Human T-helper clones induce IgG production in a subclass-specific fashion

CELLULAR

IMMUNOL4lGY

139,306-3 17 ( 1992)

Human T-Helper Clones Induce IgG Production in a Subclass-Specific Fashion’ CHIARA DEMBECH,~SABELLA QUINTI,* EMANUELACIMIGNOLI,NICOLA ALBI, ADELMO TERENZI, ROBERTOGERLI, RICCIARDAGALANDRINI, FAUSTOGRIGNANI, AND ANDREA VELARDI~ Department of Medicine, University of Perugia, and *Division of Allergy and Clinical Immunology, University of Rome, Rome, Italy Received April 9, 1991; accepted August 7, 1991

The mechanisms governing the induction of IgG subclassesby T-helper cells in humans were investigated. As preliminary bulk-culture experiments had indicated that a direct B cell contact with viable T cells was an essential requirement for optimal IgG subclassproduction, 256 CD4+ human T cell clones were preactivated with PHA and cultured in direct contact with autologous B cells. These clones induced IgG production in a strikingly subclass-specificfashion. Moreover, the distribution of subclass-specifichelper clones was very similar to the IgG subclass profile observed in serum and peripheral lymphoid tissue plasma cells (IgGl = 60%, IgG2 = 30%, IgG3 = 5-lo%, IgG4 < 5%) and unlike that observed in resting B cells (which is IgGl o 40% and IgG2 sz 50%). It would, therefore, seem that a predominance of T cells capable of delivering IgG 1-specific, as opposed to IgGZ-specific, help is an essentialfactor for the preferential induction of IgGl antibodies during B cell proliferation and differentiation. There was no relationship between IL2, IL4, IL6, and IFN-7 secretedby the T-helper clones and their IgG subclassinduction patterns. In addition, only a few supematants were able to reproduce the helper effects of the clones themselves.Therefore, direct contact of B cells with helper clones is crucial for IgG-subclass production in humans. 0 1992 Academic PI~SS, IXIC.

INTRODUCTION As the relative proportions of the four human IgG subclassesis 60-65% IgGl ,2530% IgG2, 5-10% IgG3, and ~5% IgG4 in serum and lymphoid tissue plasma cells, but ~40% IgGl, -50% IgG2, ~8% IgG3, and ~5% IgG4 among IgG+ B cells, it would seem that regulatory control for IgGl and IgG2 subclass expression varies according to the stageof B cell differentiation (l-7). The pokeweed mitogen (PWM) model, which reproduces the IgG subclassprofile IgGl > IgG2 > IgG3 > IgG4, has shown that T cell regulatory influences are responsible for the preferential differentiation of B cells precommitted to the expression of IgGl , rather than IgG2, antibodies (5). However, when B cells are cultured with PWM-induced T cell factors, instead of viable ’ This researchwas supported by a grant from the Italian Association for Cancer Research (A.I.R.C.). * To whom correspondence should be addressedat Istituto di Clinica Medica I, Universim di Perugia, Policlinico Monteluce, 06 100, Perugia, Italy. 306 0008-8749192 $3.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

