Immunology 1979 38 325

Immunoregulation by covalent antigen-antibody complexes II. SUPPRESSION OF A T-CELL INDEPENDENT ANTI-HAPTEN RESPONSE

J. P. TITE & R. B. TAYLOR MRC Immunobiology Group, Department of Pathology, The Medical School, Bristol

Acceptedfor publication 3 May 1979

body (Eichmann, 1974; Hart et al., 1974; Kohler, Kaplan & Strayer, 1974), although in one system a particular class of antibody mediated a priming effect (Eichmann & Rajewsky, 1975). A functional role for the Fc fragment has been shown in certain systems (Eichmann, 1974; Kohler, Richardson, Rowley & Smyk, 1977) and suppressor T cells have been implicated in some cases (Eichmann, 1975; Owen, Ju & Nisonoff, 1977) but not in others (Kohler, 1975). Antibody also exerts regulatory influences on humoral responses when incorporated into immune complexes. Murgita & Vas (1972) showed that administration of isologous anti-sheep red cell antibody prior to the injection of SRBC affected the subsequent antibody response, the effect differing with the sub-class of antibody injected. Other groups have demonstrated suppression with antibody (Kappler, Hoven, Dharmarajan & Hoffman, 1973; Hoffman & Kappler, 1978) or pre-formed antigen-antibody complexes (Obernbarnscheidt & Kolsch, 1978) when added to in vitro culture systems. Klaus (1978) has shown that immune complexes are extremely efficient at priming B-memory cells. These phenomena have all been demonstrated to be dependent on an intact or functional Fc region. Immune complexes and anti-idiotypic antibodies differ in that the latter are monomeric immunoglobulin molecules whilst the former will vary greatly both in size and stability according to the affinity and specificity of the antibody used to form the complex. This

Summary. Covalent antigen-antibody complexes containing the hapten fluorescein were found to suppress the thymus-independent response to that hapten. This suppression required the presence of an intact Fc fragment in the complex but could still be established in the complete absence of T cells. INTRODUCTION

The study of the regulation of the immune response is currently attracting intense interest. An early attempt to provide a framework for regulatory influences was made by Jerne (1974) who proposed that the immune system represents a functional network of lymphocyte clones which is regulated by the suppressive influences of idiotype-anti-idiotype interactions. Such a hypothesis has found some experimental support in systems in which an antigen elicits an antibody response of restricted heterogeneity, enabling anti-idiotypic antibodies to be raised (Eichmann, 1972; Hart, Wang, Pawlak & Nisonoff, 1972; Strayer, Cosenza, Lee, Rowley & Kohler, 1974). Anti-idiotypic antibody has been shown to be extremely efficient at suppressing the responsiveness of B cells producing the idiotypic antiCorrespondence: Dr R. B. Taylor, MRC Immunobiology Group, Department of Pathology, The Medical School, University Walk, Bristol BS8 ITD. 0019-2805/79/1000-0325$02.00 © 1979 Blackwell Scientific Publications

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paper describes some of the effects of stable monomeric immune complexes formed using a photosensitive affinity label 4-azido-2-nitrophenyl-(NAP) (Fleet, Knowles & Porter, 1972). Pre-treatment of mice with such complexes containing the hapten fluorescein profoundly suppressed the splenic plaque-forming cell response to the same hapten when presented in immunogenic form on a thymus-independent carrier.

MATERIALS AND METHODS

Animals CBA/H, BALB/c and (BALB/c x CBA/H)Fl mice were bred in our own laboratory from stocks obtained from L.A.C., Carshalton.

Antigens and immunization AECM-Ficoll (aminoethyl-carbamyl-methyl Ficoll) was prepared from Ficoll of mol. wt 400,000, obtained from Pharmacia fine Chemicals A.B. Uppsala, Sweden, according to the method of Inman (1975). Fl-AECM-Ficoll was prepared by the reaction of fluorescein isothiocyanate (FITC) with AECM-Ficoll in 0-1 M bicarbonate buffer at pH 9-2. The mixture was incubated for 2 h at room temperature, after which excess unreacted FITC was removed by passage down an AGl X2 ion exchange column (Biorad, California) followed by extensive dialysis against distilled water. The degree of substitution was calculated using a combination of spectrophotometric and dry weight analysis. Fluoresceinated lipopolysaccharide was prepared from lipopolysaccharide (E. coli 0.55.B5, Difco, U.S.A.) by the cyanogen bromide coupling of fluorescein ethylenediamine (Axen, Porath & Ernback, 1967). Fl I-Ficoll, and lipopolysaccharide containing approximately one fluorescein group per 100,000 mol. wt units were thus prepared. Both antigens were diluted in saline and administered intraperitoneally. Preliminary experiments revealed that a 10 yg dose of either conjugate gave an optimal anti-Fl plaque-forming cell response at 5 days. Groups of four to six mice were used and the results of the PFC assay expressed as geometric means + one standard error.

