T-cell-dependent modulation of the polyclonal B-lymphocyte responses in normal spleen cell cultures stimulated by lipopolysaccharide

©

Ann. Inst. PasteurlImmunol. 1987, 138 181-199

ELSEVIER

Paris 1987

T-CELL-DEPENDENT MODULATION OF THE POLYCWNAL B-LYMPHOCYTE RESPONSES IN NORMAL SPLEEN CELL CULTURES STIMULATED BY LIPOPOLYSACCHARIDE

by M. Bjorklund (1), L. Forni (2) and A. Coutinho (3) (I) Department oj Immunology, University oj Umea, Umea (Sweden), (2) Basel Institute jor Immunology, Basel (Switzerland), and (3) Unite d'Immunobiologie, Institut Pasteur, 75724 Paris Cedex

SUMMARY

The in vitro polyclonal B-cell proliferative and plaque-forming cell (PFC) responses to the T-independent (TI) mitogen lipopolysaccharide (LPS) are increased by the addition of normal syngeneic splenic T cells. Normal irradiated Lyt-2 - T cells also alter the IgG subclass distribution from the typical predominance of IgG3 and IgG2b PFC to the appearance of IgG 1, IgG2a and IgA PFC in T-cell-depleted spleen cell (SC) cultures. Furthermore, secondary LPS blast cultures yield increased PFC responses when co-cultured which syngeneic fresh normal T cells which, even in the absence of mitogen, induce PFC responses in such activated B cells. As LPS blasts induce normal syngeneic T cells to proliferate and significant numbers of L3T4+ blast cells are found in LPS-stimulated normal spleen cell cultures, we conclude that T cells actively participate in the regulation of these responses. The significance of these findings for the regulation of TI responses in vivo by «autoreactive» T cells is considered. KEY-WORDS:

Spleen.

T lymphocyte, IgG, LPS, Immunoregulation; B lymphocyte,

INTRODUCTION

We and others have previously shown that the nature of the stimuli driving B-Iymphocyte responses determines the isotypic pattern of the secreted

Submitted August 16, 1986, accepted December 12, 1986. Correspondence should be addressed to Dr. Antonio Coutinho, Unite d'Immunobiologie, Institut Pasteur, 28, rue du Docteur-Roux, 75724 Paris Cedex 15 (France).

182

M. BJORKLUND, L. FORNI AND A. COUTINHO

immunoglobulins (Ig) [1-5]. This is the case both in antigen-specific and in mitogen-induced polyclonal responses, and is particularly evident in selective IgG subclass expression. Thus, lipopolysaccharide (LPS) stimulation yields a high IgG3 PFC response both in vitro and in vivo [1-7], while TH cells or Twcell-derived factors yield a high IgG 1 PFC response even when added to B-blasts pre-activated with LPS [5, 7-9]. It is well established that LPS is a TI mitogen which directly activates 10-50 070 of the splenic B cells to growth and maturation to PFC [10-12]. Irrefutable evidence for T-cell-independent stimulation of B cells has been produced in culture systems where single B cells develop into clones, in the absence of any other lymphoid cell or its products [12]. It is also currently assumed that TI B-cell responses to LPS are maximal and that the addition of TH cells or Twcell-derived factors «only» cause a shift in Ig isotype patterns, more precisely, from IgG3 to IgG 1 [5, 7-9].

Routine observations of LPS-induced PFC responses repeatedly revealed differences in the IgG subclass distribution in normal SC or T-cell-depleted SC cultures. This prompted us to systematically investigate the influence of normal splenic T cells in LPS responses in terms of both proliferation and maturation to Ig secretion. The results demonstrate that normal T cells influence the B-cell response to LPS, and that LPS-activated B blasts are able to induce normal T cells to proliferate. By extrapolation to in vivo conditions, these observations suggest important mutual regulatory influences of T and B cells in immune responses and in the normal activity of the immune system. MATERIALS AND METHODS Animals. C3H/Tif and C57BL/6J were bred in our colony and used between 8 and 12 weeks of age. Mice of the same sex and age were used within each experiment. Chemicals. Ficoll-Paque was from Pharmacia Fine Chemicals, Uppsala, Sweden, and LPS from E. coli was from Difco, Detroit, MI, USA.

