Eur. J. Immunol. 1984.14: 1159-1162 Selective expression of h light chains 1159

Daniele Primi, Pierre Sanchez and Pierre-Andre Cazenave

Unit6 d’Immunochimie Analytique, Departement d’Immunologie, Institut Pasteur, Paris

Selective and polyclonal induction of high levels of h light chain-bearing immunoglobulins in BALBk and SJL mice*

Polyclonal B cell activation is V gene independent and consequently results in the unselected expression of both V h and V n genes. In an attempt to selectively and polyclonally trigger h light chain-bearing B cells, we immunized mice with low doses of lipopolysaccharide (LPS) coupled to antibodies specific for the constant region of hl chains. Analyses of the sera of treated mice revealed that LPS anti-hl selectively triggers h- but not x-bearing B cells to high rate Ig secretion. Surprisingly, immunized SJL mice (rg phenotype) mounted a h+ response comparable to that detected in mice with the rh: phenotype. Because we could definitively establish that the majority of the induced antibodies belong to the hl subtype, our results have strong implications both for the study of the repertoire of hl-bearing B cells and for our understanding of the functional mechanism(s) by which the rhl locus acts.

1 Introduction

The quantitative imbalance between n and h light chains in the mouse (respectively 95 and 5% of the total light chain pool) [1] is believed to reflect the number of germ line genes for the variable (V) part of the particular light chain. Analyses at the DNA levels have indeed revealed the existence of 100-200 V genes for n (V,) and only two for h (Vhl, Vh2) [2, 31. This paucity of h-bearing B cells has made it very difficult to selec- tively expand these cells in a V gene-independent way without affecting n chain expression [4]. Consequently, the exact con- tribution of Vh genes to antibody diversity and the size of the repertoire of h-bearing B lymphocytes still remains to be es- tablished.

Here we report that, in both BALB/c (rll phenotype) and SJL (rfl phenotype) [l, 51 mice, administration of lipopolysac- charide (LPS) coupled to antibodies specific for the constant (C) region of h chains results in a dramatic and selective enhancement of the level of h-bearing immunoglobulins (Ig) in the serum. Because we could establish the polyclonal nature of the induced antibodies as well as demonstrate selective synthe- sis of h light chains in vitro, our results represent a unique approach both for the study of the potential repertoire of h Ig and for the understanding of the mechanism(s) by which the rhl locus controls hl expression.

obtained from Dr. Rajewsky and anti-hl antibodies were purified as described [6]. Rabbit anti-h antiserum was obtained by the repeated injection of J-558 proteins. The serum was repeatedly absorbed on HO-2.2-coupled (p,x) Sepharose AH (Pharmacia, Uppsala, Sweden) and anti-hl antibodies were then isolated by affinity chromatography on Bl-8 (pJ.1) cou- pled to Sepharose AH. The specificity of the antibodies obtained has already been described*. The anti-hl MS40 hy- bridoma cells were obtained by immunizing SPE mice with hl chains isolated from BALBic 5558 (a,hl) myeloma protein as described in [7]. The purified hybridoma product has been shown to recognize hl but not h2 or h3 light chains (Sanchez, P., unpublished observation). Anti-n H.139.52.1 rat hy- bridoma cells were obtained from Dr. M. Pierres (Marseille).

2.2 Antigen and immunizations

BALBic and SJL animals were obtained from the colony of Pasteur Institute. Lipopolysaccharide (LPS) of S. thyphi- murium was purchased from Difco (Detroit, MI). Polymerized LPS-Ig complexes were obtained as previously described [8]. Mice received one i.p. injection of 5 or 10 pg of rabbit anti- mouse hl-antibodies coupled to LPS (R anti-hl-LPS), LPS in saline or 50 pg of LPS alone.

2.3 Culture conditions 2 Materials and methods

2.1 Anti-isotypic antibodies

Three different anti-h antibodies were used in these studies. Monoclonal anti-hl LS 136 hybridoma cells have been

[I 46921

* This work was supported by CNRS (LA 040.359) and the Fondation pour la Recherche Mtdicale.

Correspondence: Daniele Primi, Unit6 d’lmmunochimie Analytique, Institut Pasteur, 25 Rue du Dr. Roux, F-75724 Paris Cedex 15

Abbreviations: rll: Regulatory locus controlling the expression of the murine hl light chain R anti-ll-LPS: Rabbit anti-mouse hl anti- bodies coupled to lipopolysaccharide R-Ig-LPS: Normal rabbit im- munoglobulins coupled to lipopolysaccharide PFC: Plaque-forming cell(s)

0 Verlag Chemie GmbH, D-6940 Weinheim, 1984

Spleen cells from BALB/c mice were treated with anti-Thy-l,2 and complement and cultured at 5 x 105/ml in RPMI medium supplemented with L-glutamine, antibiotics, 10% fetal calf serum and 5 x M 2-mercaptoethanol. Total and h PFC were determined after 6 days of cultures by the protein A plaque-forming cell (PFC) assay [9] using specific rabbit anti- sera which develop either h only or total PFC. The specificity of this antisera was tested using hybridoma cells.