Preparation of a-NAP aminocaproyl-e-fluoresceinlysine (Fl-lys-cap-NAP) All operations with NAP were done in semi-darkness, e.g. single bulb in far corner of room. NAP was prepared as described by Fleet et al. (1972), and NAP-cap by a method similar to that used by these authors for

NAP-lysine. It was recrystallized from methanol, and the hydroxysuccinimide ester prepared by a method similar to that used by Becker & Makela (1975) for DNP-cap. This ester was stored in solution in pure dimetyl formamide (Pierce) in a desiccator at -20°. Assuming E:5 = 4800 in aqueous solution, its concentration was estimated as 7-2 x 10-2 M. e-Benzylidene lysine was freshly prepared by reaction of benzaldehyde with lysine in alkaline solution (Bezas & Zervas, 1961). 5 3 ml of NAP-cap succinimide (0 4 mmol) was reacted with a 1 5 x excess of s-benzylidene lysine (dissolved in 1 ml water with 0 I ml triethylamine) for 40 min at room temperature. The solution was placed on ice and two volumes of cold water added. The pH was adjusted to 3 5 with dilute acetic acid, and the resulting precipitate was consolidated by shaking and collected by centrifugation. This precipitate was suspended in 0-5 ml of 2 M HCI and incubated at 370 for 10 min to remove the benzylidene group. Some insoluble material was removed by centrifugation. After placing on ice again, the pH was adjusted to between 3 and 4 to bring down a precipitate of a-NAP-cap-lysine at its isoelectric point. This was collected by centrifugation, resuspended in the same volume of water and dissolved with addition of minimal HCl, reprecipitated, and finally dissolved in 0-1 M borate buffer Ph 9 to 1 x 10-2 M. An aliquot was reacted with a 1-5 x excess of fluorescein isothiocyanate (Koch-Light) for 2 h at room temperature. The desired compound, Fl-lys-cap-NAP, was separated from free fluorescein by precipitation with acid and solution in alkali, three times, in a volume of about 10 ml. Estimates of purity, made by electrophoresis in barbitone-buffered agarose at -30 V/cm, showed that a-NAP-cap-lys was essentially free of other coloured compounds, while Fl-lys-cap-NAP (which was itself almost non-fluorescent) still contained a minor contamination with a fluorescent compound, presumably fluorescein. The concentration of the Fl-lys-cap-NAP was estimated from the optical density at 495 nm, assuming EM = 78,000, as for fluorescein. (The contribution from NAP would have been relatively small, and was ignored since there is likely to be some hypochromism due to the intramolecular interactions which resulted in quenching of fluorescence.)

Preparation of immune complexes The preparation of covalent antigen-antibody complexes will be described elsewhere (Taylor, in preparation). Briefly, complexes were made with immunoadsorbent purified anti-NAP antibodies. Such

Immunoregulation by covalent Ag-Ab complexes II antibodies were mixed with an approximate four-fold excess of Fl-lys-cap-NAP and illuminated for 15 min using a 60 W light bulb. The mixture was then passed through a small column containing DNP-lysinecoupled Sepharose 4B and AG 1 x 2 ion exchange resin to remove excess unreacted antibody and FI-lys-capNAP respectively. The immune complex in the eluate from such a column was adjusted to the appropriate concentration according to the molar concentration of fluorescein measured by the OD at 492 nm. The complex was administered intravenously, usually 14 days before challenge. Pepsin digestion Rabbit anti-NAP antibodies were dialysed into 0-1 M sodium acetate at pH 4-5, and crystalline pepsin (Koch-Light, Bucks) was added at 2 mg/100 mg protein. After incubation of the mixture for 18 h at 370, it was adjusted to pH 8-0 and dialysed extensively against borate-buffered saline (pH 8 3). The resultant digest was then passed down a Staphylococcus protein A Sepharose 4B column (Pharmacia) and the eluate collected. Such a digestion completely removed the haemolytic activity of the antibody against NAP-coupled sheep red blood cells in the presence of complement.