C' FCS FITC Ig LPS mIg+ nw

= = = = = = =

rabbit complement. foetal calf serum. fluorescein isothyocyanate. immunoglobulin. lipopolysaccharide. membrane' immunoglobulin-positive. nylon wool.

PFC SC SE TD TH TI

= = = = = =

plaque-forming cell. spleen cell. standard error. T-dependent. T helper. T-independent.

T-CELL PARTICIPATION IN LPS RESPONSES

183

Cell preparations. Cell suspensions were prepared from multiple syngeneic donors and cultured in RPMI-I640, supplemented with 10mM Hepes, antibiotics, 5 x 10- 5 M 2-mercaptoethanol and 5-10 % foetal calf serum (FCS; Gibco-Biocult, Glasgow, Scotland). Erythrocytes were routinely removed with haemolytic Gey's solution [13]. Spleen cells (SC) were enriched for T cells by passage through a nylon wool (nw) column [14], with a 15-20 % recovery. Lyt-2- T-cell-enriched populations were obtained by treatment of nw passed SC with a monoclonal anti-Lyt-2.2 antibody (HO-2.2) [15] and rabbit complement (C' ; Buxted Rabbit Co. Ltd., Sussex, UK). This treatment removed some 25 % of the nw non-adherent cell population. T-cell-depleted SC were prepared by treatment with the monoclonal anti-Thy-l.2 antibody J1j [16] and C'. This treatment removed some 40 % of the SC. T cells were irradiated with 2,000 rad immediately before use in test cultures. LPS-activated B blasts were obtained from 48-h cultures (1-4 x 106 cells/mt) of anti-Thy-l.2 and C'-treated SC in plastic bottles (No 3013, Falcon Plastics, Oxnard, CA, USA) containing 10 ml and 50 Ilg/ml LPS, and separated on Ficoll-Paque before further use. Cultures were set up in O.2-ml aliquots in flat-bottom microplates (No 3040, Falcon) at the indicated cell concentrations. Results shown are always means of triplicate cultures; as standard errors (SE) never exceed 15 %, they are not shown, for simplicity. Assays. Proliferative responses were determined by incorporation of tritiated thymidine (The Radiochemical Centre, Amersham, GB) (l IlCi per well = 37 kBq per well, for 4 h). Isotype-specific plaque assays for all secreting cells were performed using proteinA-coated red cells and rabbit anti-mouse-Ig developing antisera, as earlier described in detail [17]. After the appropriate absorptions, the antisera were titrated and their specificity assessed in plaque assays using myeloma or hybridoma cells of known classes. Immunofluorescence. Cell suspensions were stained as previously described [18] using FITC-Iabelled sheep anti-mouse Ig antibodies, monoclonal anti-Thy-l (J1j), anti-L3T4 (H-129-19.69) [19] and anti-Lyt-2 (53-6.72) [20], either labelled with biotin and detected with FITCavidin or revealed by an FITC-labelled monoclonal mouse anti-rat K antibody (MARK-I) [21]. The stained cell suspensions were analysed in a FACS analyser (Becton and Dickinson, Sunnyvale, CA., USA) for fluorescence, and the positive cells were gated and analysed for volume with an appropriate computer program (CONSORT 30). RESULTS

The presence of normal T cells increases B-cell proliferation in response to LPS. Routine analysis of LPS-induced PFC responses often showed different IgG subclass patterns in untreated and T-cell-depleted spleen cell cultures. We therefore decided to systematically investigate this phenomenon and perform the appropriate depletion/reconstitution experiments.

184

M. BJORKLUND, L. FORNI AND A. COUTINHO

A fixed number of T-cell-depleted SC (2 x 104) were cultured in the presence of LPS either alone or together with various numbers of nw-purified T cells. As shown in figure 1, the addition of T cells significantly increased the proliferative response in a dose-dependent manner in comparison with that of T-cell-deprived cultures. These effects could, however, be attributed to either «filler» effects of T-cell populations or true helper effects of competent cells. This could be tested by enrichment for helper-cell precursors in the T-cell populations used. As also shown in figure 1, previous depletion of Lyt-2+ cells from the T-cell population yielded, on a per cell basis, much higher activity. NotablY,lower numbers of Lyt-2- T cells were required for optimal responses, directly arguing against mere « feeder» or other effects

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T-cell-depleted SC (2 X 10 4) were co-cultured with untreated (e) or Lyt-2- (0) syngeneic (C57BL/6J) nw-passed SC in the presence of LPS. Medium controls did not yield proliferative responses. T-cell-enriched populations responded to LPS at 6 x 10 4 and 8 x 104 cell numbers; these responses did not exceed 15 070 of, and are withdrawn from, the responses shown here. C = controls: D = LPS-activated T-cell-depleted SC (2 x 104): • = LPS-activated normal SC (4 X 104).