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T cells plus PWM, overall IgG plasma-cell production is markedly reduced and IgG2, not IgGl, is the predominant subclass (8). Soluble helper factors with IgG isotype specificity have been identified in the mouse: IFN-y produced by Th 1 cells induces B cells to secrete IgG2a, while IL4 produced by Th2 cells provokes the production of IgGl (9-12). However, neither anti-IFN-y nor anti-IL4 antibodies are able to completely block IgG secretion (12). These findings support the concept that regulatory signals other than secreted lymphokines, possibly related to direct T cell interaction with B cells, may be instrumental in the induction of IgG isotypes by T-helper cells. The aim of the present study was to investigate the mechanisms that govern Thelper cell induction of IgG isotypes in humans. The specific question addressedwas: why more IgG 1 than IgG2 antibodies are normally produced in serum and lymphoid tissue plasma cells in spite of the predominant numbers of IgG2-bearing precursor cells ? (l-7). As preliminary bulk-culture experiments confirmed that a direct B cell contact with viable T cells is an essential requirement for optimal IgG subclass production, a more direct experimental approach was sought by producing a large number of CD4+ clones and testing their ability to induce autologous B cells to produce IgG. It was found that T-helper clones induced IgG production in a strikingly subclassspecific fashion and that the distribution of the subclass-specifichelper clones was very similar to the IgG subclass profile observed in serum and lymphoid tissue plasma cells, and unlike that of resting B cells. It would, therefore, seem that the distribution of T cell clones is a major factor in inducing more IgGl than IgG2 antibodies in human serum, despite a predominance of IgG2-bearing precursor cells. We were unable to demonstrate any relationship between IL2, IL4, IL6, and IFNy secretion by T-helper clones and the IgG production pattern. Furthermore, addition of culture supernatants to purified B cells failed to reproduce the subclass-specific effects mediated by the clones. MATERIALS AND METHODS Preparation of B and T Cells PBMC were isolated by Ficoll-Hypaque density-gradient centrifugation and partially depleted of monocytes by plastic adherence. T cells were purified by E-rosetting and Ficoll-Hypaque. B cells were purified by removing E-rosette-forming cells by FicollHypaque, and then lysing the residual T cells in the E-negative fraction with antiCD3 (OKT3, Ortho, Raritan NJ) mAb plus complement. These B cell-enriched preparations contained 50-70% CD20+ B cells, 30-40% CD56+ NK cells, 5- 10% CD1 5+ monocytes, and ~0.2% CD3+ cells, as identified by indirect immunofluorescence with B 1 (Coulter), Leu 19, LeuM 1, and Leu4 (Becton-Dickinson) mAb, respectively. Cell Cultures To assessthe role of secretedlymphokines vs direct T cell-B cell interaction in IgG subclassproduction, purified B cells ( 1 X 106)were cultured with either PWM-induced T cell supernatants or various combinations of recombinant IL2, IL4, IL6, and IFNy in the presenceof PWM. For control cultures, 1 X lo6 T cells were added to B cells in the presence of PWM. PWM-induced T cell supematants, produced by culturing 1 X 106/ml T cells with PWM, were collected at the end of 48 hr of culture and added