2.4 Radioimmunoassays

Proteins were labeled with 1251 by the chloramine-T method. Two types of radioimmunoassays were used for these studies.

* Sanchez, P., Primi, D., Levi-Straws, M. and Cazenave, P.-A. (sub- mitted).

0014-298018411212-1159$02.50/0

1160 D. Primi, P. Sanchez and P.-A. Cazenave Eur. J. Immunol. 1984.14: 1159-1162

The first one consisted of a radioactive binding inhibition. Briefly, the indicated dilutions of sera were added to 383-8 (p,n)- or TNP-15 (p,Al)-precoated plastic wells together with '251-labeled anti-x antibodies or rabbit anti-hl. After an over- night incubation the plates were washed and bound radioactiv- ity was measured in a gamma counter. The second assay con- sisted of a two-step radioactive binding test. The indicated dilutions of sera were added to plastic microwells precoated with 100 pl of one of the following protein solutions: dini- trophenylated bovine serum albumin (DNP-BSA; 10 pg/ml), purified rabbit Ig (1 pgiml) or MS40 (1 pg/ml). The plates were

6

lmaa v8an 1 m 0 I / y x l -

1/3B1w V8Mo r/Wa liM0 inm ii96oo iimca

Sera dilutions Sera dtlutions

Figure 1. BALBic mice received one i.p. injection of S ( 6 0 ) or 10 (V---V) pg of LPS copolymerized with purified R anti-mouse hl antibodies in saline or left untreated (Q-0). Mice were bled 10 days after the injection and the sera tested for their property of inhibiting the binding of '251-labeled rabbit anti-hl antibodies to insolubilized TNP-15 (p,kl) monoclonal protein (A) or the binding of '=I-labeled rabbit anti-x antibodies to insolubilized 383-8 (p,x) monoclonal anti- bodies (B). Sera were also tested for their property of favoring the binding of the '251-labeled monoclonal anti-hl antibody MS40 to the insolubilized homologous protein (C).

incubated overnight at 4 "C and bound h l or n antibodies were then detected by incubating the plates with either '251-labeled MS40 monoclonal anti-hl protein or H.139.52.1 monoclonal anti-x antibodies.

3 Results and discussion

Groups of BALB/c mice, matched for age and sex, were either immunized by i.p. injection with 5 or 10 pg of rabbit anti- mouse hl antibodies coupled to LPS (R anti-hl-LPS) or left untreated. Ten days later the sera of these animals were ana- lyzed for the level of n and h light chains in a radioactive binding inhibition assay. As shown in Figs. 1 A and B, immunization with R anti-hl-LPS resulted in a dose-depen- dent increase in the level of h but not n light chains in the sera. In order to rule out the possibility that the inhibition observed was due to the presence of circulating immunogen or to anti- idiotypic antibodies directed against rabbit Ig, the sera of con- trol and immunized mice were also analyzed in a two-step binding assay for their property of favoring the binding of the '251-labeled monoclonal anti-hl antibody (MS40) to the insol- ubilized homologous protein in a radioactive sandwich assay. The results shown in Fig. 1C therefore establish that the marked increase in the level of h chains observed in the sera of immunized mice is due to the active synthesis of hl-bearing Ig.

To investigate the polyclonal nature of the induced hl-bearing Ig, and to exclude the possibility that the response observed is selectively directed against rabbit Ig isotypes, we injected groups of BALB/c mice either with 10 pg of R anti-hl-LPS, 10 pg of LPS coupled to normal rabbit immunoglobulins (R Ig-LPS) or with 50 pg of LPS (polyclonal dose). Sera were collected before and 7 days after immunization and tested for the level of n and h light chains, as well as for the level of x-

: 3000 5 8

0

4000

1000

1/960 1/240 1/60

.-.-. 1/960 1/2m 1/60 1/25200 1/@4M) l/2800 1/25200 1/8400 1/2800 1/960 1/240 1/60 1/960 1,240 1/60

sera dilutions 11469221 sera dilutions sera dilutions

Figure 2. Groups of BALB/c mice were immunized i.p. with 10 pg of rabbit anti-hl-LPS in saline (top), 10 pg of rabbit Ig-LPS (middle) or SO pg of LPS (bottom). Animals were bled before ( 6 0 ) and 7 days after immunization (V-V). Sera were analyzed for the levels of )i (A) and x (B) light chains as described in the legend to Figure 1. Anti-DNP-hl (C) and x (D) as well as anti-rabbit Ig antibodies bearing hl (E) or x (F) light chains were analyzed in a two-step radioactive assay using monoclonal anti-x and anti-hl proteins as described in Sect. 2.4.

