@/well) were cultured in 96-well flat-bottom microtiter plates (Nunc, Roskilde, Denmark) with irradiated 2500rad) cloned T cells or primed LN T cells (3 x 104/well and 1 x 105/well,respectively) in 200 pl final volume of RPMI1640 media, supplemented with 4 m~ L-glutamine, 1 mM sodium pyruvate, 10 mM Hepes, antibiotics, 5% FCS and 5 X M 2-ME (culture medium) for 48 h at 37”C, 5% COz in a humidified atmosphere. In the experiments on “bystander” B cell activation Igx-la+ B cells from August rats or Igx-lb+ B cells from August.lb rats (positive control) were cultured at 5 x lo4cells/well in the presence of 5 x 104-10 x lo4 irradiated (lo00 rad) August.lb splenocytes as specific stimulator cells. In the controls irradiated August splenocytes were used. Cell proliferation was measured by adding 1 pCi = 37 kBq [3H]dThd (sp. act. 5 CUmmol,V/O “Isotope”, Leningrad, USSR) to each well for the last 18-20 h of incubation. The cells were then harvested on glass fiber filters using Cell Harvester 550 (Flow Laboratories) and counted on RackBeta liquid scintillation counter (LKB Wallac, Turku, Finland). All cultures were set up in triplicates. Results are presented as mean cpm [3H]dThd incorporated by triplicate cultures or as stimulation indexes (SI). (SI = mean cpm in the presence of antigerdmean cpm in control). In the inhibition experiments anti-RT-lBC and anti-Igx-lb antibodies were added to the culture wells at final concentrations 2 pg/ml and 10-30 pg/ml, respectively, simultaneously with the cell preparations.

2.6 Ig synthesis induction in vitro Spleen cells from August.lb, August and (August.lb x August)F1 rats (3 x 106/well)were cultured in 24-well tissue culture cluster (Nunc) with irradiated (2500 rad) cloned T cells in 2 ml of culture medium for 10 days at 37”C, 5% C02 in humidified atmosphere. The SN were then collected, and the amount of Ig(1gx-lb), Ig(1gx-la) and total Ig was tested using solid-phase RIA.

2.3 Cell preparation

2.7 LD analysis

Cell suspensions were prepared in RPMI 1640 medium supplemented with 5% heat-inactivated FCS and 50 pg/ml penicillirdstreptomycin (Flow Laboratories, Irvine, Scotland). Erythrocytes were removed from splenocyte suspensions by centrifugation on a density gradient of FicoIlHypaque (e = 1.086). B cells were purified from SC population by panning on polystyrene petri dishes coated with rabbit anti-rat Ig antibodies as described [14]. Cell fractionation on Percoll density gradient as well as the isolation of Igx-lb- and PPD-primed LN T cells from August rats have been described previously [141.

The frequency of B cells from August.lb rats capable of stimulating Igx-lb-specific cloned Tcells and the frequency of B cells capable of proliferating in response to the same T clone were determined in LD assay.The cultures were set up in 96-well U-bottom microtiter plates. In the first case, irradiated (lo00 rad) stimulator spleen B cells from August.lb or August (control) rats were mixed at titrated densities with irradiated (2500 rad) August splenocytes as filler cells, to maintain a constant number (lW/well) of stimulator plus filler cells. To each well 3 X 104 responder cloned T cells were added and incubated in 200 pl final volume of culture medium for 48 h at 37 “C in a humidified atmosphere of 5% COz in air. In the second case, LD of unirradiated responder August. l b (Igx-lb+) and August (Igx-la+) B cells (control) were set up as described for stimulator B cells, but the cloned T cells (4 X l@/well optimal stimulating dose) were irradiated (2500 rad). Cell proliferation was measured by adding 0.5 mCi [3H]dThd of high sp. act. (50 CUmmol). Cultures were set up using a minimum of five different responder concentrations with

2.4 T cell clones Derivation and maintenance of Igx-lb-, PPD- and OVAspecific August rat T cell clones were described previously [15]. All the clones used were of helperhnducer phenotype (W3/13+, W3/25+, OX8-) and their Ag-specific response was restricted by RT-1B (I-A) molecule.

