Clin. exp. Immunol. (1987) 70, 649-657

Inhibition of T cell-dependent human B cell proliferationand B cell differentiation by polyspecific monomeric IgG

W. STOHL & L. MAYER* Rockefeller University, New York, and * Mount Sinai School ofMedicine, New York, USA

(Acceptedfor publication 13 July 1987)

SUMMARY

A commercially available polyspecific, monomeric IgG preparation suitable for intrave-nous administration (IgSRK; Sandoglobulin) can inhibit pokeweed mitogen (PWM)-induced proliferation of peripheral blood mononuclear cells (PBMC) by a small, butstatistically significant, amount compared to control cultures. Such inhibition could notbe demonstrated when PBMC were stimulated with the T cell mitogen phytohaemaggluti-nin. Surface phenotype analysis of the PWM-stimulated cells indicated that in IgSRK-containing cultures, the proportion of B cells was decreased and the proportion ofT cellswas increased compared to control cultures. This alteration in T: B ratio was not due toantigenic modulation of B or T cell markers from their surfaces. In addition, IgSRKinhibited the proliferation of T cell-depleted PBMC cultures stimulated by B cellproliferation factors (BCPF) but not by fixed protein A-bearing Staphyococcus aureusstrain Cowan I. The capacity to inhibit B cell proliferation was independent ofand distinctfrom its capacity to inhibit B cell differentiation, since IgSRK inhibited the differentiationof a B cell differentiation factor (BCDF)-sensitive line by BCDF (which contains noBCPF activity). IgSRK inhibited PWM-induced generation of cytoplasmic Ig+ cells buthad no effect on Ig secretion from mature Ig-secreting cells. Taken together, these findingssuggest that IgSRK (which contains the IgG fraction from pooled plasma from 2,000healthy donors) can inhibit T cell-dependent or T cell factor-dependent B cellproliferation and B cell differentiation.

Keywords monomeric IgG B cell proliferation B cell differentiation

INTRODUCTION

In addition to specific antibody production in response to a given antigen, there is a polyclonal,antigen-non-specific immunoglobulin (Ig) response in man to naturally-occurring polyclonalactivators, such as Epstein-Barr virus (EBV) (Rosen et al., 1977), Staphylococcus aureus strainCowan I (SAC) (Saiki & Ralph, 1981), and Gram-negative bacteria (Chen et al., 1981; Gross et al.,1983). Immune complexes and aggregated Ig have both been shown to inhibit in vitro human B celldifferentiation induced by various antigen-non-specific stimuli (Moretta et al., 1977; Le Thi Bich-Thuy& Brochier, 1979). Furthermore, we have recently demonstrated that polyspecific, monomericIgG suitable for intravenous administration can inhibit pokeweed mitogen (PWM)-inducedgeneration of Ig-secreting cells (as measured by plaque-forming cells (PFC)) in a dose-dependentfashion (Stohl, 1985). Such an inhibitory effect could also be demonstrated with isolated monomericIg from single normal donors and donors autochthonous to the cells being stimulated in culture

Correspondence: Dr William Stohl, Clinical Immunology and Rheumatic Diseases Section, University ofSouthern California, 2025 Zonal Ave. HMR 711, Los Angeles, CA 90033, USA.

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(Stohl et al., 1986). The target of inhibition appeared to be the B cell itself with only minor, if any,roles in this system for T cells, monocytes, or Leu-l 1 + cells (Stohl, 1986).

The mechanism by which Ig inhibits B cells was not elucidated in the above studies. A netdecrement in PFC would be realized by any of three mechanisms: (1) inhibition of the expansion(proliferation) of the PFC precursors; (2) inhibition of B cell maturation into cytoplasmic Ig (clg)-containing cells; (3) inhibition of secretion of intracellular Ig by the cIg+ cells. Evidence is presentedin this report that polyspecific, monomeric IgG has an inhibitory effect both on T cell-dependent orT cell factor-dependent B cell proliferation and maturation to the cIg+ state, but it has noappreciable inhibitory effect on Ig secretion from mature B cells.