Eur. J. Immunol. 1984.14: 1159-1162 Selective expression of h light chains 1161

and h-bearing antibodies capable of binding either DNP or normal rabbit Ig. As shown in Figs. 2 A and B normal rabbit Ig coupled to LPS did not induce an increase in the level of x or h chains, while polyclonal doses of LPS resulted in a four- fold increase in the level of n chains, the level of h was too low to be quantified. These results therefore establish that the increase of h1 chains observed after injection of R anti-h-LPS can not be ascribed to the presence of anti-isotypic antibodies. The polyclonal nature of the antibodies obtained in our experi- ments is further proved by the high level of h l ' anti-DNP antibodies which is observed in the sera of R anti-hl-LPS- but not R Ig-LPS-treated animals (Figs. 2C and D).

We have previously reported that Ig coupled to LPS induce a vigorous complementary response [8], and consequently is was important to analyze the light chain class of the specific anti- rabbit antibodies presumably induced by our immunizations. The results shown in Figs. 2 E and F demonstrate that normal rabbit Ig coupled to LPS induced a similar level of 11' anti-R Ig antibodies. In contrast the h l component of this specific response was found enhanced in the sera of mice immunized with R anti-hl-LPS (Figs. 2E and F). Thus, R anti-hl-LPS not only induces the selective polyclonal expression of h-bearing Ig but also seems to help the h l compartment in the specific antibody response.

The possibility of selectively enhancing the expression of a given light chain was also studied in vitro. The results shown in Table 1 were obtained by determining the number of h PFC and by quantifying the level of hl-bearing antibodies in the culture supernatants of splenocytes containing various doses of either LS 136-LPS or LPS alone. The data demonstrate that preferential enhancement of hl chain synthesis can also be achieved in vitro and in the complete absence of T cells. This

Table 1. Polyclonal secretion of h-bearing Ig in vitroa)

Antigen Dose PFUculture Concentration of anti- of light chains

ge* in the (Pfw) supernatants

( P r W ) Total A Ratio x Ig h Ig K / h

totan ratio

- Ls 136-LPS Ls 136-LPS LS 136-LPS LS 136-LPS LPS LPS LPS LPS

- 0.50 0.25 0.12 0.06 0.50 0.25 0.12 0.06

1720 7 542 6 880 3000 4000

23 400 7400 4000 4600

140 12.2 0.65 NDb) - 2400 3.1 5.41 1.90 2.15 2450 2.8 3.25 2.66 1.22 1260 2.3 2.70 1.50 1.80 780 5.1 1.66 0.68 2.44

1515 15.4 8.86 0.78 11.35 590 12.5 8.24 0.54 15.25 240 12.6 7.40 0.39 18.87 120 38.3 4.12 0.25 16.48

Spleen cells from BALBic mice were treated with anti-Thy-1.2 and complement and cultured at 5 x 105/ml in RPMI medium sup- plemented with L-glutamine, antibiotics, 10% fetal calf serum and 5 X lV5 M 2-mercaptoethanol. Total and h PFC were determined after 6 days of culture by the protein A PFC technique. The con- centrations of A- and x-bearing Ig were determined in an inhibition assay as described in the Sect. 2.4. Not detectable.

.--.

lj5120' 1im' 1;m Sera dtlutmns

Figure 3. Analysis of the level of x- and h-bearing Ig in the sera of normal (-0) or R anti-hl-LPS (10 pg)-treated (V---V) SJL mice. Sera were collected 10 days after immunization and analyzed as described in the legend to Fig. 1.

therefore indicated that the phenomenon studied is completely thymus independent.

Taken together these results demonstrate that selective poly- clonal induction of h-bearing Ig is indeed possible and there- fore open the possibility of gathering more information on the mechanism responsible for the defective expression of h chains observed in several inbred strains of mice.

