A noncognate interaction with anti-receptor antibody-activated helper T cells induces small resting murine B cells to proliferate and to secrete antibody

Eur. J. Immunol. 1988.18: 395-401 Polyclonal B cell activation by anti-receptor antibody-activated T helper cells 395

Trevor Owens’

Walter and Eliza Hall Institute, Melbourne

A noncognate interaction with anti-receptor antibody- activated helper T cells induces small resting murine B cells to proliferate and to secrete antibody*

Culture of small resting allogeneic B cells (of an irrelevant haplotype) with two clones of T helper (Th) cells that were activated by the F23.1 anti-T cell receptor antibody led to the activation of B cells to proliferate and to secrete antibody. Th cell supernatants by themselves had no effect on resting B cells (even in the presence of intact €23.1 antibody), but could induce antibody secretion by anti-Ig-preactivated B cells. Both F23.1’ clones (E9.D4 and 4.35F2) and one F23.1- clone (D2.2) could synergize with supernatants from activated E9.D4 T cells to induce B cell activation. F(ab‘), fragments of F23.1 induced E9.D4 to activate B cells as efficiently as intact F23.1, and B cell populations that had been incubated with F23.1 were not activated when cultured with E9.D4, although T cells recognized cell-presented F23.1 and were weakly activated. Reduction of the density of F23.1 adsorbed to plastic resulted in weak T cell activation, and these T cells did not induce B cell responses. Haptenated B cell populations, although recognized by E9.D4, were not activated. Separation of T and B cells by a 0.4-pm membrane prevented T-dependent B cell activation, although Th cell-derived B cell-activating lymphokines would be assayed across these membranes. These results suggest a polyclonal noncognate B cell activation that depends on physical contact between B cells and activated T cells. The requirement for a cognate interaction of T h with B cells for the production and delivery of B help can therefore be overcome by activating Th cells with high densities of T cell receptor ligands.

1 Introduction

The prevailing model for the collaborative interaction between T helper (Th) and B cells is that of a cognate interaction whereby Th cells are activated by antigen presented in association with class I1 major histocompatibility complex (MHC) molecules on the B cell surface [l]. This activation induces the production of lymphokines that in turn lead to activation of the antigen-presenting B cell. The overall specificity of the interaction is preserved by the requirement for antigen recognition in order to activate lymphokine production by Th cells [2], and by the fact that resting B cells do not respond to Th cell-derived B cell-activating factors [3-51. “Bystander” B cell activation is considered to result from the proximity to an activated Th cell of B cells that have been preactivated through encounter either with antigen, or with other surface immunoglobulin (s1g)-ligating agents [ l , 6, 71. The production by one Th cell subset [8] of lymphokines that can activate resting B cells directly [e.g. interleukin 4 (IL4)] can be accomodated

[I 61661

* J. F. A. P. Miller’s laboratory, in which this work was carried out, is supported by the National Health and Medical Research Council of Australia.

’ Recipient of a Centennial Fellowship from the Medical Research of Canada.

Correspondence: Trevor Owens, Montreal General Hospital Research Institute, 1650 Cedar Avenue, Montreal, Quebec H3G 1A4, Canada

Abbreviations: ABA: Azobenzenearsonate CM: Culture medium Con A: Concanavalin A ELISA: Enzyme-linked immunosorbent assay FCS: Fetal calf serum IFN: Interferon IL: Interleukin LPS: Lipopolysaccharide LFA-1: Lymphocyte functional antigen-1 MHC: Major histocompatibility complex SC: Spleen cell(s) sIg: Surface immunoglobulin TcR: T cell receptor for antigen T,: T helper (cell)

within this model because the action of IL4 is optimized through synergy with antigen or anti-Ig [9, lo].

The collaborative activation of Th and B cells therefore involves a number of cellular interactions. Attempts to examine these interactions individually are complicated by the restriction of T cells to recognition of cell-associated antigen, and by the ability of B cells to present antigen [ll-131. Anti- bodies that recognize the T cell receptor for antigen (TcR) can be used to activate T cells independently of cellular interactions [14] and this should in principle allow such dissection. I have used the anti-TcR antibody F23.1 [15] to activate antigen-specific cloned Th cell lines in co-culture with allogeneic small resting B cells. These B cells were activated to proliferate and to secrete Ig, in the absence either of any sIg ligating agent or of a MHC-restricted interaction with the Th cells. Activation required co-culture with T cells, and supernatants from activated T cells had no effect. Results indicate that a physical interaction between activated Th cells and B cells was required to induce those B cells to become responsive to B cell-activating factors. This was dependent on a high level of activation of the Th cells, which was achieved by using high densities of F23.1. The requirement for cognate interaction between T and B cells can therefore be overridden by a strong T cell activating stimulus, and so is not in itself necessary for B cell activation.