Eur. J. Immunol. 1990. 20: 833-839 24-36 replicates of each. Cultures giving less than a 3-fold increase of mean cpm [3H]dThd uptake over the control level were considered as negative. Statistics were performed according to Henry et al. [20].

Ig-specific T-B cell interaction

Table 1. Igx-lb+ B cell proliferative response to irradiated Igx1b-recognizing T cell clonesa)

Irradiated T e l l clone added

August B cells (Igx-la)

August.lb B cells (Igx-1b)

2100

22 100 68500 18 300 38 300 2400 4 100 3000 2 500

2.8 RIA Ig(1gx-lb), Ig(1gx-la) and total Ig levels in culture SN were measured by solid-phase RIA. The wells of U-bottom polyvinylchloride plates (Flow Laboratories) precoated overnight with 50 pl of 20 pg/ml solution of rabbit anti-rat antibodies in BBS were saturated by addition of 100 pl of 1% BSA solution per well for 1h. The plates were washed three times and serial dilutions (50 pl/well) of culture SN were incubated in the wells for 2 h at room temperature. By measurement of Igx-lb allotype in the SN, the residual free binding sites of immobilized rabbit anti-Ig rat antibodies were blocked by addition of 50 p1 of 200 pg/ml Ig(1gx-la) for 1 h. The plates were washed three times and incubated with 1251-labeled rat anti-Igx-lb mAb (lo5 cprn in 50 pl/well). After 1 h the wells were washed four times and the bound radioactivity was counted on “1213 Minigamma” counter (LKB). By measuring Igx-la allotype or total Ig, 1251-labeled mouse anti-Igx-la and anti-C, mAb (105 cpm/well) were added to the wells, previously blocked by incubation with 200 p g / d Ig mouse. Standard curves to calculate the amount of Ig(1gx-lb), Ig(1gx-la) and total Ig in SN (pg/ml) were plotted using serial dilutions of IgGzb(Igx-lb) and IgGzb(Igx-la), prepared from August.1b and August rat sera, respectively, as described previously [131.

T4 (Ie-lb)b) T24 (I@-lb) T25 (Igx-lb) T55 (Ie-lb)

3.1 Igx-lb-recognizing T cell clones induce proliferative response of Igx-lb+ B cells Earlier it was shown that the irradiated Igx-lb+ B cells from August. l b rats elicited a strong proliferative response of MHC-restricted Igx-lb-specific T&ducer cell clones [151. In this study we observed that irradiated Igx-lb-recognizing T cell clones were able to induce a vigorous proliferation of Igx-lb+ B cells, but not of Igx-la+ B cells (Table 1). The control OVA- and PPD-specificT cell clones did not exhibit any stimulatory effect on Igx-lb+ B cells as compared to Igx-la+ B cells. Similar to theT cell clones, primed LN T cells from August rats irradiated at 2500 rad also induced a proliferative response of Igx-lb+, but not of Igx-la+ B cells (Table 2).The control PPD-specific primed LN T cells did not stimulate both Igx-lb+ and Igx-la+ B cells. Thus, we can conclude that the observed Igx-lb+ B cell proliferation is mediated by Tcell recognition of the Igx-lb determinant on the B cell surface. This conclusion is confirmed by the results of the study of anti-RT-1B mAb effect on the B cell response. It was found that anti-RT-1B mAb (YR5/24) completely inhibited Igx-lb+ B cell activation by the Tcell clone (90% inhibition) in contrast to both poly- and monoclonal anti-Igx-lb antibodies (0%-2% inhibition). Previously, in the study on anti-RT-1 mAb inhibition of Igx-lb-specific T cell clone response to irra-