MATERIALS AND METHODS

Cells. Heparinized venous blood was collected from normal volunteers or patients meeting theAmerican Rheumatism criteria for systemic lupus erythematosus (SLE) (Tan et al., 1982).Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll-Hypaque density gradientcentrifugation (Pharmacia Fine Chemicals, Piscataway, NJ). In some experiments, the non-T cellswere isolated by rosetting with neuraminidase-treated sheep erythrocytes as described (Hoffmann&Kunkel, 1976) and harvesting the non-rosetted fraction. The B lymphoblastoid line 8866P whichsecretes IgG (originally obtained from Dr Hans Spiegelberg, Scripps Clinic, La Jolla, CA) has beenmaintained in culture in this laboratory and was used as a source of differentiated Ig-secreting cells.

Reagents. Ig for intravenous use (IgSRK; Sandoglobulin) was generously supplied by Sandoz,East Hanover, NJ. This lyophilized product was reconstituted according to the supplier'sspecifications resulting in an Ig stock solution of 30 mg/ml in a 5% sucrose-normal saline vehicle.This preparation consists of >90% monomeric IgG with <4% dimers, <0 3% aggregates, andtraces of IgA, IgM, and Ig fragments. The distribution of IgG subclass corresponds to that found innormal serum (Romer et al., 1982). 'Monomeric IgSRK' was prepared by ultracentrifugation of thestock IgSRK solution at 45,000 rev/min for 120 min. Only the top half of the supernatant washarvested. 'Aggregated IgSRK' was prepared by heating the 'monomeric IgSRK' at 63°C for 15minutes until the sample just began to turn opalescent. Human serum albumin (HSA)(Worthington Biochemical Corp., Freehold, NJ) was stored at a stock solution of 30 mg/ml in thesame sucrose-saline vehicle. Monoclonal antibodies OKT4A, OKT8, and OKT 1I were purchasedfrom Ortho Diagnostic Systems, Raritan, NJ. Anti-Leu-l lb was purchased from Becton-Dickinson, Mountain View, CA. B1, B2, and B4 were purchased from Coulter Immunology,Hialeah, FL. Anti-T3 was produced in our laboratory as culture supernatant from the OKT3 cellline obtained from American Type Culture Collection, Rockville, MD. Fixed protein A-bearingSAC (Pansorbin) was purchased from Calbiochem, La Jolla, CA.

Cytofluorographic analysis. Surface antigens were detected by indirect immunofluorescence andanalysed on a Cytofluorograf 30-H (Ortho Instruments, Westwood, MA) as previously described(Stohl & Kunkel, 1984). Cells were first stained with the appropriate mouse monoclonal antibodyand then labelled with fluorescein isothiocyanate-conjugated F(ab')2 sheep anti-mouse Ig (CappelLaboratories, Cochranville, PA). The electronic gate was set to include only lymphocytes, and 5,000cells were counted per sample.

Assessment of lymphocyte proliferation. PBMC were cultured in 96-well flat-bottomedmicrotitre plates (Linbro, Flow Laboratories, McLean, VA) at 1 x 105 cells/0-2 ml/well in a 5% CO2humidified 37°C incubator. Culture medium consisted of RPMI 1640 medium (Flow Laboratories)supplemented with 10% fetal bovine serum (Hazelton Dutchland, Denver, PA), 2 mm glutamine(Gibco, Grand Island, NY), and 100 U/ml penicillin/streptomycin (Gibco). To control for thesucrose present in the IgSRK and HSA preparations, an equal volume and concentration of sucrosewas added to the medium controls to maintain total volume and total sucrose content constant.PWM (Gibco) at 2 yg/ml final concentration and phytohaemagglutinin (PHA) (Gibco) at 10 yg/mlfinal concentration were added as indicated. In some experiments, 2 x 105 non-T cells were culturedas above with supernatants from T-T hybridomas demonstrating B cell proliferation factor (BCPF)activity (Mayer& Kunkel, 1984) at the indicated concentrations. Eighteen hours before terminating