2 Materials and methods

2.1 T cells

The [azobenzenearsonate (ABA) + Iak]-specific cloned CBA T cell line E9.D4 and the I-Ek-alloreactive cloned A.TH T cell line 4.35F2 (both L3T4+, F23.1’) have been described else- where [16, 171. The L3T4+, F23.1- CBA clone D2.2 is (ABA+Iak) specific. All three clones correspond to T h l

0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1988 0014-2980/88/0303-0395$02.50/0

396 T. Owens Eur. J . Immunol. 1988.18: 395-401

helper T cells, as defined by Mosmann et al. [8]. E9.D4 trans- cribes and secretes the lymphokines IL2, IL 3, granulocyte- macrophage colony-stimulating factor (GM-CSF) and interferon-gamma (IFN-y), but does not transcribe or secrete IL 4 or interleukin 5 (Kelso and Owens, Williamson, Owens and Pearse, Kelso and Gough, unpublished results). Clones 4.35F2 and D2.2 secrete IL2, IL3 and IFN-y upon activation ([16], Owens, unpublished, and Fazekas de St. Groth, personal com- munication). Cloned T cells were routinely screened for mycoplasma by Hoechst 33 258 staining. T cells were maintained in culture in RPMI 1640 (Flow Labs., Rockville, MD) sup- plemented with fetal calf serum (FCS; Flow, lo%), L- glutamine (Calbiochem, San Diego, CA; 2 mM) and 2-mer- captoethanol (Sigma Chem. Co., St. Louis, MO, 5 x M), in the presence of irradiated (3000 rds from an Atomic Energy Canada, Ottawa, 6oCo source) antigenic spleen cells (SC), and optimal concentrations of a partially purified IL 2-containing supernatant from concanavalin A (Con A; Ca1biochem)- stimulated rat SC [18]. Prior to assay, T cells were purified by centrifugation on Ficoll-Hypaque (Pharmacia, Uppsala, Swe- den) and cultured for 3 to 10 days in culture medium that contained 5% FCS (CM), without antigen or IL2.

2.2 Antibodies

The F23.1 (mouse anti-TcR Vp8) [15] and E4.2 (rat anti-mouse p) [19] antibodies were affinity purified by protein A chromatography (Pharmacia), and the 30H12 (rat anti-Thy- 1.2) [20], 53-6-7.2 (rat anti-Ly-2) [20], F7D5 (mouse anti-Thy- 1.2) [21] and GK1.5 (rat anti-L3T4) [22] antibodies were used as dilutions of culture supernatant. E4.2 was conjugated at 1 mg/ml to cyanogen bromide-activated Sepharose 4B (Phar- macia). Pepsin (Sigma) digestion of F23.1 to yield F(ab’)2 fragments was carried out by the method of Lamoyi and Nisonoff [23]. F(ab’)* fragments were purified by protein A chromatography.

2.3 Assay for T cell activation

Intact or F(ab’)z fragments of F23.1 were adsorbed to flat- bottom tissue culture 200 pl microwells (Nunc, Roskilde, Denmark) as previously described [ 161. Optimal activation of lymphokine production is induced by F23.1 adsorbed at 1-3 pg/ml to tissue culture plastic [16]. Inclusion of SC as fillers enhances these responses by 2 to 5-fold (Owens, unpublished). SC were chemically coupled with the ABA hapten as described [16]. Responses to optimal doses of plastic-adsorbed F23.1 and of antigen (lo6 haptenated SC) are equivalent, whereas responses to lo5 haptenated SC are equivalent to those to F23.1 adsorbed to plastic in the 10-20 qg/ml concentration range ([16], see also Fig. 4). Production of other lymphokines (IL2, GM-CSF, IFNy) varies similarly with F23.1 concentration (Kelso and Owens, submitted for publication, and Owens, unpublished). In some experiments, T cells were stimulated by T-depleted SC populations that had been incubated overnight on F23.1-coated plastic. IL3 titers in supernatants from 24-h cultures containing 2 x lo4 T cells and stimulated as described for each experiment were measured by bioassay on the R6X-E4.8.9 IL3-dependent cell line, as described [16, 241. Titers were calculated by linear regression analysis of log transformations of proliferation data [16]. The lower limit of detection for this assay was about 0.01 Units/ml, and maximal IL3 production was of the order of 20-200 Units/

ml. Supernatants from activated T cells were collected from 24-h cultures of lo6 T cells/ml on F23.1-coated (3 pg/ml) plastic, then centrifuged and stored at 4°C. Supernatants contained less than 5.0 qglml Ig as measured by enzyme-linked immunosorbent assay (ELISA).

2.4 B cells

Small resting B cells were prepared by depletion of T cells from erythrocyte-depleted cell suspensions from CBA, BALB/c or C57BL/6 spleens (Walter and Eliza Hall Institute SPF colony, Kew, Victoria), using a cocktail of anti-Thy-1.2 (F7D5), anti-L3T4 (GK1.5) and anti-Ly-2 (53-6-7.2) antibodies, and rabbit complement. After depletion (verified by counting), SC were fractionated by centrifugation on gradients of Percoll (Pharmacia) at densities of 1.07, 1.075, 1.08 and 1.09 kgll. The fraction of density 1.08-1.09 kg/l was used in all experiments. These SC were Thy-1.2-, 90-95% sIg+ and Ia- dull in comparison with lipopolysaccharide (LPS)-stimulated B cells were homogeneously small [mean volume 103-115 femtoliters (fl)], compared to unfractionated SC (mean volume 119-124 fl) [analyzed by flow cytometry (Coulter, Hialeah, FL)], and they did not respond to Con A (not shown). To prepare preactivated B cells (see Table 4), SC were T depleted as described, but no further fractionation was carried out. Cells were incubated at 1.5 x 106-2.0 x 106/ml in CM overnight with E4.2-coupled Sepharose (Pharmacia), then collected and Sepharose beads removed by sedimentation through FCS.