1300 2800 3500 2600 3900 2900 2700

V15 (OVA) Y19 (OVA) P16 (PPD) P41 (PPD)

a) B cells from August.lb and August (control) rats were cultured at 1@cells with 3 X 104-4 x 104 irradiated (2500 rad) T cell clones in the wells of 96-well flat-bottom plates. Proliferation was measured after 48 h of culture. The data are presented as the mean cpm [3H]dThdincorporated by triplicate cultures.SD was < 10% of the mean. b) Antigen specificity of Tclones is shown in parentheses. Table2. Igx-1-immune LN T cells from August rats induce proliferative response of Igx-lb+ B cells from August.lb ratsa)

Irradiated T cells immune to

IgG (Igx-lb) PPD

3 Results

835

Media

Proliferative response (cpm) of B cells from August (Igx-la) August.lb (Igx-lb) 2300 2470

860

7150 2600 6.10

a) B cells from August.lb and August (control) rats were cultured at 105 cells with 3 X 105 irradiated (2500 rad) LN Tcells from August rats immune to IgG (Igx-lb) or PPD (control) in the

wells of 96-well flat-bottomplates. Proliferation was measured after 72 h of culture. The data are presented as the mean cpm [3H]dThdincorporated by triplicate cultures. SD was < 20% of the mean. diated Igx-lb+ B cells we found that anti-RT-lBC,but not anti-Igx-lb antibodies, were inhibitory [15]. 3.2 “Bystander” effect in B cell proliferation induced by Igx-lb-specificT cell clones

The next important question was whether B cells are activated by soluble nonspecific factors produced by irradiated Igx-lb-recognizing T cells in response to specific antigen, Igx-lb+ B cells, or whether the B cell activation required the direct T-B cell contact. In order to answer this question we studied the “bystander” activation of Igx-la+ B cells in the presence of the irradiated (2500 rad) T cell clones and irradiated (lo00 rad) August.lb SC as a source of specific antigen (Table 3). The particular level of the “bystander” response of Igx-la+ B cells was found to be dependent on the Igx-lb-recognizing T clone used, but never exceeded the level of specific Igx-lb+ B cell response to corresponding T cell clones. (The “bystander” effect

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Eur. J. Immunol. 1990.20: 833-839

S. M. Mazel, A. Y Rudensky und V. L. Yurin

Table 3. Bystander proliferation of Igx-la+ B cells from August rats induced by irradiated Igx-lb-specificTclones in the presence of irradiated Igx-lb+ SC from August.lb ratsa) Proliferative response of B cells from August.lb (Igx-lb) August (Igx-la) August.lb August August.lb August

Irradiated SC added Irradiated T cell clones added T4 (I@-lb)b’ T6 (I@-lb) T13 (IF-lb) T24 (IF-lb) T55 (Igx-lb) V19 (OVA) P41 (PPD)

10600 8600

6400

8 700

3 800 10200

@ J

28100

4

9800 22700

4

8

0

1500

1200 4 500

4400

11500 0

1200 7000

observed was 0%-60% of the magnitude of the Igx-lb+ B cells response.) This result suggests only a moderate role of nonspecificfactors in B cell activation. Interestingly, the SN from Igx-lb-specific Tcell clones (T24,T55 and others) after stimulation with irradiated Igx-lb+ B cells did not exhibit any stimulatory effect on both Igx-la+ and Igx-lbf B cells (data not shown).Thus, for optimal B cell activation mediated by the Igx-lb-recognizing T cell clones the specific T-B cell contact is required.

3.3 Ig production of Igx-lb+ B cell is activated by Igx-lb-recognizingT cell clones Table 4 shows the effect of Igx-lb-recognizing T clones on the production of Igx-lb and Igx-la allotypes in the 10-day cultures of SC from August.lb, August and (August.lb x August)F1 rats. The B cell population of Igx-l-heterozygous F1 rats is represented by a 1: 1 mixture of Igx-lb+ and Igx-la+ B cells. Using sensitive allotype-specific solidphase RIA, we have demonstrated a dramatic increase in Table 4. Selective induction of Ig(1gx-lb) synthesis in SC cultures by Igx-lb-recognizingT cell clonesa) SC from Igx-1 allotype of Ig detected (pg/ml)

Irradiated T clones added T4 (Igx- 1b)b, T24 (I@-lb) T55 (IF-lb) P41 (PPD) Media

August Igx-la

August. 1b Igx-lb

0.5 0.3 0.7 0.6 0.4

4.6 6.7

9.3 0.7 0.5

August.la/lb Igx-la Igx-lb

0.2 0.15 0.6 0.3 0.2

1.9 3.5

_.