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to B cells at a final concentration of 25%. Recombinant-IL2 (Glaxo Institute for Molecular Biology S.A., Geneva, Switzerland) was added to B cells at a concentration of 50 U/ml, rIL4 (Genzyme, Boston, MA) at 100-400 U/ml, rIL6 (JanssenBiochimica, Beerse,Belgium) at 50 U/ml and rIFN-y (Glaxo) at 500 U/ml. To explore the role of T cell membrane-associated molecules in B cell differentiation, paraformaldehydefixed (0.05% for 90 set) T cell blasts were added to PWM-stimulated B cells in the presence or absence of T cell supematants. In other experiments, equal numbers of PWM-activated B and T cells (1 X 106/ml) were cultured either together or separated by a porous membrane in a Transwell culture system (Costar, Cambridge, MA). Alternatively, cell-cell contact was blocked by adding a 1:10 dilution of anti-LFA 1 mAb hybridoma supematant (anti-CD 11a/CD 18, kindly donated by Dr. Alessandro Moretta, University of Genova, Italy) to PWM-stimulated B and T cells. At the end of a 7-day culture, supematants were collected for IgG subclass determination by ELISA and cells spun onto slides for the detection of IgG subclass-positive plasma cells by cytoplasmic immunofluorescence staining. T Cell Cloning PBMC that formed rosettes with sheep erythrocytes (E+ cells) were isolated from three normal subjects by Ficoll-Hypaque. After osmotic lysis of sheep erythrocytes with 0.83% ammonium chloride, E+ cells were depleted (>98%) of CD8+ cells by anti-CD8 (OKT8, Ortho, Raritan, NJ) monoclonal antibody treatment plus complement-mediated lysis. CD4+ cell-enriched preparations were plated 0.5 to 0.12 cells per well in groups of 48 to 96 microcultures in U-bottomed microwells containing lo5 irradiated (5000 rad) spleen feeder cells and activated with 0.5% v/v PHA (Difco Laboratories, Detroit, MI) ( 13).Recombinant IL2 (generously provided by the GLAXO Institute for Molecular Biology S.A., Geneva, Switzerland) was added at a final concentration of 25 U/ml after 24 hr. Plates were incubated at 37°C in a humidified atmosphere with 5% COZ. Microcultures were supplemented on Day 7 with lo5 irradiated feedercells and IL2. Further IL2 was added on Day 14. Control wells contained irradiated feeder cells, PHA and IL2 but no responder cells. After 14 to 18 days of culture, each microwell was assessedmicroscopically for cell growth. Indirect immunofluorescence analysis of the clones, with anti-Leu3 mAb (Becton-Dickinson, Mountain View, CA), confirmed that they were CD4+. Frequencies of proliferating cells were calculated by the x2 method from the Poisson distribution relationship between the number of cells plated per culture and the logarithm of the percentage of nonproliferating cultures (14). Cloning efficiency ranged from 65 to 83%. Cell Culture Systemfor Induction of IgG by CD4’ Clones To induce IgG production by CD4+ clones, we adopted a culture system originally designed to induce IgE production by human T cell clones ( 15). However, this system also allows the production of sizeable amounts of IgG. Following extensive washing to remove residual IL2, CD4+ T cell clones were activated with PHA ( 1% v/v). After 24 hr, clonal supematants were removed for subsequent analysis of secretedlymphokines and helper effect for IgG production (seebelow). Following an additional washing step, lo5 aliquots of the clones were cultured in triplicate in U-bottomed microtiter wells together with lo5 autologous B cells at a final volume of 200 ~1. In parallel

IgG SUBCLASS-SPECIFIC HELP BY HUMAN CJX+ CLONES

309

experiments, clonal supernatants were added to 1O5purified B cells at a final concentration of 25% in the presence of PWM. After 10 days, culture supematants were collected for analysis of IgG content by ELISA. Cytoplasmic Immunofuorescence Subclasses

for the Detection of Plasma Cells of the Four IgG

Distribution of IgG subclassesexpressedby plasma cells was determined on acetic acid ethanol-fixed cytocentrifuge preparations by two-color immunofluorescence ( 16). The mouse monoclonal antibodies specific for each one of the four human IgG subclasses,used for immunofluorescence, were JL-5 12 (Serotec, Birmingham, UK), SG11 and SL-16, anti-IgGl antibodies; GOM-1 and SH-22, anti-IgG2 antibodies; HP6050 and SJ-33, anti-IgG3 antibodies; and SK-44 and HP-6025, anti-IgG4 antibodies (ICN Biomedicals, Costa Mesa, CA) ( 17). Rhodamine-conjugated goat anti-mouse immunoglobulin antibodies, extensively adsorbed against human immunoglobulins (Southern Biotechnology Associates, Birmingham, Alabama) were employed as developing reagents for the mouse monoclonal antibodies. Total IgG+ plasma cells were identified by counter-staining the cytopreps with FITC-conjugated goat anti-human IgG (Southern Biotechnology Associates). The proportions of IgG’ cells which expressedeach of the four IgG subclasseswere determined by counting at least 200 IgG+ cells in each sample. ELISA for IgG Subclasses