2.5 Assay for B cell activation

Irradiated (1500 rds, 6oCo) E9.D4 that had been cultured in the absence of antigen and IL2 for at least 3 days were titrated into flat-bottom microwells (Nunc) in CM. T cell supernatants and anti-p beads were then added, where indicated. Cultures were initiated by the addition of lo5 B cells. LPS (Sigma; S. abortus) was added at 20 pg/ml as a positive control for B cell activation. Proliferative responses were measured after 2 days. Cultures were pulsed for 6 h with 0.5 pCi = 18.5 kBq [3H]thymidine ([3H]dThd; Amersham, GB), then collected on glass fiber filters using a Titertek (Flow Labs.) cell harvester, and the incorporated radioactivity measured by scintillation spectrometry. Antibody secretion was measured after 4-days culture. Plates were washed twice with RPMI 1640 containing 1% FCS, then incubated for 16 h in 100 pl CM. Fifty microli- ter aliquots were collected and cultures were washed twice, resuspended in 100 p1 CM and incubated for a further 4 h. The Ig content of 50-pl samples from individual cultures then was measured by ELISA on goat anti-mouse Ig-coated plates [16]. The relationship between AdI4 values and Ig concentration was linear in the range 3.0 to 100 qglml. Every assay included a standard titration of purified antibody [usually F23.1, although the relationship between A414 and Ig concentration was similar for IgM, IgGza and IgGl (not shown)]. The number of Ig- secreting B cells was determined in some experiments by measurement of protein A plaques [25] using a rabbit anti-mouse IgM antiserum, from the same cultures whose Ig secretion had previously been assayed by ELISA. In some experiments, T and B cells were separated during culture by a 0.4-pm membrane, using Transwell tissue culture clusters (Costar). In these experiments, 2 X 105-3 x lo5 T cells and lo5 B cells were cultured in a final volume of 700 pl. After 4 days, cells were


collected from both wells and Transwell inserts, washed and resuspended in 200 p1 microwells, in 100 pl CM. These cultures were then processed for ELISA as described above.

3 Results

3.1 T-dependent activation of B cell proliferation

High density, small resting B cells did not proliferate in response to supernatants from activated E9.D4 (Table 1). Sepharose-coupled rat anti-mouse p (E4.2) antibody stimulated B cells to proliferate, and this was enhanced by the inclusion of E9.D4 supernatant (Table 1). Co-culture of C57BLl6 (H-2b) small resting B cells with the (ABA + Iak)-specific clone E9.D4 did not result in any proliferative response (Table 1). However, when these cells were cultured on F23.1- coated microwells, a strong proliferative response ensued, despite there being no recognition of C57BL/6 SC by E9.D4 [17] (Table 1).

3.2 T-dependent activation of antibody responses

Small resting B cells of three haplotypes, when co-cultured with E9.D4 on F23.1-coated plastic, were induced to secrete Ig, detected by ELISA 4-5 days after the initiation of culture (Fig. 1). Although E9.D4 does not respond to either unhaptenated CBA SC or to other common laboratory H-2 haplotypes [17], both syngeneic (CBA, H-2k) and allogeneic (BALBlc, H-2d, and C57BW6, H-2b) B cells were activated (Fig. 1). Ig secretion was estimated by comparison with a standard curve, included in every assay. In Fig. 1, optimal stimulation by E9.D4/F23.1 of CBA and B cells induced the production in a 4-h assay of 14.7 and 6.8 ng Iglculture, respectively, whereas LPS stimulation of the same B cells induced 32.1 (CBA) and 22.3 (C57BLl6) ng Ig/culture. Co-culture on F23.1-plastic with an F23.1- clone (D2.2), or with E9.D4 in the absence of F23.1, did not stimulate B cells (Fig. 1B). Activation of E9.D4 by F(ab')z fragments of F23.1 also induced the activation of co-cultured small resting C57BL/6 B cells to Ig secretion and to proliferation (not shown), suggesting that this noncognate B cell activation was not dependent on Fc-mediated binding of F23.1 to B cells (see also Table 3 and Fig. 3).

A similar relationship between LPS and F23.1-activated E9.D4 as stimuli was observed when the number of Ig-secreting B cells in such cultures was assessed by a protein A plaque assay (Table 2). The number of B cells detected as protein A PFC in optimally stimulated cultures was between 3-5% of the

Culture

B cellsb) B cell + SW) B cells + anti-@ BcelIs+SN+anti-p B cells i LPS E9.Wo B cells + E9.D4

No F23.1 I 11

1040 ( 5 ) 1250 (30)

34730(3040) 33090 (400)

1350 (70) 1660 (180) 10740 (1440) 16810 (17tu))

47040(3310) 53460 (280) 50 ( 5 ) 120 (70)

1150 (10) 1410 (20)