63 Gi 0.3

SC from August, August.lb and (August X August.lb)F1 rats (3 X l@/well) were cultured in 24-well tissue culture plates with irradiated (2500 rad) cloned Tcells (3 x 105) in 2 ml of culture medium at 3 7 T , 5% CO2 in a humidified atmosphere. After 10-14 days SN were collected and the amount of Ig(1gx-lb), Ig(1gx-la) was tested using solid-phase RIA. The data are presented as pg/ml of Ig of Igx-lb or Igx-la allotype in culture SN. Antigen specificity of Tclones is shown in parentheses.

~

2000 2800 9800 2700 6800~ 1200 7200

a) B cells, 5 x 104-5 x lo4, from August (Igx-la) and Auzust.lb rats were cultured with 3 x 104-16 x 104 irradiated (2500 rad) Tclones in the presence of 5 x 104-10 x 104 irradiated (lo00 rad) SC from August or August.lb rats in the wells of 96-well flat-bottom plates. Proliferation was measured after 48 h of culture.The data are presented as the mean cpm [3H]dThd incorporated by triplicate cultures. SD was < 10% of the mean. b) Antigen specificity of Tclones is shown in parentheses.

the Ig(1gx-lb) level in culture SN of F1 and August.lb SC stimulated with irradiated Igx-lb-recognizing T clones. In contrast, the level of Ig (Igx-la) production in F1 SC cultures was not elevated. In the control cultures the levels of both Ig(1gx-lb) and Ig(1gx-la) were approximately the same in the presence of “nonspecific” OVA- and PPD-reactive T cell clones and in the absence of T cell clones (Table 4). A comparable 2-3-fold increase of both Ig(1gx-la) and Ig(1gx-lb) was observed when corresponding antigen, OVA and PPD, was added with OVA- and PPD-specific clones to the control cultures (data not shown). Thus, Igx-lb-recognizing T cell clones promoted not only Igx-lb+ B cell proliferation, but also a highly specific increase of Ig(1gx-lb) production.

3.4 Small Igx-lb+ B cells can be activated by Igx-lb-recognizingclones Since the major part of normal B lymphocyte population is represented by small high-density B cells, it appears important to reveal possible involvement of the latter in direct Igx-lb-specific T-B cell interactions. In these experiments the responses of Percoll-fractionated August. l b splenic cells of low, intermediate and high density to irradiated Igx-lb-specific T clone were studied and, vice versa, the T clone responses to corresponding irradiated cell fractions were measured (Fig. 1A and B). As can be seen in Fig. 1, irradiated Igx-lb-specific Tclone elicited a high response of small B cells (SI 19.0) and, vice versa, irradiated small B cells efficiently stimulated T cell clone response (SI 16.0). The levels of both responses were comparable t o those observed when unfractionated B cells were used. Thus, small B cells are able to provide a specific signal stimulating aTcell clone and to accept a signal from the same T clone to become activated by themselves. 3.5 The frequency of Igx-lb+ B cells involved in Igx-lb-specific T-B cell interaction In order to estimate the frequency of August.lb spleen B cells capable of becoming activated upon interaction with Igx-lb-recognizing Tcell clone (Igx-lb+ responder B cells) and the frequency of August.lb splenic B cells stimulating