Polystyrene microtitration plates with 96 flat-bottomed wells (Nunc Lab., Roskilde, Denmark) were coated with 100 ~1 of a predetermined concentration of goat antimouse IgG, Fc-specific, antibodies (Sigma Chemical Co., St. Louis, MO) in carbonate buffer 0.05 M, pH 9.6. After three washes with PBS-Tween 20 (0.05%), plates were coated with predetermined concentrations of monoclonal antibodies to human IgG subclasses.The anti-human IgG-subclass monoclonal antibodies used in this study were: JL5 12 (anti-IgGl), GOM- 1 (anti-IgG2), ZG4 (anti-IgG3), and RJ4 (anti-IgG4) (Oxoid) ( 17). After an additional washing step, plates were filled with PBS containing 0.1% gelatin and incubated at 37°C for 1 hr and washed again with PBS-Tween. One hundred microliters of undiluted and diluted l/5 and l/50 culture supematants were added to wells in duplicate. Each plate included the standard W.H.O. reference serum 67/97 (Janssen Biochimica, Beerse,Belgium) at six different concentrations for constructing a titration curve. Plates were incubated overnight at 37°C and washed again in PBS-Tween. One hundred microliters of alkaline-phosphatase-conjugated goat anti-human IgG (Zymed Laboratories, San Francisco, CA) diluted l/1000 were added to wells and incubated at 37°C for 1 hr. After three washeswith PBS-Tween and one with 0.1 A4 Tris-HCI, pH 8.6, bound conjugate was developed by adding 100 ~1 of paraphenylnitrophosphate (1 mg/ml) (Sigma) in Tris-HCl assubstrate.Optical densities were read on microplate reader (Titertek Multiskan, McLean, VA) after 15 and 30 min. 4ssays for IL2, IL4, IL6, and IFN-y Production

Clonal microcultures were washed several times and resuspended at a cell concentration of 1 X 106/ml in 200 ~1 culture medium containing 0.5% (v/v) PHA. Super-

DEMBECH ET AL.

310

natants were removed after 24 hr and used at a final concentration of 25% for determination of lymphokine activity. The IL2-dependent mouse CTLL cell line was used as an indicator system for determination of IL2 activity (18). Clonal supernatants were added to 5 X lo3 indicator cells in a volume of 200 ~1.IL2 activity was assessed by [3H]thymidine uptake by CTLL cells after 6-hr pulsing at the end of a 24-hr culture period. IL4 was measuredwith an enzyme-linked immunosorbent assaykit, “InterTest4” (Genzyme, Boston, MA). IL6 we measured as hybridoma growth factor activity on the IL6-dependent mouse-mouse hybridoma cell line, 7TDl (kindly donated by Dr. J. Van Snick, Ludwig Institute for Cancer Research, Brussels Branch, Belgium) ( 19). Two-thousand 7TD 1 cells were incubated with the clonal supernatants in a 200~1 volume. Cell numbers were determined by a calorimetric assay at the end of a 5day culture period. IFN-y was measured by a cytopathic effect reduction assay,using vescicular stomatitis virus and FLAM cells, as indicator system (20). Calculation of the lymphokine activities was based on standard sources of recombinant IL2 and IFN--y (GLAXO Institute for Molecular Biology S.A., Geneva, Switzerland), IL4 (Genzyme, Boston, MA), and IL6 (JanssenBiochimica, Beerse,Belgium). RESULTS

Role of SecretedLymphokines vs Direct T Cell-B Cell Interaction in IgG Subclass Production The addition of T cell supernatants or recombinant IL2, IL4, IL6, and IFN-y (alone and in combination) to PWM-stimulated B cells induced poor IgG secretion (Table 1). Some increase in IgG production was observed when fixed T cell blasts were added to PWM-stimulated B cells and further enhanced by the simultaneous addition of T TABLE 1 IgG SubclassProduction in PWM-Stimulated B Cell Cultures Additions to PWMstimulated B cell cultures None T cell supematant IL2, IL4, IL6, IFN-yd Fixed T cell blasts Fixed T cell blasts plus T cell supematant Viable T cells Viable T cells plus anti-LFA 1 mAb’

% of IgG plasma cells expressing* Total IgG rig/ml” 8Ok

20'

100 f 120 f

25 27

24Ok

38

IgGl 35k 25+ 32k 22+

IgG2 4 5 4 3

65 + 15 f 6lk 76 f

7 8 6 5

IgG3
ks4


320t 42 2600 rt 200

35+ 4 81+ 12

61+ 9



11+3

7+5

3+2


ND

ND

ND

ND

L?As determined by ELISA. b As determined by two-color immunofluorescence. c Mean values +- SE obtained in three independent experiments. dRecombinant cytokines were added alone and in combination. For the sake of simplicity, only the combinations are shown. ’ 1:10 dilution of hybridoma supematant.