0 D2.2 Filled: F23.1-P

E9.D4 (CellslCulture x ~ O - ~ ) T celIslCulture ( x I O - ~ ) [1616611

Figure 1. Ig secretion by syngeneic and allogeneic B cells through co- culture with F23.1-activated cloned Th cells. High-density B cells from the indicated haplotypes were cultured at 105/culture with titrations of irradiated (1500 rds) E9.D4 or D2.2, in the presence or absence of F23.1 antibody, coated to tissue culture plastic at 3 pgiml. Super- natants from 4-h secretion cultures at 5 days were assayed for their Ig content by ELISA. Results are shown as mean absorbances from duplicate cultures f SEM (shown by bars, where greater than the radius of the symbols). (A) Activation of CBA and BALB/c B cells by clone E9.D4 stimulated by F23.1. (O), CBA. (W), BALBlc. Open symbols, no F23.1. (B) Activation of C57BL/6 B cells by E9.D4, and lack of effect of the F23.1- clone D2.2. (O), E9.D4. (W), D2.2. Open symbols, no F23.1. (A) and (B) describe different experiments.

initial input at day 0. However, measurement of the frequency of responding B cells by single-cell clonal analysis has shown that between 10-25% of B cells were activated [26]. These responses cannot therefore be ascribed to a minor subpopula- tion of B cells, but represent a polyclonal B cell activation.

3.3 Induction of B cell activation by T cell supernatants

B cell activation required co-culture either with activated T cells or with T cells plus the product of T cell activation. Supernatants from E9.D4 that had been activated by F23.1- plastic did not stimulate B cells to proliferate (Table 1) or to secrete Ig, even if assayed in F23.1-coated microwells (Table 3). In fact, no density fraction of B cells, including those that did not enter the Percoll gradient, responded by Ig secretion to T cell supernatant in the absence of sIg ligation.

1910 (240) 730 (80) Nld' NT N T NT NT NT N7' NT

38510 (4%)) 34170 (1900) 70 (20) 90 (5 )

a) Adsorbed from 3 pg/ml in PBS. b) 105/culture. Results show

[3H]dThd incorporation (cpm), as means of duplicate cultures k SD in parentheses.

c) E9.D4 supernatant, 1/6 dilution. d) Not tested. e) E4.2 [19], coupled to Sepharose

beads. f) 1500 rds, 2 x l@/culture.

398 T. Owens Eur. J. Immunol. 1988.18: 395-401

Table 2. Comparison of ELISA and plaque counting as assays for B cell response

E9.D4‘) PFUculturebl Ad,, ,,,,,b) Ig production‘) x 10-~ x 10-2 (ngkulture)

6 30.00 (3.10) 1.320 (0.010) 8.1 2 15.40 (2.50) 1.010 (0.070) 5.3 0.6 1.50 (0.01) 0.060 (0.010) 0.8 0.2 < 0.50 (0.20) 0.030 (O.oO0) < 0.03

Lps 49.90 (1.70) 1.690 (0.200)d’ 17.7*] Nil < 0.50 (0.20) 0.040 C0.W) < 0.03

a) E9.D4 (1500 rds) were prepared as described for Fig. 1 and titrated in microwells that had been coated with F23.1 at 3 pg/ml.

b) B cells were prepared from C57BL/6 spleens as described in Sect. 2.4, and cultured at 105/well with E9.D4. After 5 days Ig secretion was measured in a 4-h assay as described in Sect. 2.6, after which the number of PFC was measured using a protein A plaque assay [25] (SD in parentheses).

c) Calculated from a standard curve of AdIdnrn vs. qglml Ig (F23.1). d) &14nrn read and Ig content calculated from a 50% diluted super-

natant.

Preactivation of B cells by ligation with Sepharose-coupled anti-y induced their response to E9.D4 supernatants (Tables 1 and 3). However, inclusion of T cells (either E9.D4 or D2.2) with B cells and supernatant-induced Ig secretion (Fig. 2), albeit not as strongly as did F23.1-activated T cells.

3.4 Induction of B cell activation by other Th cells

The F23.1’ I-Ek-allospecific clone (4.35F2) induced the activation of C57BL/6 small resting B cells to Ig secretion in an exactly similar manner to E9.D4 (not shown). This clone could also synergize with supernatant from activated E9.D4 to induce antibody secretion, similar to both E9.D4 and D2.2. Supernatants from F23.1-activated 4.35F2 had equivalent activity to those from E9.D4 (not shown). In other experiments, activation of all three clones and of ex-vivo T cells with anti-T3 antibody produced effects exactly similar to those of F23.1 (Owens, unpublished). Taken together with the results in Figs. 1 and 2, this suggests that the ability to induce polyclonal B cell activation through a noncognate interaction was not confined to rare or unusual clones.