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IgG-mediated inhibition of B cells

the cultures, 2 1iCi 3H-thymidine (Schwartz-Mann, Orangeburg, NY; specific activity 19 Ci/mmole) were added to each well. The cultures were harvested on to glass fibre filters with anautomated cell harvester, and ct/mmn were measured by liquid scintillation. Results are expressed as

the mean of triplicate cultures, and standard deviations were generally <20% of the mean.Inhibition was calculated by the formula

((ct/mmn control -ct/min expenmentai)/ct/min control) X1000-Assessment of B cell differentiation. PBMC were cultured in 24-well plates (Linbro) at1 x 106

cells/ml/well in the presence ofPWM as above and harvested at day 6. Cells were fixed and analysedby immunofluorescence forcIg using a rhodamine-conjugated rabbit anti-human total Ig(IgG + IgA +IgM) antiserum (Cappel)(Fu et al., 1974). At least 200 cells per sample were counted.Inhibition was calculated by the formula

((cg + control -c1g + experimental)/cIg+ control) X10000.In some experiments, cultures were assayed for PFC by the reverse haemolytic plaque assay

using protein A-coupled sheep erythrocytes (Gronowicz etal., 1976). Total PFC were detectedusing an unconjugated rabbit anti-human total Ig (Cappel). In other experiments, supernatantsfrom human T-T hybridomas (MOW, MOP, MTP) demonstrating B cell differentiation factor(BCDF) activity (Mayer et al., 1984) were added (0.5% final concentration) to 104 cells/0-1 ml/wellin triplicate from the BCDF-responsive EBV-transformed line, CN (Sherris & Mayer, inpreparation), in flat-bottomed microtitre plates. On day 3, the cultures were assayed for PFC asabove. Results are expressed as the mean of duplicate determinations. Inhibition was calculated bythe formula

((PFC control - PFC experimental)/PFC control) X100%.Parallel cultures were labelled with 3H-thymidine and processed for scintillation counting as above.

Statistical analysis. The Student t test for paired data was used. P values <0-05 were consideredsignificant.

RESULTS

Inhibition ofB cell proliferation by IgSRK. It has previously been demonstrated that IgSRK at aconcentration of 300 pg/ml could inhibit the generation of PFC in PWM-stimulated PBMCcultures by a mean 76% (Stohl, 1985). To determine whether the mechanism of this inhibition wasdue to inhibition of proliferation (Jelinek & Lipsky, 1983), PBMC from11 normal donors werestimulated with PWM in the presence of IgSRK or control additives (Table 1). Althoughstatistically significant (P <0-02), IgSRK induced only a mean 17% inhibition in proliferation asassessed by3H-thymidine uptake, and <20% inhibition was observed in six of theI1 donors tested.HSA had no net effect on proliferation (mean 3% inhibition; P not significant). Similar results wereobtained by counting the numbers of viable cells recovered from culture (data not shown). In eightseparate dose-response experiments, IgSRK inhibited proliferation a mean 18% at 300 pg/ml(P=0-06) and a mean 36% at 1000 Mg/ml (P<0-01). Thus, even at a dose 3-3-fold greater than thatpreviously demonstrated to inhibit PFC generation by 76% (Stohl, 1985), proliferation could stillbe inhibited only by 36%. Of importance is that comparable inhibitions were observed with the'monomeric IgSRK' and 'aggregated IgSRK' preparations (data not shown), suggesting that theobserved inhibition was not due to contaminating complexes of aggregated Ig.

This modest inhibition of PWM-induced proliferation could not be duplicated on PHA-stimulated PBMC. IgSRK had no effect on PHA-induced proliferation at any time point tested inany of three donors tested (data not shown).

A possible explanation for these findings was that the IgSRK-mediated inhibitory effects onproliferation were directed preferentially against the B cell population (which accounts for only asmall portion of the PWM-induced response and essentially none of the PHA-induced response).