Eur. J. Immunol. 1990. 20: 833-839

a

b

c

Ig-specificT-B cell interaction

1’

d

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4 Discussion The aim of this study was to further investigate the direct T-B cell interaction mediated by Tcell recognition of B cell IgR determinants. In the preceding studies [14, 151, we have shown that B cells from August. l b rats (RT-lC;Igx-lb) were the most potent APC of self IgR determinants, i.e. Igx-lb allotype, and stimulated high proliferative response of both polyclonal and cloned Igx-lb-specific T cells from congenic August rats (RT-lC;Igx-la). The most important finding in these studies was that Tcells, in fact, recognized Igx-lb allotypic epitope(s) of Ig peptide of B cell self IgR, presented in the complex with RT-1B molecule [15].These observations are extended by the experiments described herein which demonstrate that Igx-lb-specific T-B cell interaction, mediated by Tcell recognition of the processed self IgR/MHC class I1 complexes, results in specific and highly efficient B cell activation, i.e. proliferation and induction of Ig(1gx-lb) synthesis.

Previously, the efficient B cell activation has been shown to be promoted by the T cell recognition of other specific ligands on the B cell surface: allogeneic and haptenmodified or intact syngeneic MHC class I1 molecules, minor histocompatibility antigens and H-Y antigen [21-251. In addition, it is known that even conjugation of Th and B cells through their Ag-specific receptors leads to induction of B cell proliferation and differentiation [26, 271. B cell activation could also be induced by direct Th-B cell a b C d contact mediated not via TcR but via other T cell surface Figure 1. (A) Proliferative response of Percoll density gradient- structure(s) [28, 191, e.g. proliferation of small B cells could fractionated B cells from August.lb (open bars) and August be mediated by CD2-LFA-1 interaction. In this case B cell (control, closed bars) rats to irradiated (Igx-lb-recognizingT24 activation could be inhibited by anti-LFA-1 but not by clone (4 X 104 cell/well). 1 x 105 responder B cells were added to anti-h4HC class I1 antibodies [29]. O n the other hand, each well. (a) Unfractionated SC; (b) fraction I; (c) fraction 11; several investigators have reported non-cognate B cell (d) fraction 1II.The data are presented as the mean cpm [3H]dThd activation and differentiation triggered by soluble nonspeincorporated by triplicate cultures. SD was < 10% of the mean. cific factors (IL4, B cell activating factors, B cell growth The corresponding stimulation indexes (SI) are written over the factors, etc.) produced by activated Tcells both in mice and bars. (B) Igx-lb-specific T24 clone response to irradiated B cells (l@/well)from August.lb (open bars) and August rats (control, in humans [30-331. For instance, L. Leclerq et al. [31] closed bars) fractionated on Percoll density gradient). 3 X 10“ demonstrated that SN of cloned GAT-specific T cells responder T24 cells were added to each well [see legend to induced both proliferation and differentiation of small B cells into PFC in an Ag-independent and Ia-unrestricted (41. manner . Igx-lb-specific T cell clone response (Igx-lbf stimulator B cells) LD analysis was performed. In both cases the number of responding wells increased as the number of Igx-lb+ B cellskulture increased. When the log of the fraction of nonresponder cultures vs. B cell doses was plotted (Fig. 2A and C) the corresponding responses were linear, suggesting that Igx-lb+ B cell is the only limiting element in these assays. The calculated frequencies of the “Igx-lb+ responder” and “Igx-lb+stimulator” cells among B SC were found to be roughly the same: 1/10. Similar frequencies were obtained by LD analysis of unfractionated SC from August.lb rats (data not shown). In the control, by the L D analysis of “nonspecific” B cell activation by Igx-lb-recognizing T cell clone, the frequency of responding Igx-la+ B cells from August rats was found to be approximately 1/6000 (Fig. 2B). These data suggest that under “limiting” conditions the Igx-lb+ B cell-stimulating Igx-lb-specific T clone is activated by itself via direct Igx-lb-specific MHC-restricteti T-B interaction.