T-CELL PARTICIPA TION IN LPS RESPONSES

185

mediated by cells such as macrophages, which contaminate both cell populations to a comparable extent. Interestingly, 4 x 104 normal SC still yielded a better proliferative response to LPS than that of reconstituted mixtures of T and B cells, showing that optimal T-cell modulation of proliferative responses is fulfilled in the normal untreated SC population. In order to ascribe the T-cell-dependent increases in proliferative response to the B-cell population, 2,OOO-rad-irradiated or normal T cells were titrated into B-cell cultures. The results presented in figure 2 show that addition of irradiated T cells increased the B-cell proliferative response to LPS in comparison with that of B cells cultured alone, demonstrating radioresistant helper effects. However, lower cell concentrations of non-irradiated T cells were required for maximal response, indicating that a part of the helper activity is radiosensitive, or else that the T-cell population itself might, in part, contribute to the proliferative response.

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2. - Normal T cells increase B-cell proliferation to LPS.

See legend to fig. 1; 3 X 10 4 T-cell-depleted SC were cultured in the presence of LPS either alone (x) or with untreated (e) or irradiated (2,000 R) (0) syngeneic (C3H/Tif), nwpassed SC.

186

M. BJORKLUND, L. FORNI AND A. COUTINHO

LPS-activated B blasts induce T cells to proliferate.

T-cell participation in LPS-induced responses could result from activation of normal T cells in these cultures either by the mitogen or by the activated B cells themselves. We therefore investigated the proliferative activity of splenic T cells confronted with LPS-activated B-cell blasts in the presence or absence of LPS. These experiments could also test the hypothesis of T-cell contribution to the overall proliferation detected in LPS-stimulated cultures of normal spleen cells. A fixed number of normal T cells were cultured with various numbers of syngeneic irradiated LPS blasts. As shown in figure 3, LPS blasts indeed induced a proliferative T-cell response, also indicating that T-cell participation in LPS cultures was not limited to filler or feeder effects. Addition of LPS

FIG.

3. - LPS-blasts induce normal T cells to proliferate.

Two-day C3H/Tif LPS blasts were irradiated (2,000 R) and re-cultured with syngeneic nwpassed SC (1.2 x 10 5) in the presence (e) or absence (0) of LPS. Proliferative responses were measured on days 4 and 5 of secondary culture. None of the cell populations responded to LPS when cultured alone.

T-CELL PARTICIPATION IN LPS RESPONSES

187

had little effect, showing that the response was not due to B-cell contamination of the T-cell population and, most important, that T cells are not « LPSreactive», but rather directly induced by the B-cell blasts. Such T-cell responses to LPS blasts and the effector functions of the responding T cells have been the object of further investigations and are reported elsewhere [22]. In order to directly assess the extent of secondary T-cell activation in LPSstimulated cultures, we have investigated which types of lymphocytes undergo blast transformation in cultures of normal or T-cell-depleted spleen cells. As shown in table I, dual parameter analysis for size and surface expression of Ig, L3T4, Lyt-2 and Thy-l reveals, even at relatively early times in the culture, a considerable involvement of T lymphocytes in the response to LPS. Thus, up to 10 070 of all activated blasts in normal SC cultures 3 days after stimulation with LPS are L3T4+ T cells. These numbers are greatly reduced in cultures of T-cell-depleted spleen cells, providing the appropriate control for specificity in these determinations and supporting the above results on the performance of such cultures where cell proliferation is notoriously reduced. The presence of T cells increases the B-cell PFC response to LPS. The observation leading to the experiments undertaken in this report was a differential IgG subclass distribution in T-cell-depleted versus normal SC cultures activated with LPS. To confirm this and to investigate whether T cells could also influence the magnitude of the PFC response to LPS, a constant number of B cells were cultured in the presence of LPS and various numbers of T cells, which were either irradiated or not and either depleted of Lyt-2+ cells or not. Any of these four cell populations added to the cultures caused an increased PFC response in comparison with that of B cells alone and, as for B-cell proliferation, this increase was dose-dependent. The highest PFC responses were obtained by addition of irradiated Lyt-2 - T cells, and the kinetics of the IgM, IgG3, IgG2b and IgG2a PFC responses of such cultures are shown in figure 4. Compared with B cells cultured alone, the addition of irradiated Lyt-2 - T cells yielded increased PFC responses throughout the culture period and for all classes (except for IgG3 on day 4), indicating that more B cells are induced to Ig secretion in the presence of T cells. T cells alter the IgG subclass distribution in the LPS response. Polyclonal LPS activation results in the production of IgM [23, 24], later followed by other isotypes [25,26]. The PFC isotype distribution in vivo [6] is compatible with that observed in vitro [4, 5] and consists of a selective stimulation of IgM, IgG3, IgG2b and IgG2a (in this order). In view of this, it is noteworthy that the presence of Lyt-2- T cells in LPS cultures mostly enhances IgG2a PFC, a relatively TD isotype [4, 27, 28] (fig. 4). In comparison with B cells cultured alone, at the peak of responses, IgG2a PFC were increased to-fold, while IgG2b and IgG3 PFC were not greatly augmented (2.3-