Table 3. Small resting B cells do not respond to E9.D4 supernatant

E9.D4-SN B cell respoiise (A,,, (Udilution) Control M3. I-pIastic’) Anti-u-

Sepharose“

2 0.190 (0.020) 0.140 (0.010) 1 .Y40 (0.060)

18 0.070 (0.020) O.Z(X1 (0.060) 1.860 (0.070)

Nil 0.120 (0.050) 0.120 (0.060) 0.060 (0.010)

6 0.090 (0.010) 0.290 (0. loo) 1.830 (0.030)

54 0.140 (0.0oOl 0.230 (0.080) 1.680 (0.llW)

a) CBA B cells (ld/culture) assayed after 5 days. Results are presented as means ? SD of duplicate cultures.

b) Adsorbed at 3 pg/ml. c) E4.2 [19], coupled to Sepharose beads.

oNo T cells n 0 illed: +supernatant

1lDilution Supernatant T cells/Culture ( x ~ O - ~ )

Figure 2. Activation of small resting allogeneic B cells by T cell supernatants in co-culture with T cells. High-density B cells were prepared from C57BLi6 spleens as described in Sect. 2.4. These were incubated at los cells/culture in microwells as follows. (A) with titrations of supernatant from F23.1-activated E9.D4, either alone (0) or with added T cells [2 X lo4 1500 rds-irradiated E9.D4 (0) or D2.2 (A) per culture]; (B) with titrations of 1500 rds-irradiated E9.D4 (0) or D2.2 (0) with (filled symbols) or without (open symbols) a 1/6 dilution of supernatant from F23.1-activated E9.D4. Cultures were incubated for 5 days and then the Ig content of supernatants after a 4-h secretion was measured by ELISA. Each point represents the mean of duplicate cultures f SEM.

3.5 F23.1 “bridging” does not mediate B cell activation

High-density C57BL/6 B cells were incubated overnight on F23.1-coated tissue culture wells, then washed and cultured with irradiated E9.D4. In some cases, F23.1 and control anti-

IL3 (Uiml)

9 B, No F23.1 Q RF23.1-P 4 BF23.1 + S N 8 ! 2 ’ p+*: 4,4

<0.01 0 Nil 0.2 0.6 2 6

E9.D4 (CeWculture x 10 -4)

Figure 3. Effect of bridging T cells and B cell populations with F23.1. High-density B cells, prepared from C57BL/6 spleens as described in Sect. 2.4, were incubated Overnight at 1 X 106-2 X lo6 celldm1 on F23.1-coated (3 pghl) wells. These and B cells from control wells without F23.1 were collected, washed, and incubated with titrations of irradiated (1500 rds) E9.D4, with or without a 1/6 dilution of supernatant from activated E9.D4, or in F23.1-coated microwells. After 5 days Ig secretion was measured by ELISA, from a 4-h secretion assay. Each point represents the mean of duplicate cultures, the bars represent SD. (0): Control B cells; (8): control B cells, co-cultured with E9.D4 on F23.1-coated assay wells; (+): F23.1-incubated B cells; (0): F23.1-incubated B cells, incubated with E9.D4 and supernatant. T cell activation was assayed in parallel, by measurement of IL3 titers in supernatants from replicate cultures containing 2 x lo4 E9.D4 and lo5 B cells. Values (units IL3/ml) are shown for each group. NA: Not applicable.


bodies could be demonstrated on these populations by immunofluorescence (not shown). Presentation of F23.1 by these cells to E9.D4 induced a weak but significant IL3 response (Fig. 3). E9.D4 did not respond to control B cells (Fig. 3), or to B cells that had been incubated with other anti- T cell antibodies (not shown). Both control and F23.1-treated B cells remained competent to respond to T cell-derived sig- nals (F23.1-activated E9.D4, and E9.DCsupernatant with E9.D4, respectively) (Fig. 3). Despite the ability of F23.1- presenting B cell populations to induce lymphokine secretion by E9.D4, B cells in these populations were neither activated to proliferate (not shown) nor to secrete Ig (Fig. 3).

3.6 F23.1 dose response for the induction of B cell activation

The level of response of E9.D4 was modified by reduction of the density of F23.1 on the microwells. Co-cultured CBA B cells were not activated below 20 qglml F23.1, although IL3 production by E9.D4 was still detectable (Fig. 4). Similarly, although E9.D4 was detectably activated by E9.D4 ABA-coupled CBA SC, B cells in the haptenated SC were not induced to Ig secretion. These B cells were activated when cultured with E9.D4 on F23.1-plastic, coated at the optimal concentration of 3 pg/ml (Fig. 4). The response to 3 pg/ml F23.1-plastic was enhanced in the presence of lo5 ABA-SC, consistent with there being two stimuli for E9.D4 [F23.1 and (ABA + lak)], one of which (€723.1) is enhanced in the presence of fillers (see Sect. 2.3).

These results suggest that noncognate, polyclonal B cell activation required that T cells be activated above some critical threshold. Where B cells were activated through culture with T cells and supernatant, the highest su ernatant concentra-

T cells. In direct co-activation culture, up to 6 x lo4 optimally tions were equivalent to those from 10 P optimally activated

IL3 (Ulml)

p-.J 22.1 13.6

3.3 115.3 * uglml

+ 100 nglml 4 20nglml 9 4nglml -8- CBA-ABA 4 CBA-ABAfF23.1

0.2 0.7 -

Nil 0.2 0.6 2 6

E9.D4 (Cellslculture x 10 -4)