Thus, several mechanisms could be responsible for the Igx-lb+ B cell activation observed in our experiments: non-cognate B cell triggering by soluble factors produced by activated Igx-lb-specific T clones, direct T-B cell interaction mediated via non-polymorphic T cell surface molecules, or cognate interaction mediated by TcR binding to Igx-lb determinant presented by B cell.The strict antigenic specificity of the B cell activation observed and its inhibition by anti-MHC class I1 but not by anti-Igx-lb antibodies suggest that, in our experimental system, B cell activation depends on T cell recognition of the specific ligand, i.e. processed self IgR peptideMHC class I1 complex on the B cell surface.The observed proliferation of Igx-lb+ B cells was not only an in v i m effect produced by long-term cultured cloned T cells because irradiated Igx-lb-primed LN T cells from August rats also induced response of Igx-lb+ but not of Igx-la+ B cells.The inability of activated Igx-lb-specific T cell clone SN to induce both B cell proliferation and Ig synthesis and, on the other hand, relatively low but significant proliferation of bystander Igx-la+ B cells suggest that apparently nonspecific factors

Eur. J. Immunol. 1990. 20: 833-839

S. M. Mazel, A.Y. Rudensky und V L.Yurin

838

(C)

60

40

10 I

I

!O- 5

I l

l

1

I

5 10 15 20 25 30 NUMBER OF Ig x-lb*B CELL/WELL

3 6 9 NUMBER OF Ig x-la’B

0

I

l

l

0

l

l

I

12 15 18 CELLIWELLX~O-~

I I

I

I

I

I

I

I

I

10 15 20 25 30 NUMBER OF IRRADIATED Ig x-lb*B CELLlWELL

0

5

I

Figure 2. The frequencies of SC from August.lb (A) and August (B) rats responding to Igx-lb-recognizingTcell clone. Limiting numbers of isolated Igx-lb+or Igx-la+ B cells were stimulated with 4 X 105irradiated T24 cells in the presence of 105 irradiated SC from August rats in the wells of 96-well microtiter plates in a final volume of 0.2 ml medium per culture.Thefrequencies of responding wells were estimated by [3H]dThdon day 3 of culture.Thedotted line (---) shows the number of cells at which 37% of all replicate cultures do not respond. (C) The frequencies of spleen B cells from August.lb (A) rats capable of being stimulated by Igx-lb-recognizing Tcell clone. Limiting numbers of irradiatedIgx-lb+B cells were used to stimulate3 x 105T24 cells in the presence of 105 irradiated SC from August rats as filler cells in the wells of 96-well microtiter plates at a final volume of 0.2 ml medium per culture.The frequencies of responding wells were estimated by [3H]dThdincorporated on day 3 of culture.The dotted line (---) shows the number of cells at which 37% of all replicate cultures do not respond.

produced by T cells are acting presumably at short distances in the cell contact sites. As evidenced by A. Kupfer and co-workers [34,35],Tcells undergo re-orientation of microtubule-organizing center and Golgi apparatus in the TcR contact site with antigen-presenting B cell. W.-J. Po0 et al. [36] demonstrated that under physiologic conditions (low antigen concentration) T cells produced lymphokine by “directed exocytosis” in the Tcell-APC contact sites. Thus, during specific T-B cell interaction a labile functional structure in the cell contact sites is formed. This structure, which could be termed immunosynapse, analogously to neurosynapse, enables the selective signal transfer from one interacting cell to another by local lymphokine production in the cell contact sites andor by receptor crosslinking. Apparently, this kind of events leads to bidirectional activation of Tand B cells observed in our experiments by Ig-specific MHC-restricted T-B interaction. The lack of bystander effect by Igx-lb-specific T clone-mediated Ig synthesis induction in Igx-lb+/Igx-la+ B cell population, in contrast to observed proliferation of bystander Igx-la+ B cells, suggests that there is a clear difference in the B cell activation and differentiation requirements: the latter is absolutely dependent on specific T-B cell interaction mediated by TcR. This result correlates well with similar observations made by others, when bystander B cells exhibiting even higher proliferative response are not differentiated into nonspecific PFC [37,38]. In general, the data demonstrated the prime role of specific T-B cell contact in B cell proliferation and differentiation observed in our experimental system. High efficiency of Ig-specific T-B cell interaction is illustrated by the fact that small Igx-lb+ B cells could be easily triggered by Igx-lb-recognizing Tclone. Furthermore, the LD analysis shows the frequency of B cells capable of participating in this interaction is very high: every tenth cell in Igx-lb+ B cell population is activated. One order lower frequency of B cells capable of being directly stimulated by autoreactive Ia-specific T clones was observed by T. Saito