IgG SUBCLASS-SPECIFIC HELP BY HUMAN CD4+ CLONES

311

cell supematants. However, IgGl never predominated physiologically and IgG2 was always the main subclass. Finally, IgG production was again poor and there was a relative predominance of IgG2 plasma cells when PWM-activated T and B cells were cultured separated by a porous membrane in a Transwell culture system (Fig. 1). Moreover, it became virtually undetectable when cell adhesion was prevented by the addition of anti-LFA 1 (anti-CD 11a/CD 18) mAb to PWM-activated mixed B and T cell cultures (Table 1). These results suggest that direct T cell-B cell interaction is essential for optimal induction of IgG. Human T-Helper Clones Induce Production of IgG in a Subclass-Specijic Fashion

The mechanisms governing IgG isotype production were, then, investigated more directly by generating 256 CD4+ T cell clones and, following activation with PHA, coculturing them with autologous B cells. Spontaneous IgG production by purified B cells was ~50 rig/ml. As expected, the total IgG induced by individual clones was highly variable, ranging from ~50 to 10,000 rig/ml. A further striking heterogeneity became apparent when the IgG subclasscontent of the clonal supematant was evaluated. No clonal culture induced all four IgG subclasses.Instead, there was a clearcut subclass-specifichelper effect with selective induction of one or two subclassesand negligible production of the others. Analysis of 128 clones that induced significant IgG production (>600 rig/ml for IgGl, IgG2, and IgG3 subclassesand >300 rig/ml IgG4) demonstrated that 73 clones predominantly induced IgG 1, 2 1 IgG2, and 2 1 both IgG 1 and IgG2 (Fig. 2). Fewer clones induced IgG3, IgG4, or combinations of IgG2 and IgG3 or IgGl and IgG3 (Fig. 3). When random isotype-specific clones were retestedin subsequentexperiments, their helper effectswere reproducibly obtained (not shown). Therefore, single human T-helper cells induce IgG production in a subclass-specificmanner. In addition, since IgGl-specific helper clones were 62%, IgG2 29%, IgG3 6%, and IgG4 2%, there was a rig/ml

6

A

3,000

1,500

IgGl

lgG2

lgG3

lgG4

IgGl

IgGP

lgG3

lgG4

FIG. 1. Patterns of IgG subclassesproduced by PWM-stimulated B and T cells either cultured in direct contact (A) or separated by a porous membrane in a Transwell (Costar) culture system (B). When B and T cells were separated,minimal IgG secretion and a relative predominance of IgG2 plasma cells were observed. However, culturing B and T cells in direct contact promoted optimal differentiation with a physiologic (IgGl > IgG2) subclassdistribution. Total IgG secretedin culture were measuredby ELISA and the subclass distribution among IgG+ plasma cells produced in culture was determined by two-color immunofluorescence (mean + SE of three independent experiments).

312

DEMBECH ET AL. CLONES IHIUCING

PREDOMNANTLY

1000

I#01

IgO4

1003

I202

C

CLOI(S

IgG 1

lgG2

lgG3

lgG4

INDUCING PREDOMINANTLY bG2 (n : 211128)

ngfml

2000,

IgG 1

lgG2

IgO3

l2G4

FIG. 2. Human T-helper cells, analyzed at the single precursor cell level, induce IgG production in a subclass-specificfashion. Two-hundred and fifty-six CD4* T cell clones were derived from three normal donors. Following activation with PHA, clones were cocultured with autologous B cells for 10 days. Onehundred and twenty-eight clones exhibiting significant helper function for IgG production were selectedfor further ELISA analysis of the IgG subclasses.Shown are the three most frequent subclassprofiles induced by the clones. The data are given as mean rig/ml + SE of IgG subclassesinduced by each clone. The majority of the clones (73/128) induced a predominant IgGl production (A), 21 gave a preferential help for IgGl and IgG2 together (B) and 2 1 clones induced IgG2 (C).