M. BJORKLUND, L. FORNI AND A. COUTINHO

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and 1.6-fold, respectively). It is interesting to compare these IgG subclass patterns with that of normal SC containing the same numbers of B cells and «natural» mixtures of T and B lymphocytes. While all responses are higher in normal SC than in B-cell cultures, the only IgG isotype increased above the normal SC control by the presence of Lyt-2 - T cells is, again, IgG2a, on day 6 of culture. Another interesting property of Lyt-2- T cells is to prolong the PFC response in time. Thus, by day 4 of culture, the highest response of any isotype is found in the normal SC control, but by day 8, these display the lowest response with a total number of some 3,500 IgG PFC, while the B cells co-cultured with Lyt-2 - T cells still yield good responses with some

T ABLE I. -

Surface markers expressed by cells in LPS-activated cultures.

Starting cell populations Cell treatment None Anti-Thy-l.2 + C' None None Anti-Thy-l.2 + C' Anti-Thy-l.2 + C' None Anti-Thy-l.2 + C'

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M. BJORKLUND, L. FORNI AND A. COUTINHO

4 X 104 IgG PFC (fig. 4, table II). This lO-fold difference in the «late» IgG PFC response together with the Lyt-2 - T-cell-dependent increase in IgM PFC on day 6, could indicate that T cells induce a subpopulation of B cells to highrate secretion, which would otherwise not develop to plasma cells. On the other hand, the rapid decline of PFC responses in normal SC cultures, which can obviously not be assigned to deteriorating culture conditions by comparison with cultures containing Lyt-2- cells, may well be due to suppressive activities, secondarily developed from the Lyt-2+ T-cell compartment (see below). Figure 5 and table II further elucidate T-cell-dependent regulation of the IgG subclass expression in the LPS response. Figure 5 shows that, iRdependently of the magnitude of the total IgG response, IgG2a PFC are the most T-cell dependent. IgG3 PFC on the other hand, are rather inhibited by the presence of T cells but only in its relative representation and not in absolute numbers of PFC (fig. 4, table II). Interestingly, the relative IgG2b PFC response is practically unaffected by the presence or absence of T cells and time of culture, indicating either a very strict LPS-dependent regulation of this subclass or rather, in view of the strong T-cell dose-dependency of absolute numbers of IgG2b PFC (fig. 4), that this subclass is induced by both TD and TI stimuli. This is in agreement with previous findings of ours that IgG2b is well represented in response to LPS both in vivo and in vitro [4, 6, 28] as well as in the in vivo response to the typical TD antigen sheep erythrocytes [27] and in the in vitro polyclonal response to TH cells [5].

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See also table II. Panel A, Band D: T-cell-depleted SC (2 X 104) were cultured in the presence of LPS and (A) syngeneic Lyt-2- T cells (6 x 104); (B) syngeneic untreated T cells; (D) alone. Panel C: LPS-activated normal SC (4 X 10 4). Black columns = IgG3; horizontally hatched columns = IgG2b; diagonally hatched columns = IgG2a.