Figure 4. Dose response for F23.1-plastic as inducer of T-dependent polyclonal B cell activation. Microwells were coated with F23.1 at the indicated concentrations. Irradiated (1500 rds) E9.D4 were titrated in these wells, and 105 high-density CBA B cells added. In some groups, B cells had been chemically coupled with the ABA hapten. After 5 days, the Ig secreted in a 4-h assay was measured by ELISA. Each point represents the mean of duplicate cultures, the bars represent SD. (1): Unhaptenated B cells, 3 clg/ml F23.1; (+): unhaptenated B cells, 10 q g h l F23.1; (0): unhaptenated B cells, 20 qglml F23.1; (0): unhaptenated B cells, 4 qg/ml F23.1; (m): haptenated B cells, no F23.1; (0): haptenated B cells, 3 pg/ml F23.1. Activation of E9.D4 was assayed in parallel, measuring IL3 titers in supernatants from replicate cultures that contained 2 X 104 E9.D4. The lower limit of detection in this assay was 0.01 U/ml.

Table 4. Separation of B from Th cells by a membrane prevents B cell activation

Tnatment Ig secretion (A4,4,)1) I IIb'

Highdensity B cells Control Well 0.027 (0.003) O.O4Ic)

lased 0.037 (0.012) 0.030 E9.D4 + F23.1-I"'' WeU 0.312 (0.005) 0.337

Insert 0.034 (0.002) 0.030 E9.D4supernatant Well NT' + E4.2-sepharosc Insert 0.435 NT

Anti-Ig preactivated B cellsb) Control Well 0.214 0.061

Insert 0.188 0.067 E9.W +F23.1-P Insert 0.715 o.%o E9.D4 supernatant Well 1.692 NT E9.D4snpernataat Insert 1.933 NT

T cells (2 X 105-3 X l@/culture) were cultured in 700 pl wells, and B cells (ld/culture) either in wells with T cells, or in inserts, separated from T cells by a 0.4 pm membrane, l mm above the surface of the well. Results are shown as absorbance at 414 nm from 4-h incubations f SD, unless otherwise indicated. Values shown are from 16-h incubations. Single point readings were corroborated by assaying supernatants from 4-day, 16-h (4-5 days) and 4-h (day 5) incubations. F23.1 adsorbed to plastic at 3 pg/ml. Not tested.

activated T cells were present. On the other hand, T cell activation was markedly reduced when either antigen (ABA + Iak) or anti-TcR (F23.1) densities were suboptimal. In some experiments, a weak but significant B cell response was induced by T cells that were not intentionally stimulated. This correlated with the use of T cells that remained in a residually activated state, through not having been cultured in the absence of antigen or of IL2 for as long as in other experiments.

3.7 Requirement for cellular interaction

To address the question of whether physical interaction between activated T cells and small resting B cells was required for B cell activation, the two were separated during culture by a 0.4-pm membrane. B cells were cultured in an insert 0.4-pm membrane-bottom well 1 mm above the plastic surface on which E9.D4 cells were activated by F23.1. Although activated E9.D4 cells produced B cell-activating lymphokines that induced Ig secretion by anti-p-preactivated B cells in the inserts, small resting B cells were not activated (Table 4). As before, B cells were activated by direct co-culture with the F23.1-activated T cells (Table 4).

In other experiments, anti-LFA-1 antibodies inhibited the induction of allogeneic B cell proliferation by E9.D4 (not shown). Maximal inhibition of proliferation was about 50%. However, the effect of anti-LFA-1 on antibody responses was highly variable, and in many experiments (including those in which anti-LFA-1 inhibited proliferative responses), there was no effect on the induction of Ig secretion. While LFA-l-mediated interactions contribute to the activation of B cell proliferation [27] and to the formation of T-B conjugates [28], they

400 T. Owens Eur. J. Immunol. 1988.18: 395-401

clearly do not play a major role in the T-dependent B cell activation described here.

The possibility that production of a labile T h cell-derived factor required T-B contact for its initiation was addressed by gener- ating E9.D4 supernatants in the presence of SC, both unfractionated and T-depleted, and by transferring supernatants after 1, 2, 3 or 4 days of culture (without interim storage or freezing) from cultures in which polyclonal B cell activation was induced, into fresh B cell cultures. In no case was there any transfer of polyclonal B cell-stimulating activity (not shown). That E9.D4 might induce B cells to become responsive to supernatants through their physical contact was tested by co-culture of both without stimulus, then removal of T cells and assay of B cell response. Preliminary experiments indicate no effect on B cells (unpublished).

4 Discussion

The consensus view on T-dependent B cell activation is that although Th cells operate via the production of nonspecific soluble factors (lymphokines), resting B cells are refractory to these lymphokines unless they have been triggered through encounter with antigen [l]. In this context, the results presented here are unexpected. Small resting B cells that did not respond by either proliferation or Ig secretion to supernatants from activated T cells were nevertheless induced both to proliferate and to differentiate through co-culture with T cells that were activated by an anti-TcR antibody. There was no sIg- ligating agent present in culture, and the T cells were both MHC allogeneic and nonalloreactive to the B cells. Explana- tions such as mycoplasma contamination of T cells, Th-B cell bridging and/or B cell triggering by F23.1, or direct-acting lymphokines in supernatants have been excluded as causes for activation. The possibility that the Th cells mediating this activation are rare or unusual also seems unlikely. Moreover, preliminary results suggest that anti-TcR-activated ex-vivo L3T4' T cells can also activate allogeneic B cells in like manner (Owens, unpublished).