near perfect concordance between their relative distribution and the subclass distribution pattern observedin serum and lymphoid tissuesplasma cells. It would, therefore, seemthat the contrasting IgG subclassdistribution patterns for B cells (IgG2 > IgGl) and serum or plasma cells (IgGl > IgG2) are a direct consequence of the relative predominance of IgG 1-specific over IgG2-specific helper clones. Lymphokine Activities and Helper Efects in Supernatantsfrom IgG Subclass-SpeciJic Helper Clones The lymphokine production evoked by CD4+ IgG subclass-specifichelper clones as well as by CD4’ clones which did not induce significant IgG are shown in Table 2. There were no differencesin the lymphokine patterns of high and low IgG-producing clones, nor among IgG subclass-specifichelper clones. The IgG subclass profiles induced by adding supernatants from subclass-specific clones to B cells in the presenceof PWM are shown and compared with those induced by the helper clones themselves in Table 3. Although some clonal supernatants provoked the production of IgG subclass profiles similar to those induced by their corresponding clones, the majority were unable to support IgG production. Again, there

313

IgG SUBCLASS-SPECIFIC HELP BY HUMAN CD4+ CLONES

A

CLOHS

CLONES INDUClNG PREDOMNANTLY lgG4 (n = 2/128)

B

INDUCING PREDOMINANTLY JgC3 (n : 3/128)

nglml

rig/ml 1000 ,

1000

5;im~ k _pa soar IgG 1

C

lgG2

lgG3

lgG4

D

CLONES INDUCING PREDOMlNANTLY IgG2 + lgG3 (n = 3/128)

rig/ml

_Gn IgG2

IgGi

lgG2

lgG3

IgG4

CLONES INDUCING PREDOMINANTLY IgGl + lgG3 (n = 2/128)

rig/ml 1500

I

IgG 1

IgGl

750

I

lgG4

0

~

fii

lgG2

lgG3

__

/jjiz:;:y IgGl

lgG4

FIG. 3. Human T-helper cells, analyzed at the single precursor cell level, induce IgG production in a subclass-specificfashion (continued). The experimental design is the same as in Fig. 2. Shown here are less frequent IgG subclass patterns induced by each clone (mean f SE). Three clones induced IgG3 (A) and three clones IgG4 (B). in addition, three and two clones, respectively, induced combinations of IgG2 and IgG3 (C), or IgCl and IgG3 (D).

were no significant differences in the lymphokine production patterns of supematants which supported IgG production and those which did not (not shown). DISCUSSION The aim of this study was to gain an insight into the mechanisms that govern the production of the four IgG subclassesin man, with the ultimate goal of acquiring a better understanding of the pathogenesisof both congenital and acquired IgG immune defects (2 1,22). We wished to know why, in spite of the slight predominance of IgG2 over IgG 1 bearing precursor cells, more IgG 1 than IgG2 antibodies are normally produced ( l-7). This problem was initially approached by carrying out a set of experiments with PWM-stimulated purified B cells as a model system. When PWM is added to mixed B and T cells it mainly induces IgGl antibodies and an overall IgG subclass profile identical to that observed in vivo, i.e., IgGl > IgG2 > IgG3 > IgG4 (5). The addition of recombinant lymphokines (IL2, IL4, IL6, and IFN-7, alone or in combination) involved in B cell proliferation and differentiation (23) to purified B cells failed to induce significant IgG secretion, and the predominant subclass was always IgG2. Although the addition of fixed T cell blasts plus T cell-derived supematants to purified B cells provoked a slight increase in IgG secretion, IgG2 remained the major subclass. IgG secretion was again low and IgG2 was the most represented subclass when PWM-activated B and T cells were separated by a porous membrane in the

DEMBECH ET AL.