TABLE

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IgO subclass

1I. -

T-cell influence on IgG PFC isotype numbers and distribution in the response to LPS

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070

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2,213 ± 167 6,800 ± 113 11,440± 1,697 20,453

10.8 33.2 55.9

5,840 ± 1,082 5,013 ± 938 10,240 ± 1,131 21,093

27.7 23.8 48.5

9,493 ± 2,322 7,413 ± 514 12,853 ± 3,841 29,759

31.8 24.9 43.2

3,147 ± 1,225 1,600 ± 423 2,133 ± 721 6,880

45.7 23.3 31.0

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G3 G2b G2a Sum

21,120 ± 3,439 13,312 ± 1,874 39,936 ± 4,344 74,368

28.4 17.9 53.7

24,832 ± 6,667 7,936 ± 362 10,752 ± 0 43,520

57.1 18.2 24.7

34,091 ± 9,500 10,923 ± 3,596 19,712 ± 3,942 64,726

52.7 16.9 30.5

13,184 ± 1,629 5,880 ± 0 4,437 ± 461 23,501

56.1 25.0 18.9

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18,640 ± 160 8,704 ± 2,557 12,160 ± 1,991 39,504

47.2 22.0 30.8

16,880 ± 453 5,291 ± 2,981 6,272 ± 543 28,443

59.3 18.6 22.1

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2,453 ± 683 ± 341 ± 3,477

378 266 391

70.5 19.6 9.8

6,840 ± 597 ± 640 ± 8,077

396 411 640

84.7 7.4 7.9

192

M. BJORKLUND, L. FORNI ANDA. COUTINHO

IgG1 and especially IgA PFC expression are known to be highly T-cell dependent [3-5, 30-32] and consequently, these isotypes are rarely or not at all detected after stimulation with LPS. IgGl PFC responses are usually detected in vitro and in vivo, but these are of low magnitude and vary between experiments and with strains of mice (data not shown). IgA PFC responses are refractory to LPS in vivo [6] and until now, we have never detected any IgA PFC responses in vitro, althougl'l we have routinely assayed for all isotypes in numerous LPS control cultures. Analysis of LPS-dependent switches to IgA under clonal conditions have confirmed the extreme rarity of these events [29]. The more striking are, therefore, the data shown in figure 6, where these two classes can be induced by the presence of increased numbers of normal splenic T cells. Interestingly, the best IgGl PFC response

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T-CELL PARTICIPATION IN LPS RESPONSES

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was obtained with irradiated Lyt-2- T cells, while IgA PFC responses required non-irradiated Lyt-2 - T cells, indicating more stringent requirements for T-cell help. Normal T cells also regulate the responses of pre-activated LPS blasts. In view of the above observations showing that normal T cells respond to LPS blasts, it was interesting to examine T-cell influences on preactivated B cells. This could also provide indications as to the site of action of the regulatory T-cell influence. T-cell-depleted SC were activated with LPS for 2 days, and the resulting purified blasts were extensively washed and subcultured with or without T cells in the absence or presence of LPS. Figure 7

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Normal T cells induce pre-activated LPS blasts to secrete [g. Two-day LPS blasts (3 x 10 4) were re-cultured alone or in the presence of syngeneic (C3H/Tif) nw-passed SC (9 X 104). Additions to secondary cultures: e = T cells and LPS; 0 = T cells; + = LPS. Medium controls did not yield PFC responses. The T-cell population responded to LPS with some 2,500 IgM PFC (but no IgG PFC) at day 5 of culture; this number is withdrawn from the appropriate (e) responses shown here. FIG. 7. -

194

M. BJORKLUND, L. FORNI AND A. COUTINHO

shows that also under such conditions, there is a drastic T-cell-dependent increase in the magnitude of the PFC response. Optimal responses to LPS are obtained on day 4 of subculture, thus showing the same day-6 «peak» as for fresh Band T cells (fig. 4). The presence of T cells increases the IgM PFC response 3-fold, while IgG responses are 5-fold higher. The Ig classes preferentially induced by the T cells are IgGI, IgG2a and IgG3, in that order. Under these conditions, T-cell influences on the IgG subclass pattern are again most obvious for the IgG I subclass, which is not represented at all in control cultures, although the increase in the IgG3 isotype is noteworthy. In the absence of LPS, there is aT-ceIl-induced PFC response, consisting mainly of the IgM class, with little IgG3 and IgG2a, further underlining the active part that T cells take in the LPS response. The absence of IgG2b in this mitogen-independent response is surprising, and it should be considered within the particular protocol for time of exposure to LPS and T cells used there. DISCUSSION