Adult-thymectomized, lethally irradiated, bone marrow reconstituted (A TXBM) mice ATXBM mice and non-thymectomized controls (XBM) were prepared. (BALB/c x CBA/H)F, were thymectomized at 6 weeks of age or left as controls. Six weeks later they were subjected to 850 rad irradiation from a '37Cs source, and reconstituted intravenously with 5 x 106 bone marrow cells pre-treated with a rabbit anti-mouse brain serum plus complement as described by Elson & Taylor (1974). The resultant ATXBM and XBM mice were used approximately 6 weeks later.

327

ying doses of immune complexes, containing fluorescein, formed with rabbit antibodies (R-FI complex) and challenged 14 days later with Fl-Ficoll. A dosedependent suppression of both the direct and indirect anti-Fl PFC response was observed; significant suppression was obtained even with 0-1 pg of complex (Fig. 1). The suppression was not due to a change in kinetics of the response to Fl-Ficoll, as suppressed mice exhibited low responses even up to 10 days after challenge (Fig. 2). In order to ascertain the time of onset of suppression, mice were pre-treated with R-Fl complex at various times before challenge with Flficoll. Figure 3 shows that significant suppression was not obtained until 3 days after administration, and that the effect became more marked by 7 days. At day 1 after administration of complex, instead of suppression, a large increase in responsiveness was noted: a phenomenon which is being further investigated. To test whether this effect was true of other fluorescein T-independent carriers (especially carriers thought to stimulate the putatively immature Bl.l population; Mosier, Zitron, Mond, Ahmed, Scher & Paul, 1977) complex-treated animals were challenged with F1-LPS. Table 1 shows that the response to F1-LPS was markedly reduced by pre-treatment with R-Fl complex, indicating that the effect is not restricted to the B1.2 T-independent B-cell population. Control ; Response 9

104-

CL

103 -\

PFC assay

Assays for plaque-forming cells were performed as previously described (Tite, Marshall-Clarke & Playfair, 1978) except that fluorescein-coupled Fab fragments of a hyperimmune rabbit anti-SRBC serum were used instead of DNP-Fab. RESULTS Suppression of the response to FI-Ficoli by covalently bound immune complexes (BALB/c x CBA/H)FI mice were pre-treated with var-

102

I

I

0-1 1.0 10 Dose of complex (jigq)

30

Figure 1. Dose curve of rabbit-Fl complex. (BALB/cx CBA/H)Fi mice were pre-treated intravenously with the doses of rabbit-Fl complex indicated, 14 days before intraperitoneal challenge with 10 ug Fl-Ficoll. The PFC assay was performed 5 days after challenge. Open symbols, direct PFC; closed symbols, indirect PFC.

328

J. P. Tihe & R. B. Taylor 105 [ I

-M

Control x

Response

I

a

0) 0)

Li.

102

10 I

I

Interval before challenge (days)

I

0 2 4 6 8 10 Interval after challenge (days)

Figure 2. Kinetics of normal and suppressed responses to Fl-Ficoll. (BALB/c x CBA/H)Fi mice, that had been pretreated 7 days previously with 10 pg rabbit-Fl complex or left as controls, were challenged intraperitoneally with 10 pg Fl-Ficoll and killed for the PFC assay as indicated. Normal mice, circles; suppressed mice, squares; open symbols, direct PFC; closed symbols, indirect PFC.

Figure 3. Kinetics of induction of suppression. (BALB/c x CBA/H)Fi mice were pre-treated intravenously with 10 pg rabbit F1 complex at various times prior to challenge with 10 pg Fl-Ficoll as indicated. The PFC assay was performed 5 days after challenge. Open symbols, direct PFC; closed symbols, indirect PFC. suggests a functional role for the Fc fragment of the

immune complex in the establishment of the suppression.