314

TABLE 2 Lymphokine Production by Human CD4+ Clones” IL2

IL4

Clones inducing

IL6

IF-N-y

(mean U/ml + SD)b

IgGl (73/256) IgG2 (2 l/256) IgGl + IgG2 (21/256) IgG3 (3/256) IgG4 (3/256) IgGl + IgG3 (2/256) IgG2 + IgG3 (3/256)

23 IL 36 14 +- 20 14 +- 20 II+- 8 18 +20 24 + 28 lo? 4

7+ 5+ 4+ 6& 5+ 12? 6+

12 14 4 2 4 6 4

11 f 16 10a 5 16 f 21 16a 4 16 f 12 14* 0 ilk 5

39 f 39 40 + 42 51 f42 40 + 35 30f 14 20+ 0 56 z?80

Clones not inducing significant IgG product (128/256)

16?23

4+

5

13* 10

48 z!c39

a Clonal microcultures were stimulated with PHA. Supernatants were removed after 24 hr. Lymphokine assayswere performed as indicated under Materials and Methods. Evaluation of the lymphokine activities (U/ml) in the clonal supematantswas basedon standard sourcesof recombinant lymphokines which contained known amounts of international standard units. b Statistical analysis by a Student’s t test for independent samples revealed no significant differences in lymphokine production between the sets of clones.

Transwell culture system. Finally, almost no IgG production was detected when cellcell contact was inhibited by adding an anti-LFA 1 (anti-CD I 1a/CD 18) mAb to PWMstimulated mixed B and T cell cultures. These results favor a direct cell-cell interaction TABLE 3 Helper Effects of T Cell Clones and Their Supematants on IgG-SubclassProduction” T Cell clones + B cells IgGl

IgG2

IgG3

Clonal supernatants + B cells IgG4

IgG 1

WW IgG 1-specific IgG2-specific IgG3-specific

3500 2600 5600 <80 180 180 160 250 200

100 320 300 4000 3500 2000 130 300 50

IgG2

IgG3

IgG4

(Wml) 60 35 50 35 50 <5 1000 3000 2000

<5 10 <5 <5 <5 10 <5 <5 10

7300 <90 <90 <90 500 <90 300 <90 100

190 <50 230 <50 <50 90 60 300 60

<5 120 220 t5 c5 170 <6 2000 10

70 <5 170 <5 <5 12 120 70 50

n Supematants from 128 IgG subclass-specificclones were added to purified B cells in the presence of PWM. The IgG subclassesproduced in culture were evaluated by ELISA at the end of a IO-day period. Shown are the helper effectsof a representative set of subclass-specifichelper clones and their supematants (w/ml).