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Table 1. Effect of IgSRK on PWM-induced proliferation*

Donor Medium IgSRK HSA

R.B. 61,144 65,677 (-7) 68,274(- 12)S.K. 79,375 68,105 (14) 65,896 (17)D.B. 45,413 31,962 (30) 42,132 (7)J.J. 16,785 15,888 (5) 15,881 (5)I.C. 33,468 39,954(-19) 34,388 (-3)E.R. 42,879 35,126 (18) 34,004 (21)B.R. 42,295 36,559 (14) 51,917 (-23)L.V. 25,694 16,114 (37) 20,554 (20)R.F. 27,493 9,279 (66) 35,410(-29)B.S. 24,228 17,835 (26) 22,397 (8)P.P. 48,756 35,673 (26) 44,716 (8)

Mean 40,685 33,834 (17) 39,597 (3)

* PBMC were cultured with PWM and IgSRK(300 pg/ml) or HSA (300 pg/ml) or control mediumfor 6 days. 3H-Thymidine (2 pCi) were added to thecultures during their final 18 hours of incubation.Cells were harvested on to glass filter papers, andct/min were determined by liquid scintillation count-ing. Data are expressed as ct/min. Numbers inparentheses indicate inhibition compared to themedium control. A negative value indicates augmen-tation.

t P<002.t P not significant.

PBMC from five donors were stimulated with PWM in the presence of IgSRK or control additivesand analysed for surface markers as a function of time in culture. Day 2 was chosen as one time

point since the inhibitory effects of IgSRK on PWM-induced B cell differentiation are complete by48 h (Stohl, 1985). Day 6 was chosen as the other time point tested since PWM-inducedproliferation was routinely assessed on day 6 of culture. The percentages of cells bearing markersspecific for B cells (BI, B2, B4) were significantly reduced in those cultures containing IgSRKcompared to the medium control both at day 2 and day 6 of culture (Table 2). This was not due to

modulation ofthe B 1, B2, or B4 antigens from the B cell surfaces (data not shown). No reductions in

numbers ofB cells occurred in the cultures containing HSA. The decrease in numbers of BI +, B2 +,and B4+ cells by day 6 in all cultures (including controls) compared to days 0 or 2 has previouslybeen noted and corresponds to concomitant loss of these surface markers with terminal B cell

differentiation (Stashenko et al., 1981; Nadler et al., 1983). In addition, IgSRK also had a smallerbut significant inhibitory effect on the percentages of Leu- 1I + cells. By contrast, the percentages of

cells bearing markers present on all T cells (TI 1, T3) were increased in the IgSRK-containingcultures compared to the control cultures. Thus, the small inhibitory effect of IgSRK on PWM-

driven PBMC proliferation (Table 1) seemed to be due to a preferential inhibitory effect on B cell

proliferation.To directly address this point, IgSRK or control additives were added to T cell-depleted cultures

which were stimulated with two different BCPF (Mayer & Kunkel, 1984). IgSRK inhibited BCPF-induced proliferation by 46-90% (Table 3). In contrast, HSA had little inhibitory or enhancingeffect. This pattern of inhibition was reproduced in additional experiments (data not shown).

The above observations cannot be explained by a toxic effect of IgSRK on B cells in that the T

cell-independent SAC-induced proliferation ofT cell-depleted cultures was not inhibited by IgSRK(300 pug/ml) in three separate experiments (mean ct/min with IgSRK, 17,508; mean ct/min without

652

IgG-mediated inhibition of B cells 653Table 2. Effect of IgSRK on cell surface markers*

Day 2 Day 6

SurfaceMarker Day 0 Medium IgSRK HSA Medium IgSRK HSA

TII 80 9+8-8 81 1 +±72 84 5+6 8t 80 7+7 6 91 4+±77 93-9+4 7 88 6+6-6T3 71 6+5 3 70 1 +6 0 75 4i7-5+ 71-2+6-1 87-6+6 5 91-8+442+ 88 4+3 9T4 48-1+57 505+7 1 527+84§ 502+84 497+158 523+199 498+19-8T8 204+85 19 1+58 194+72 193+5-9 33-1+166 346+193 350+19-1