These results can be reconciled with existing observations on B cell activation by consideration of the F23.1 dose response for noncognate polyclonal activation (Fig. 4). Suboptimal densities of F23.1 adsorbed to plastic did not induce E9.D4 to activate co-cultured B cells. Similarly, the suboptimal T cell responses that were stimulated by lo5 haptenated T-depleted SC did not induce B cell activation. However, anti-Ig-triggered B cells in haptenated populations were activated by E9.D4 (data not shown). This is consistent with results from other laboratories [ l , 3, 29, 301 in that small resting B cells in haptenated SC populations were not activated simply through their recognition by activated Th cells (Fig. 4), but required another signal (e.g. sIg ligation) to respond.

That noncognate polyclonal B cell activation occurs when Th cells are highly activated was also suggested by DeFranco et al., who showed an antigen concentration dependence for the induction of non-MHC-restricted activation of small resting B cells by cloned Th cells [31]. A similar dependence on antigen concentration for the polyclonal induction of Ig secretion by small resting B cells is apparent in a report by Tite et al. [32], who also showed that cloned Th cells, activated by a soluble anti-clonotypic antibody could not induce the activation of small resting allogeneic B cells to Ig secretion [32]. The

level of T cell activation was not assessed. Tedder et al. [27] described by a polyclonal B cell proliferative response that resulted from co-culture with human T cells activated by soluble anti-CD2 antibody. Antibody secretion was not measured, and since Th and B cells were from the same donor, the human and murine systems are not exactly comparable.

A model to account for the results presented here would pro- pose that physical interaction with a Th cell that has been activated above some threshold induces small resting B cells to respond to late-acting growth and differentiation factors. Such a model is difficult to test, given that activated T cells also produce lymphokines. Furthermore, although supernatants could not transfer activation, and separation by a membrane prevented activation, a role for Th cell-derived lymphokines cannot formally be ruled out by these results. A steep gradient of effect for a labile factor could lead to its action being detectable only when T and B cells were actually in contact. Results of experiments referred to in Sect. 3.7 argue against the related possibility that production of such a lymphokine might require T-B contact. However, a model that involves cell-cell interaction is consistent with some other observations on the interactions of Th with B cells. Both Sanders et al. [28] and Kupfer et al. [33] have shown the formation of conjugates between Th cells and B cells, in the absence of antigen recognition by Th cells. A similar antigen-independent interaction may be involved in the B cell activation described here, with the distinction that the Th cells in this study were activated independently of T-B interaction. The correspondence between the degree of T cell activation and the ability to mediate noncognate help might suggest a structural change in the T cell surface, an aspect that should be amenable to further examina- tion.

The experiments described in Tables 1 and 3 demonstrate the ability of Th cell supernatants to synergize with anti-y, and the results generally can be interpreted as showing synergy of T-derived factors with preactivated B cells. Preliminary experiments (Owens, unpublished) indicate that the response to supernatants is predominantly due to their IL2 content. It has been suggested that IL2-producing Th cells cannot help B cells, due to their inability to produce IL4 [34]. However, IL2 can stimulate preactivated B cells to proliferate and to secrete Ig [35], and the failure of ILZproducing Th cells to help B cells in the experiments of Killar et al. [34] may relate more to inefficient sIg ligation of B cells than to a T cell deficiency. If antigen were presented to B cells in a more ordered array in vivo than the soluble protein conjugates that are used in many in vitro systems, this would induce B cell responsiveness to late-acting factors produced by both types of Th cells (although IL 4-producing T h cells would be more effective under conditions where antigens were limiting or suboptimally presented).

These results bear directly on the role for MHC-cognate T-B interaction in the generation of the humoral immune response. The noncognate polyclonal B cell activation described here may only be detected under conditions for T cell activation that are supraoptimal by phsyiological standards. An equivalent activation of Th cells in vivo would require that antigen be encountered at an improbably high density. It has been argued that Th cell-derived lymphokines operate only over a short range, and are not found at high concentration distal from activated Th cells in vivo [36]. These considerations would impose a requirement that the partners in T-B collaboration be physically juxtaposed to optimize the production and deliv-


ery of help. The observation that hapten-carrier linkage optimizes in vivo antibody responses [37] experimentally demonstrated this requirement. The concentration and presentation of antigen in association with class I1 MHC by B cells enables the cognate interaction that optimizes their activation to Ig secretion. The results presented here show that this serves as a device to optimize T cell help, but is not intrinsi- cally necessary for B cell activation.

I would like to thank Ladina DiRago for excellent technical assistance. I am grateful to Anne Uelso for many helpful discussions and for com- ments on the manuscript. I also thank Jacques Miller, Gus Nossal and Michael Julius for helpful discussions. I would also like to thank Pauline Gallagher for polyacrylamide gel electrophoresis of antibody digests, Alan Harris for provision of flow cytometry facilities, and Frank Battye, Justin Beale and Mark Cozens for performing FACS analyses. The 4.35F2 clone was made available to me by Barbara Fazekas de St. Groth.