The present results establish the induction of normal splenic T cells in LPScultures and the modulation by such T cells of the B-cell proliferative and PFC responses to LPS. It has been demonstrated that LPS stimulates 10-50 % of all splenic B cells [10-12] to proliferate and differentiate into Ig-secreting PFC in the absence of T cells [12]. This implies that growth and maturation of B lymphocytes can be truly TI. However, as shown here, this does not imply that T cells, if present, do not influence B-cell responses to TI mitogens and antigens. The same applies to other accessory cells, such as macrophages, which were not considered in the present experiments. The presence of T cells in LPS-activated cultures leads to significantly increased proliferative responses to be ascribed to both the B-cell and the T-cell compartments: T-cell-depleted SC yield elevated responses when co-cultured with irradiated normal T cells and, inversely, irradiated B blasts induce normal T cells to proliferate. In this context, some of the observations deserve a few comments. First, addition of T cells enriched for helper cell precursors (by depletion of Lyt-2+ cells) to T-cell-depleted SC caused better proliferative responses to LPS than did addition of untreated T cells. Yet, maximal proliferative responses to LPS were obtained in the normal SC controls. Thus, the normal untreated SC population appears to contain the optimal balance between the cell compartments which participate in the proliferative response to LPS. Second, non-irradiated T cells were more efficient than after irradiation in enhancing SC responses to LPS. This difference can be partially attributed to the B-blast-mediated induction of normal T cells. On the other hand, the fact that irradiated normal T cells could indeed augment the B-cell proliferative response suggests the presence of in vivo preactivated T H cells and/or the production of lymphokines in a radioresistant manner. Interestingly, T cells responding in these conditions are not LPS-reactive, but rather self-

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reactive lymphocytes, since the addition of LPS inhibited the T-cell proliferative responses to syngeneic LPS-blasts (fig. 3). We have previously described the LPS-blast-mediated induction of normal syngeneic T cells to helper and killer functions [22]. As shown here, not only MLR culture conditions, but also activation of normal SC by LPS, leads to the induction of T cells. As much as 12.4 010 of the viable cells in a day-3 low-density LPS-culture were L3T4+, and half of these were large-sized, indicating the ability to yield competent help. Only a small portion was Lyt-2 positive, but while the L3T4+ cells decreased in number from day 3 to day 6, Lyt-2+ cells doubled in number during the same period of time. A model for the cellular interactions in LPS-activated normal SC cultures could be as follows: first, resting immunocompetent B cells are activated by LPS to undergo blast formation and proliferate. Next, normal T cells are induced by the activated B blasts (or macrophages) to proliferate, undergo blast transformation and develop effector functions. Activation may be due to a variety of reasons, e.q. increased levels of la-antigen expression by activated B cells, or new idiotypic interactions arising from mixing T and B cells from different syngeneic donors. It is not our purpose here to analyse these mechanisms. Alternatively, in vivo activated T cells are kept stimulated and preferentially survive under these conditions. Finally, these autoactivated T-cell blasts display regulatory functions, such as help and, later in time, suppression of activated B-cell blasts. Thus, the presence of normal untreated T cells can be expected to not only enhance the optimal proliferative and PFC responses by inducing Ig secretion by B cell blasts which otherwise would not mature to plasma cells, but also to actively participate in the termination of the «late» response to LPS. The kinetics of the B-cell responses studied here indicate that this is the case. Thus, previous irradiation of normal or Lyt-2T cells to B-cell cultures prolonged the response to LPS in time in comparison with that of B cells or normal SC control. It has previously been shown that the nature of the stimuli determines the IgG subclass pattern of the PFC response [1-5] and the present studies confirm earlier in vitro [4, 5, 28, 33] and in vivo [6] investigations showing the selective IgG3, IgG2b and IgG2a expression after stimulation with LPS. Most striking, however, is the strong T-cell dependency seen for the IgG2a, IgGI and IgA PFC responses. The presence.of Lyt-2- cells enhanced the IgG2a PFC response to LPS to-fold over control cultures with B cells alone, and induced the highly T-cell-dependent IgG 1 and IgA isotypes [30-32], undetectable in B-cell cultures. The induction of IgA PFC was quite unexpected, as IgA PFC are refractory to LPS in vivo [6] and had never been obtained in vitro. The presence of normal T cells in LPS cultures enhanced PFC responses of all isotypes, even the typical «TI-isotypes» IgG3 and IgG2b. This might result from the increased proliferation in reconstituted cultures and/or from a direct effect of T cells or T-cell factors on IgG3 production in the presence of LPS [7]. It should be noted that the levels of enhancement of the «TD» PFC isotypes are kinetically correlated with the levels of L3T4+ versus Lyt-2+ T-cell activation in culture. Thus, IgG2a PFC, for instance, are preferentially