Leu-1 12 6+±36 11 4+5 0 9 8+4 7t 113±+4 9 6-1 +4 5 4±1+2 7l1 5 4+±34

Bi 63+38 9-1+47 54+3-5t 89+43 37+±31 15+l15§ 3-7+24B2 132+67 97+43 58+3-0 97+39 3-6+ 2-9 17±+2-0+ 34+24B4 140+60 11-3+52 65+39t 110+5 1 48+35 17+1 5§ 43+22

* PBMC from five normal donors were immunostained by indirect immunofluorescence with theappropriate monoclonal antibodies after 0, 2, or 6 days in culture with PWM and IgSRK (300 pg/ml) or HSA(300 pg/ml) or control medium. Cells were analysed cytofluorographically including only lymphocytes withinthe electronic gate. Data are presented as percentages of positively stained cells + I s.d.

t P < 0 005 compared to medium control.+ P< 0 02.§ P<005.P=0 076.P<0 0l.

Table 3. Effect of IgSRK on BCPF-induced proliferation*

BCPF medium IgSRK HSA

BCPF1 30% 19,722 1,961 (90) 17,059 (14)30o 12,956 2,133 (84) 14,204(- 10)0. 30o 14,209 1,548 (89) 12,446 (12)

BCPF2 300° 15,306 3,435 (78) 13,649 (11)3%), 9,020 4,657 (48) 9,398 (-4)0. 30 10,826 5,816 (46) 10,295 (5)

Medium 1,221 1,517 (-24) 1,548 (-27)

* Peripheral non-T cells were cultured in triplicatewith the indicated concentrations (v/v) of the different Bcell proliferation factors (BCPF) in the presence ofIgSRK (300 pg/ml) or HSA (300 pg/ml) or controlmedium. 3H-thymidine was added to each well 18 h beforeharvesting the culture on day 4.

Numbers in parentheses indicate percentage inhibi-tion compared to medium control.

IgSRK, 14,476). Taken together, IgSRK appears to be inhibitory to T cell- or T cell factor-dependent B cell proliferation.

Inhibition ofB cell maturation bya IgSRK. The possibility remained that the previously describedinhibition of PWM-induced PFC generation by IgSRK was not due to any effect on B cellmaturation per se, but rather was due to an inhibition in the expansion of the PFC precursors. Todirectly assess the capacity of IgSRK to influence B cell differentiation, 104 cells from the BCDF-responsive EBV-transformed line, CN, were incubated with three distinct BCDF (Mayer et al.,

1984) in the presence of IgSRK (300 ,ug/ml) or controls. As seen in Table 4, the BCDF augmented by6-fold to 10-fold the PFC generation by the EBV-transformed line. In the presence of IgSRK, thisaugmentation was severely inhibited with the resulting PFC numbers being 74-82% lower thanthose generated in medium control cultures. Cultures containing HSA exhibited no inhibition ofthis BCDF-induced increase in PFC generation.

This inhibition was not due to an effect on proliferation. None of the BCDF augmented thespontaneous proliferation of the EBV-transformed line, and IgSRK exhibited no inhibitory effecton 3H-thymidine uptake (Table 4). In addition, cell viability as judged by trypan blue exclusion wasnot decreased in IgSRK-containing cultures (data not shown).

It still remained possible that the secretion of Ig from mature cells was being inhibited by IgSRKrather than the maturation of the B cells into cIg+ cells. This was not the case. IgSRK (300 Pig/ml)had no effect on spontaneous PFC in SLE PBMC (Table 5), nor did it have any effect on Ig secretionby Ig-secreting 8866P lymphoblastoid cells (data not shown). However, IgSRK did inhibit PWM-induced generation of cIg+ cells by a mean 62% in three separate experiments (data not shown),which is similar to the reported 760/o inhibition in PWM-induced PFC generation (Stohl, 1985).