Received May 14, 1987; in final revised form November 30, 1987.

5 References

1 Julius, M. H., Immunol. Rev. 1987. 95: 177. 2 Herold, K. C., Lancki, D. W., Dunn, D. E., Arai, K. and Fitch, F.

W., Eur. J. Immunol. 1986. 16: 1533. 3 Julius, M. H., Von Boehmer, H. and Sidman, C. L., Proc. Narl.

Acad. Sci. USA 1982. 79: 1989. 4 Howard, M. and Paul, W. E., Annu. Rev. Immunol. 1983. I : 307. 5 Parker, D . C., Imrnunol. Rev. 1980. 52: 115. 6 Anderson, J., Schreier, M. H. and Melchers, F., Proc. Narl.

Acad. Sci. USA 1980. 77: 1612. 7 Ratcliffe, M. J. H. and Julius, M. H., J . Immunol. 1983.131: 581. 8 Mosmann, T. R., Cherwinski, H., Bond, M. W., Giedlin, M. A.

9 Klaus, G. G. B., Nature 1986. 324: 16. and Coffman, R. L., J . Immunol. 1986. 136: 2348.

10 Rabin, E. M., Mond, J . J., Ohara, J. and Paul, W. E., J . Exp.

11 Chesnut, R. W. and Grey, H. M., J . Immunol. 1981. 126: 1075. 12 Shimonkevitz, R., Kappler, J . , Marrack, P. and Grey, H., J . Exp.

13 Lanzavecchia, A., Nature 1985. 314: 537.

Med. 1986. 164: 517.

Med. 1983. I58: 303.

14 Marrack, P. and Kappler, J., Adv. Immunol. 1986. 38: 1. 15 Staerz, U. D., Rammensee, H.-G., Benedetto, J. D. and Bevan,

16 Owens, T. and Fazekas de St. Groth, B., J. Immunol. 1987. 138:

17 Owens, T. and Miller, J. F. A. P., J . Immunol. 1987. 138: 1687. 18 Thomas, W. R., Smith, F. I . and Miller, J . F. A. P., J . Exp. Med.

19 Boyd, A. W. and Pike, B. L., Eur. J. Immunol. 1982. 12: 184. 20 Ledbetter, J. A. and Herzenberg, L. A, , Imrnunol. Rev. 1979. 47:

63. 21 Lake, P., Clarke, E. A., Korshidi, M. and Sunshine, G. H. , Eur.

J . Immunol. 1979. 9: 875. 22 Dialynas, D . P., Wilde, D. B., Marrack, P., Pierres, A., Wall, K.

A., Havran, W., Otten, G., Loken, M. R., Pierres, M. and Fitch, F. W., Immunol. Rev. 1983. 74: 29.

23 Lamoyi, E . and Nisonoff, A , , J. Imrnunol. Methods 1983.56: 235. 24 Fazekas de St. Groth, B. , Thomas, W. R., McKimm-Breschkin, J.

L., Clark-Lewis, I . , Schrader, J . W. and Miller, J. F. A. P., Znr. Arch. Allergy Appl. Immunol. 1986. 79: 169.

25 Gronowicz, E., Coutinho, A. and Melchers, F., Eur. J . Immunol. 1976. 6: 588.

26 Riedel, C., Owens, T. and Nossal, G. J. V., Eur. J . Immunol. 1988. 18: 403.

27 Tedder, T. F., Schmidt, R. E., Rudd, C. E., Kornacki, M. M., Ritz, J. and Schlossman, S. F., Eur. J. Immunol. 1986. 16: 1539.

28 Sanders, V. M., Snyder, J. M., Uhr, J. W. and Vitetta, E. S. , J . Immunol. 1986. 137: 2395.

29 Ratcliffe, M. J . H. and Julius, M. H. J., Eur. J . lmmunol. 1982. 12: 634.

30 Zubler, R. H. and Kanagawa, O., J. Exp. Med. 1982. 156: 415. 31 DeFranco, A . L., Ashwell, J. D . , Schwartz, R. H. and Paul, W.

32 Tite, J . P., Kaye, J. and Jones, B., Eur. J . Imrnunol. 1984.14: 553. 33 Kupfer, A., Swain, S. L. and Singer, S. J., J . Exp. Med. 1987. 165:

1565. 34 Killar, L., MacDonald, G . , West, J . , Woods, A . and Bottomly,

K., J. Immunol. 1987. 138: 1674. 35 Zubler, R. H., Lowenthal, J . W., Erard, F. , Hashimoto, N.,

Devos, R. and MacDonald, H. R. , J. Exp. Men. 1984.160: 1170. 36 Mitchison, N. A. and Ratcliffe, M. J. H., in Hoffman, G. W.,

Nepom, G. and Levy, J. G. (Eds.), Paradoxes in Immunology, CRC Press, Boca Raton 1986, p. 163.

M. J. , J . lmmunol. 1985. 134: 3994.

2402.

1981. 153: 1124.

E., J . Exp. Med. 1984. 159: 861.

37 Mitchison, N. A., Eur. J . Immunol. 1971. I : 18.

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A noncognate interaction with anti-receptor antibody-activated helper T cells induces small resting murine B cells to proliferate and to secrete antibody