The expression of hl chain has been attributed to a gene rhl which acts in cis with the structural gene [l, 51. Thus mice exhibiting the rg phenotype ( i .e. SJL) express a serum level of h l Ig 50-100 times lower than that observed in rh: strains (i.e. BALB/c). Surprisingly, however, we observed that injection of R anti-hl-LPS in SJL mice resulted in a dramatic increase of the level of h chains in the sera, and this level was comparable to that of immunized BALBic mice (Fig. 3). As even in this case the response could be detected with the monoclonal anti- h l antibody MS40 (Fig. 3 c ) , these results unequivocally establish the potential capacity of mice exhibiting the rg phenotype to produce normal levels of h l Ig and raise impor- tant questions concerning the mechanism(s) of action of the rh locus. Functionally, this locus has been described to reduce both the number of spleen cells expressing surface hl molecules and the serum level of hl [l, 5, lo]. Recently, how- ever, we demonstrated that the number of LPS-sensitive pre- cursors of hl-secreting B cells is similar in the splenocytes of both BALB/c and SJL mice but the level of h l mRNA is greatly reduced in LPS blasts of SJL animals as compared to the mRNA detected in BALB/c blasts*. The results presented here clearly confirm the existence of normal numbers of hl- bearing B cells in mice with the r: phenotype but raise the question of how transcriptional events can be modulated by our protocol of immunization. Even more intriguing is the fact that in in vitro experiments we have previously observed that LPS alone at polyclonal doses is unable to rescue the defective expression of h l chains in SJL splenocytes. Active suppression has recently been demonstrated to selectively affect light chain transcription [ l l ] and therefore it can be proposed that the reduced level of h mRNA found in SJL mice is also the result of chronic suppression which can be bypassed by R anti-hl complexes. Although at the present time we do not have any

* Sanchez, P., Primi, D., Levi-Strauss, M. and Cazenave, P.-A. (sub- mitted).

1162 D. Primi, P. Sanchez and P.-A. Cazenave Eur. J . Immunol. 1984.14: 1159-1162

alternative proposal we do not favor this explanation since it appears difficult to explain how specific control of h l expres- sion can be linked to the structural Cxl gene. We believe how- ever that the finding that SJL mice can be induced to express normal level of h l chains should greatly facilitate our under- standing of their impairment under normal conditions. In con- clusion our data have strong implications for the study of the repertoire of h-bearing B cells and for the contribution of VJ, genes to Ig diversity. The possibility of extending these studies to the selective triggering of Ig with a given heavy chain iso- type is certainly attractive and currently under study.

We are grateful to Dr. J . C. Mazit! for preparing the MS40 hybridorna. The excellent editorial assistance of Ms Mich2le Berson is gratefully acknowledged.

Received August 2, 1984.

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The American Association of Immunologists is sponsoring a course in advanced immunology to be held at the Lindenwood College, St. Charles, MO, June 16-23, 1985. This course will delve into recent advances in immunobiology, immunogene-

4 References

1 Geckeler, W . , Faversham, J. and Cohn, M., J . Exp. Med. 1978.

2 Tonegawa, S . , Nature 1983. 302: 575. 3 Blomberg, B., Traunecker, A., Eisen, H. and Tonegawa, S. , Proc.

Natl. Acad. Sci. USA 1981. 78: 3165. 4 Weiss, S. , Lehmann, K., Raschke, W. C. and Cohn, M., Proc.

Natl. Acad. Sci. USA 1984. 81: 211. 5 Epstein, R., Lehmann, K. and Cohn, M., J . Exp. Med. 1983.157:

1681. 6 Reth, M., Imanishi-Kari, T. and Rajewsky, K., Eur. J . Irnrnunol.

1979. 9: 1004. 7 Amor, M., Guenet, J. L., Bonhomme, F. and Cazenave, P.-A,,

Eur. J . Irnrnunol. 1983. 13: 312. 8 Primi, D. and Gazenave, P.-A,, J . Irnrnunol. 1982. 129: 1124. 9 Gronowicz, E., Coutinho, A. and Melchers, F., Eur. J . Irnrnunol.

10 Takemori, T. and Rajewsky, K., Eur. J. Irnrnunol. 1981. 11: 618. 11 Milburn, G. L., Parslow, T. G., Golbenberg, C., Granner, D. K.

148: 1122.

1976. 6: 588.

and Lynch, R. G., J . Mol. Cell. Irnrnunol. 1984. 1: 115.

tics, and molecular and clinical immunology as they relate to immune regulation. Topics will include: MHC and immuno- globulin gene organization; T and B cell growth and differenti- ation; macrophage function; helper and suppressor T cell clones and hybrids; T cell receptors; MHC restriction, Ir gene control and regulatory T cells; effector mechanisms and immunologic analysis and manipulation in human disease states.

This course is intended primarily for advanced graduate stu- dents, post-doctoral and clinical fellows, who want to expand their knowledge of modern immunology. This is not a survey course although minimal review materials will be introduced. The registration fee will be $ 225.00. Dormitory space at the Lindenwood College has been arranged. Room and board for the week will be approximately $ 250.00. To apply for the course, please submit a letter documenting your background in immunology, stating your reasons for wanting to attend, a current researchheaching activities and a brief c.v., no later than March 1, 1985 to:

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