Requirement for a functional Fc fragment for complexmediated suppression The role of the Fc fragment of the immune complex in suppression was tested in two ways. First, complex was prepared using F(ab')2 fragments of rabbit antiNAP antibodies and compared with the whole R-Fl complex; equivalent amounts of hapten were administered in each case. Table 2 shows that removal of the Fc fragment prevented the establishment of the suppressed state, suggesting a role for Fc receptor mediated interactions in the induction of suppression. Second, complex was made from purified fowl antiNAP antibodies (F-Fl complex) and administered to mice. A small but non-significant reduction in response was observed, which again did not approach that observed with the entire rabbit complex (Table 3). As chicken antibodies are thought not to interact with mouse Fc receptors (Kappler et al., 1973) this also

Requirement for T cells in the induction of suppression In order to establish whether T cells were necessary for the induction of the suppressed state, an experiment Table 1. Effect of complexes formed with rabbit antibody and fluorescein (R-Fl) to Fl-LPS

Pre-treatment

10 pg R-Fl complex Nil

Direct PFC/spleen

Indirect PFC/spleen

3,986 (3,533-4,491) 16,249

3,651 (2,980-4,470) 25,322

(13,494-19,341)

(19,930-31,571)

(BALB/c x CBA/H)Fi mice were injected intravenously as indicated above 14 days before intraperitoneal challeinge with 10 pg Fl-LPS; the PFC assay was performed 5 days later.

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Immunoregulation by covalent Ag-Ab complexes II Table 2. Requirement for the Fc fragment in complex-mediated suppression Exp I Pre-treatment 10ug R-FI complex 6 pg* RF(ab')2 Fl complex Nil

Exp 2

Direct PFC/spleen

Indirect PFC/spleen

Direct PFC/spleen

1,002 (706-1,422) 7,175 (6,508-7,707) 9509

490 (323-735) 7,077 (6,568-9,015) 11,034

142 (91-221) 10,829 (7,707-15,214) 17,326

Exp 3

Indirect PFC/spleen

Direct PFC/spleen

Indirect PFC/spleen

81 757 1,036 (57-114) (897-1,187) (678-845) 11,614 16,481 18,033 (8,103-16,647) (15,521-17,500) (16,514-19,341) 11,968 21,162 19,116

(8,578-10,614) (9,996-12,088) (15,836-18,958) (10,097-14,135) (20,537-27,807) (15,994-22,471) CBA/H mice (Expt 1) or (BALB/c x CBA/H)Fi mice (Expts 2 and 3) were injected intravenously as indicated above either 14 days (Expts 1 and 2) or 21 days (Expt 3) before intraperitoneal challenge with 10 pg Fl1 j-Ficoll; the PFC assay was performed 5 days after challenge. * Equivalent dose of fluorescein to 10 pg whole complex. Table 3. Inability of complexes formed with fowl antibody (F-Fl) to mediate suppression Exp 1 Direct

Indirect

Direct

Indirect

PFC/spleen

PFC/spleen

PFC/spleen

PFC/spleen

384 (270-544) 4,964 (3,568-6,904) 7,631 (6,836-8,518)

Nil 12,964 (11,498-14,764) 15,068 (13,226-17,154)

10,938 (9,136-13,095) 15,063 (12,964-17,500)

Pre-treatment 10 pg R-Fl complex 10ug F-Fl complex Nil

Expt 2

4,188 (2,489-6,124) 8,103 (6,438-10,198)

(BALB/c x CBA/H)F1 mice (Expt 1) or BLAB/c mice (Expt 2) were injected intravenously as indicated above 14 days before intraperitoneal challenge with IO Pg Fl1 I-Ficoll; the PFC assay was performed 5 days after challenge. Table 4. Lack of requirement for T cells in complex-mediated

suppression Direct

Indirect

Mice

Pre-treatment

PFC/spleen

PFC/spleen

ATXBM

10 pg R-Fl

ATXBM

complex Nil

346 (194-616) 6,602 (4,570-9,549) 204 (109-380) 13,182

XBM

10 pg R-F1

complex XBM

nil

(12,022-14,454)

218

(131-363) 6,165 (4,168-9,120) 141

(100-199) 10,471

(10,000-10,964)

(BALB/c x CBA/H)FI ATXBM or XBM mice were injected intravenously as indicated above 7 days before intraperitoneal challenge with 10 pg Fli -Ficoll; the PFC assay was performed 5 days after

challenge.