and K. Rajewsky [39]. In the same study the frequencies of B cells responding to LPS or to KLH-recognizing T cell clone plus KLH were found to be 1/6 and 1/9, respectively [39]. Taking into account the sensitivity limitations by [3H]dThd cell labeling utilized in our study, one might expect that the frequency of both “Igx-lb+ responder” and “Igx-lb+stimulator” B cells is in fact even higher.The same frequency of B cells stimulating T clones and of B cells responding to T cells suggests that, apparently, a single interactional event led to codirectional activation of both Ig-presenting B cell and Ig-recognizingT cell. In the preceding studies, we have proposed that B cells are also capable of expressing idiopeptides, i.e. a set of clonotypic peptides related to VH and VL regions, which could be recognized by MHC-restricted Id-specific T cells [15,40]. This proposal, also made by others [41-421, was recently confirmed by S.Weiss and B. Bogen [43] in a study using B lymphoma cells. We consider the specific B cell activation and maturation mediated by MHC-restricted T cell recognition of the endogeneous Igx-lb peptide presented by Igx-lbf B cells as a model of putative Ig-driven “idiopeptidic” network interactions. It could be predicted that a single B cell clone is capable of expressing idiopeptides related to Id of its own IgR and acquiring idiopeptides related to Id of anti-Id antibodies produced by the complementary B cell clone, i.e. anti-Id B cell. A T cell clone specific for one or another set of idiopeptide/class I1 complexes would interact with and, thus, regulate both complementary B cell clones. Our preliminary results provide indirect evidence supporting this supposition. It was found that Igx-lb-recognizing Tcell clone injected together with IgG(1gx-lb) into August rats provided help for Igx-lb-specific antibody response in vivo, i.e. for anti-Igx-lb B cells. Analogously to “parallel sets” in “classical” Id networks, Ig peptide-mediated T-B cell interaction could also be responsible for non-specific Ig synthesis, both in the absence and in the presence of

Eur. J. Immunol. 1990.20: 833-839

antigenic stimulation. Obviously, only positive (helper) idiopeptidic regulation was taken into account in the proposed hypothesis. It seems reasonable, however, to also assume the existence of negative (suppressor) idiopeptidic regulation based on similar principles. It should be noted that the speculations on the possible immunoregulatory role of Ig-specific as well as Agmediated direct T-B cell interactions in unmanipulated immune system were considerably challenged recently by the data obtained by 0. Lassila and co-workers in chicken [44].These authors found that Ma are needed, in addition to APC B cells, to trigger SRBC-specific h4HC-restricted T cells. In other words, it was suggested that B cells could not stimulate resting T cells. However, in experimental systems using other animal species or other antigens, e.g. soluble proteins or B cell surface molecules, the result might not be the same. Nonetheless, results of the present study enable us to suggest that resting B cells could efficiently interact at least with putative autonomously activated Tcells specific for B cell IgR determinants, i.e. Ig peptide/MHC class I1 complexes. Evidence for the existence of autonomously activated self-reactive Th cells, capable of inducing B cell activation and maturation, as well asT, cells in normal young and adult mice, was recently provided by A. Coutinho’s group [45,46]. Further experiments in our experimental model and in systems utilizing the true Id-anti-Id T-B cell interaction are needed to verify these considerations. We thank Dr. A . S. Apt for helpful comments on the manuscript. Received July 31, 1989; in revised form October 30, 1989.

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Immunoglobulin-specific T-B cell interaction. III. B cell activation by immunoglobulin-recognizing T cell clones.

Immunoglobulin (Ig)-specific T-B cell interactions were studied in the model of T cell recognition of Ig kappa chain Ig kappa-1b allotype in Ig kappa-...
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