Table 4. Effect of IgSRK on B cell differentiation*

PFC/well ct/min

BCDF Medium IgSRK HSA Medium IgSRK HSA

Medium 210 149 (29) 289 38) 3895+897 4590+637 4938+ 1920MOW 2075 544 (74) 2640( 27) 4935+1020 6303+2214 6694+ 1100MOP 1289 245 (81) 933 (28) 4086+95 4807+314 4411 +699MTP 2045 369 (82) 2115 (-3) 3858 +276 7840+3072 4511 +916

* 104 cells from an Epstein-Barr virus-transformed B cell line were cultured in triplicate with 0 5%, B celldifferentiation factor (BCDF) and IgSRK (300 pg/ml) or HSA (300 pig/ml) or control medium. One set ofcultures was assayed for PFC on day 3. The other set was labelled with 2 yCi 3H-thymidine per well for the final18 h of culture and harvested for 3H-thymidine incorporation on day 3.

Numbers in parentheses indicate percentage inhibition compared to medium control.

Table 5. Lack of effect of IgSRK on spontaneous PFC*

PFC/well

Donor Medium IgSRK HSA

A.L. 3020 2320 (23) 2190 (27)D.W. 1695 1345 (21) 1535 (9)D.S. 620 595 (4) 660 (-6)D.Sm. 860 1110 (-29) 925 (-8)K.L. 4025 4420 (-10) 3125 (28)V.T. 2220 2415 (-9) 3830(-73)Mean 2073 2034 (2) 2044 (1)

* PBMC from SLE patients were culturedwith IgSRK (300 jug/ml) or HSA (300 jpg/ml) or

control medium for 6 days and assayed for PFC.Numbers in parentheses indicate inhibition

compared to the medium control. A negativevalue indicates augmentation.

654 W. Stohl & L. Mayer

IgG-mediated inhibition ofB cells

DISCUSSION

We have previously demonstrated that IgSRK, a polyspecific, monomeric IgG preparation, caninhibit PWM-induced PFC generation in a dose-dependent fashion (Stohl, 1985). The target of theIgSRK appeared to be the B cell itself, although minor roles for T cells, monocytes, or Leu- 11 + cellscould not be rigorously excluded (Stohl, 1986). This report extends those findings in that this IgGpreparation slightly, but significantly, inhibited PWM-induced lymphocyte proliferation (Table 1)and markedly inhibited proliferation induced by two different BCPF (Table 3). In contrast, neitherPHA-induced T cell proliferation nor SAC-induced T cell-independent B cell proliferation wereinhibited (data not shown). These findings strongly suggest an inhibitory capacity for IgSRK on Tcell- or T cell factor-dependent B cell proliferation.

Although B cell proliferation and B cell differentiation can be dissociated one from the other(Mayer et al., 1982; Okada et al., 1983), inhibition of B cell proliferation in the PWM system canresult in an inhibition in Ig produced (Jelinek & Lipsky, 1983). Previous studies demonstrating aninhibitory effect of IgSRK on PFC generation (Stohl, 1985) did not distinguish between inhibitionin expansion of the PFC precursors with intact maturation vs inhibition of the maturation of anysingle PFC precursor. The ability of IgSRK to inhibit BCDF-driven differentiation of an EBV-transformed line (Table 4) indicates that IgSRK has an inhibitory effect on B cell maturation. Thismust be independent of any effect on B cell expansion in that none of the BCDF tested augmentedthe proliferation of the EBV-line nor did IgSRK adversely affect its spontaneous proliferation(Table 4). In addition, IgSRK inhibited the PWM-induced generation ofcIg+ cells (data not shown)but had no effect on mature PFC present in PBMC from SLE patients (Table 5). The effects ofIgSRK on spontaneous PFC from normal donors was difficult to assess due to their low numbersbut could be readily assessed in SLE patients who harbour large numbers of circulating PFC(Ginsburg, Finkelman & Lipsky, 1979; Blaese, Grayson & Staenberg, 1980). These observationssuggest that IgSRK truly inhibits B cell maturation and not merely Ig secretion from Ig-containingB cells. Furthermore, IgSRK added to PWM-stimulated cultures during the last 4 days of a 6-dayculture had no effect on PFC generation (Stohl, 1985). This strongly suggests that the observeddecrement in PFC generation was not due to cytophilic Ig inhibiting the PFC assay itself.