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was performed in adult-thymectomized lethally irradiated bone marrow reconstituted (ATXBM) mice. Cells from such mice were unable to respond to ribonuclease (a T-dependent antigen) in an adoptive transfer, unless additional T cells in the form of thymocytes were supplied, whereas control XBM cells responded well in such circumstances (results not shown). ATXBM or control XBM mice were injected intravenously with 10 jug R-Fl complex or left as controls. Seven days later all groups were injected with 10 pg Fl-Ficoll. Both control groups made good anti-Fl responses to this powerful thymus-independent antigen (Table 4) but pre-treatment with complex was efficient at reducing the response to almost background levels in both ATXBM and XBM mice. This indicates that an active suppression by T cells is not operating in this system. DISCUSSION In this communication we report the effect of covalently bound antigen-antibody complexes on the immune response to a hapten presented on T-independent carriers. The hapten-specific response to the fluorescein hapten is markedly suppressed in animals pre-treated with antigen-antibody complexes. Although the kinetics of the onset of suppression would suggest that this requires an active process (Fig. 3) suppressor T cells are unlikely to be involved, as the suppressed state is readily induced in ATXBM mice (Table 4). The phenomenon is, however, dependent on the presence of an intact Fc fragment (Table 2) or an Fc fragment able to interact with mammalian Fc receptors (Table 3) in the antigen-antibody complex. This is analogous to the results reported by Kappler et al. (1973) and Hoffman & Kappler (1978), although in these cases a T-dependent response was being studied, and the suppression was reversed by the addition of a non-specific T-cell replacing factor. An exact comparison between these studies and the experiments reported here is difficult as in the previous cases a complex antigen (SRBC) was being used in an in vitro system-both being circumstances which may favour physical interference with T-B collaboration. A role for Fc-mediated interactions, either between Fl-specific B cells and accessory cells such as macrophages, or with Fc receptors on the Fl-specific B cell itself is nonetheless implicated in this study. Such interactions may then lead to direct inactivation of the T-independent B cell in a fashion analogous to that

reported by Ryan, Arbeit, Dickler & Henkart (1975) who showed inhibition of mitogenesis by antigenantibody complexes, the inhibition being dependent on the presence of an intact Fc fragment in the complex. In contrast to the T-independent response, we have found that the T-dependent response is primed by covalent antigen-antibody complexes prepared with rabbit anti-NAP (Taylor, Tite & Manzo: in preparation). This was true both for the response to haptens such as Fl, and protein antigens, such as ribonuclease. The difference in effect may be attributable to the greater susceptibility of T-independent B cells to inactivation (Cambier, Vitetta, Uhr & Kettman, 1977; Tite et al., 1978). On the other hand it may be that the complexes activate T-dependent B cells with the help of T cells specific for rabbit IgG, while the T-independent B cells, being refractory to T-cell help, become inactivated by the same stimulus. The latter hypothesis receives some support from the fact that prior induction of tolerance to rabbit immunoglobulin before the administration of rabbit anti-NAP-NAP-RNase complexes completely abrogates the priming effect of such complexes and in some cases a suppression of subsequent anti-RNase responses is observed (unpublished results). The possible implications of such observations will be discussed at length elsewhere.

ACKNOWLEDGMENTS This work was supported by the National Research Development Corporation and the Medical Research Council REFERENCES AXEN R., PORATH J. & ERNBACK S. (1967) Chemical coupling of peptides and proteins to polysaccharides by means of cyanogen halides. Nature (Lond.), 214, 1302. BECKER M. & MAKELA 0. (1975) Modification of bacteriophage with hapten-e-aminocaproyl-N-hydroxysuccinimide esters: increased sensitivity for immunoassay. Immunochemistry, 12, 329. BEZAS B. & ZERVAs L. (1961) On the peptides of L-lysine" 2. J. Am. chem. Soc. 83, 719. CAMBIER J.C., VITETTA E.S., UHR J.W. & KETTMAN J.R. (1977) B-cell tolerance. II. Trinitrophenyl human gammaglobulin induced tolerance in adult and neonatal murine B-cells responsive to thymus-dependent and independent forms of the same hapten. J. exp. Med. 145. 778. EICHMANN K. (1972) Idiotypic identity of antibodies to streptococcal carbohydrates in inbred mice. Europ. J. Immunol. 2, 301.

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