IgG SUBCLASS-SPECIFIC HELP BY HUMAN CD4+ CLONES

315

in the induction of IgG subclassesin the PWM model system (5, 8) and indicate that isolated B cells are not a suitable model for analyzing the mechanisms that control IgG isotype production in humans. For this reason, we established a large number of CD4+ clones and, following preactivation with PHA, cultured them in direct contact with autologous B cells. The objective was to ascertain whether T cells deliver their helper effect for IgG production in an isotype-specific manner, a question that so far has only been addressedin the mouse system, where cloned interferon-y-producing Thl and IL4-producing Th2 cells induce B cells to secreteIgG2a and IgG 1, respectively ( 12). Our data indicate that human IgG production is the consequenceof a combination of strikingly specific, isotype-restricted helper effectsdelivered by individual T-helper cells. CD4+ clones induced a single IgG subclassor a combination of two subclasses, and negligible production of the others. In addition, the relative distribution of the subclass-specifichelper effects paralleled the subclassdistribution of serum and lymphoid tissue plasma cells (IgGl N 60%, IgG2 x 30%, IgG3 = 5-lo%, IgG4 < 5%). Since the B cell IgG subclassdistribution is ~40% IgGl vs ~50% IgG2, the relative predominance of clonable T cells capable of delivering IgGl-specific, as opposed to IgG2-specific, helper effectswould seemto be of prime importance in the preferential induction of IgGl antibodies during B cell proliferation and differentiation. The attempts to elucidate the mechanisms whereby T cell clones deliver their isotypespecific help for IgG-subclasseswere unsuccessful.When clones were grouped according to their isotype specificity patterns, there were no significant differencesin the amounts of IL2, IL4, IL6, or IFN-y the various sets of clones produced. Supernatants from some of the clones mimicked the effect of adding the clones themselves, but other supematants exhibited little or no T cell replacing activity. In addition, there were no differences in the lymphokine activities of supernatants that supported helper function for IgG production and those that did not. In attempting to reconcile these findings, it should be noted that studies on the expression of mRNAs for IL2, IL4, and EN-7 by CD4+ clones have also failed to identify functionally distinct human CD4+ T cell subsets comparable to the mouse Th 1 and Th2 helper T cells (24). In addition, not only secretedlymphokines, but also membrane-associatedmolecules from activated T cells, have been reported to promote B cell proliferation (25). T-helper cells have been demonstrated to releasetheir lymphokines in a polar fashion directed at that portion of the B cell membrane where T cell receptor cross-linking occurs, i.e., at the site of Th cell-B cell contact (26). These phenomena may explain the somewhat surprisingly limited information obtained from our lymphokine determinations in clonal supernatants and the inconsistent helper effects mediated by the supematants. Our study did not addressthe question of whether T cell clones instruct B cells to switch from IgM to IgG synthesis (27-29) or select IgG isotype-precommitted B cells and preferentially enhance their differentiation into plasma cells (30-32). However, in PWM-activated human blood cells, the preferential stimulation of IgGl plasmacell production appears to be due to isotype-specific help acting on B cells that have been preactivated in viva, rather than T cell-directed switching (5). This mechanism also seemsto have been operative in the present experiments where PHA-activated T cell clones were cocultured with freshly isolated autologous B cells in the absence of any specific stimulus directed at the B cell.

316

DEMBECH

ET AL.

This study provides evidence for IgG subclass-specifichelp by CD4+ T cell clones in humans. However, neither the IL2, IL4, IL6, and IFN-7 production in these clones nor the T cell replacing activities of their supematants correlated with the subclassspecific helper effectsmediated by the clones themselves. We, therefore, conclude that direct contact of B cells with helper clones is crucial for IgG production in humans and that the triggering for isotype commitment is delivered by short-range soluble mediators and direct cell-cell contact. A strict requirement for a direct T-B cell contact for optimal Ig production by lectin-stimulated bulk-cultures of B and T cells has also been convincingly demonstrated by other groups of investigators (8,33,34). Our findings conhrm this observation and extend it to PHA-stimulated T cell clones cultured with B cells. However, this concept is contradicted by data which indicate that, even in the absence of T cells, recombinant cytokines such as IL2 and IL4 are able to induce striking Ig production by SAC-activated B cells (35). In attempting to reconcile these findings it should be noted that lectins, such as PWM, have no direct activity on B cells and act by triggering T-helper cells to exert their helper function on a small subpopulation of large IgD- B cells that have been physiologically preactivated by antigen in vivo (reviewed in Refs. (5) and (8)). In contrast, SAC directly activates resting B cells in vitro (36). Therefore, it would seem that in vivo antigen-activated B cells depend on costimulatory signals delivered by direct contact with &tin-activated T-helper cells in order to respond to T-helper cellderived cytokines for terminal plasma-cell differentiation. On the other hand, SAC would seemto be able to drive B cells a step further and make them directly susceptible to the action of T cell-derived cytokines, without the need for the costimulatory signals delivered by direct contact with the T cell membrane. ACKNOWLEDGMENTS We thank Drs. Sergio Romagnani and Lorenzo Moretta for suggestionsand Dr. Carlo E. Grossi for suggestionsand for critical review of the manuscript.

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