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enhanced by the presence of T cells early in culture, precisely when the numbers of L3T4+ blasts are higher. In contrast, late PFC isotype patterns are typically « TI» even in cultures that had received T cells, but no longer show a high proportion of activated L3T4+ cells and instead contain activated Lyt-2+ T cells. The regulatory effects of normal splenic T cells were further elucidated by co-culture with purified pre-activated LPS-blasts. The higWy increased PFC numbers and, particularly, the induction of IgG1 PFC show that LPS blasts can be the targets for T-cell-dependent modulation of LPS-responses and that T-cell effects on LPS cultures cannot solely be explained by recruitment of otherwise unreactive small B cells. The present observations could be a manifestation of a general phenomenon whereby any TI mitogen or antigen-induced response is modulated by autoreactive T cells [22]. A number of observations support this assumption. In vivo studies by Mongini et al. have demonstrated increases in IgG2a and, to a lesser extent, IgG2b serum levels in Nude mice in response to trinitrophenyl-Ficoll, a TI-antigen, after reconstitution with T cells [27]. Moreover, Nude mice respond in vivo to LPS with lower relative numbers of IgG2a and IgG1 PFC than do normal mice [6]. Finally, Ivars et af. have demonstrated the T-cell dependency of all non-IgM classes in the in vivo PFC responses to dextran B-512, another TI-antigen [34]. Autoactivated T cells are also likely to take part in the maintenance and regulation of the « natural» background PFC found in untreated normal mice of any strain and status [32, 33, 35]. Thus, Nude and normal antigen-free mice of the same strain display different background PFC patterns, with markedly lower levels of IgG1 and IgA PFC in Nude mice. The activity of such T cells and their induction by B blasts could also explain the well-known observation that in vivo antigen challenge results in antigen-non-specific PFC and antibody responses which actually far exceed the specific part of the response [28-36]. A T-cell-dependent correlation in isotype distributions between the specific and non-specific responses has, in fact, been observed [36]. RESUME MODULATION DEPENDANTE DES CELLULES T DES REPONSES POLYCLONALES DES LYMPHOCYTES B DANS LES CULTURES DE CELLULES SPLENIQUES NORMALES, STIMULEES PAR LE LIPOPOLYOSIDE

Les reponses polyclonales, proliferatives et maturatives in vitro, des lymphocytes B au lipopolyoside (LPS), mitogene thymus-independent, sont augmentees en presence de cellules spleniques T, syngeniques normales. Les lymphocytes T Lyt-2 - normaux irradies, ajoutes a des cultures de cellules spleniques sans lymphocytes T, modifient la distribution des sous-classes d'IgG produites en raison de la predominance en IgG3 et IgG2b, a l'apparition d'IgGl, d'IgG2a et meme d'IgA.

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D'autres evidences pour la participation active des lymphocytes T dans la regulation des reponses B au LPS ont ete apportees: 1) des cultures secondaires de blastes induits par Ie LPS sont stimules par la presence de lymphocytes T normaux syngeniques, meme en l'absence de mitogtme; 2) ces lymphocytes B actives stimulent la proliferation des cellules T normales; 3) un pourcentage considerable de lymphocytes T L3T4 + apparait dans des cultures de cellules spleniques totales. Nous suggerons l'importance de mecanismes de ce type dans la regulation des reponses «thymo-dependantes» in vivo par des lymphocytes T auto-reactifs. MOTS-CLES:

B, Rate.

Lymphocyte T, IgG, LPS, Immunoregulation; Lymphocyte

ACKNOWLEDGEMENTS

We thank Ms L. Ornehult, R. Martin and J. Badella for typing the manuscript. This work was supported in part by the Swedish Medical Research Council and DRET. The Basel Institute for Immunology was founded and is supported by Hoffman-LaRoche.

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