The inhibitory capacity of monomeric IgG is similar to that of human suppressive B cell factor(SBF). SBF is produced by B cells bearing the receptor for the Fc portion of IgG (FcyR) upon theirstimulation with antibody-sensitized erythrocytes and inhibits PWM-induced lymphocyte prolifer-ation and B cell differentiation (Suzuki et al., 1985). It would be of interest to determine whetherincubation of FcyR+ B cells with monomeric IgG could induce release of SBF. However, availablemethods for isolating FcyR+ B or T cell populations depend upon an incubation step withaggregated IgG or immune complexes (such as antibody-sensitized erythrocytes) (Winchester et al.,1979), so at present this experiment cannot be performed.

The molecular mechanism of action of IgSRK remains speculative. It is possible that IgSRK,which is prepared from a pool of 2,000 normal donors, contains antibodies to certain lymphokinereceptors. Antibodies to the IL-2 receptor have been detected in the sera of some patients with SLE(Sano et al., 1986). Low levels of such autoantibodies and/or autoantibodies against the BCPF orBCDF receptors may be present in normal individuals as well. Fluctuation in levels of these putativeautoantibodies might play a crucial role in regulating B cell expansion and/or differentiation innormal and disease states.

Alternatively, but not mutually exclusively, IgSRK may be inhibiting the B cell by crosslinkingits FcyR with some as yet undetermined surface structure. Neither the isolated F(ab')2 fragmentsnor the isolated Fc fragments of IgSRK alone are inhibitory in the PWM-driven system (Stohl,1985; unpublished observations). In the mouse, crosslinking of FcyR with surface IgM or IgD hasbeen shown to inhibit B cell proliferation induced by F(ab')2 anti-Ig or lipopolysaccharide (Phillips& Parker, 1984; Klaus et al., 1984). Although IgSRK contains no rheumatoid factor activity asmeasured by latex fixation (unpublished observations) and isolated Ig from normal seronegativedonors can inhibit PWM-induced B cell PFC generation to the same degree as can IgSRK (Stohl etal., 1986), only nanogram quantities of anti-Ig (which may be below the detectable level in a latex

655

656 W. Stohl & L. Mayerfixation assay) are needed to inhibit PWM-induced Ig secretion (Birdsall & Rossen, 1984). Even inthe absence of any rheumatoid factor activity (no anti-Fc fragment activity), such a crosslinkingmight also occur via idiotype-anti-idiotype interactions (Geha, 1981). Experiments are currently inprogress to elucidate the molecular mechanism(s) for the inhibitory properties of polyspecific,monomeric IgG and to ascertain that isolated Ig from individual normal donors effect inhibition ina manner identical to that of IgSRK.

A small but significant decrease in Leu- II+ cells was also observed in IgSRK-containingcultures compared to controls (Table 2). Leu-l I is a determinant associated with Fcy R onpolymorphonuclear cells and natural killer (NK) cells (Lanier et al., 1983), and monomeric Ig hasbeen previously shown to inhibit NK function (Sulica et al., 1982). Although NK function was notassessed in our studies, it is possible that monomeric Ig can inhibit both NK cell expansion andfunction. It is also possible that the IgSRK blocked the Leu- 1 antigenic sites from anti-Leu-1 lb orinduced Leu- 11 modulation from the NK cell surface leading to a decrease in Leu- I I + cells withouta true decrease in NK cells. Further studies are required to ascertain these points.

The authors thank Susan Mechanic and Chris Thompson for excellent technical assistance, Dr David Posnettfor performing the latex fixation studies, Dr Mark Grebenau of Sandoz for supplying the IgSRK, and PhyllisHyde for secretarial assistance. Supported in part by Public Health Grants Al 10811, AM 04761, AM 36527,AI23504 and CA 41583. Dr Mayer is a recipient of the Irma T. Hirschl Trust Award.

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