Proc. Natl. Acad. Sci. USA Vol. 86, pp. 282-286, January 1989 Immunology B-lymphoma cells process and present their endogenous immunoglobulin to major histocompatibility complex- restricted T cells (antigen-presenting cells/idiotype/MOPC315) SIEGFRIED WEISS* AND BJARNE BOGEN*t *Basel Institute for Immunology, Grenzacherstrasse 487, CH-4005 Basel, Switzerland; and tInstitute of Immunology and Rheumatology, University of Oslo, Fr. Qvamsgt. 1, 0172 Oslo 1, Norway Communicated by Niels K. Jerne, September 29, 1988 ABSTRACT Antigen-presenting B-lymphoma cells were bearing the A2315 idiotope should be exceedingly rare in transfected with the gene encoding the immunoglobulin A2 light normal BALB/c mice. To obtain a clonal population of chain of MOPC315 cells (A2315). The A2 chain is expressed on idiotope-expressing B cells we have transfected antigen- the cell surface of the transfectants together with the endoge- presenting B-lymphoma cells with the A2 gene from nous heavy chain. The transfectants present an idiotope of the MOPC315. Here we show that the transfectants express the A2315 light chain to class 11-restricted T-cell clones. Recognition A2315 light chain together with the endogenous heavy chain on by the T cells requires processing of the A2315 light chain. From the cell surface. At the same time they present a processed these data we conclude that B-lymphoma cells constitutively form of the idiotope of A2315 to the I-Ed-restricted T cells. process and present their immunoglobulins. Secretion and reuptake of the light chain was not necessary for the presen- tation. Thus, B cells bear two types of idiotypes on their MATERIALS AND METHODS membrane, a native form as surface immunoglobulin and a Proteins. A2315 light chain of BALB/c myeloma protein processed form in the context of products of the major M315 (a, A2) was purified as described (16). FRN, YRN, histocompatibility complex. FSN, and FRT synthetic peptides were kindly provided by K. Hannestad (Institute for Medical Biology, Troms0, Nor- B cells use immunoglobulin on their cell surface as receptors way) and J.-P. Briand (Institut de Biologie Moleculaire et for native antigen. Every immunoglobulin molecule bears a Cellulaire, Strasbourg, France). The FRN peptide represents collection of antigenic determinants, the so-called idiotype, the A2315 amino acid sequence from positions 91 to 108. YRN, which is localized in the variable region of the molecule. FSN, and FRT peptides are identical to FRN except for the Idiotypes and anti-idiotypic antibodies were postulated by amino acid exchanges indicated by the one-letter code in Jerne to form a regulatory network in the immune system (1). position 94, 95, or 96, respectively (6). This network most likely includes also T cells, since T cells Antibodies. K24.199 (anti-I-Ad fJ.v) (17) and 13/4 (anti-I-Ed) are essential for clonal expansion of B cells. (18) IgG2a monoclonal antibodies were purified from ascites In contrast to B cells, T cells can recognize only processed fluid on staphylococcal protein A-Sepharose. Fluorescein- forms of antigen in association with products of the major conjugated monoclonal anti-D3.137 (anti-I-Ad) (19) was histocompatibility complex (MHC) on the surface ofantigen- kindly donated by M. H. Julius (Basel Institute for Immu- presenting cells (APCs). Some B cells can function as APCs nology). (2). In 1981 J0rgensen et al. (3) proposed that B cells process CeU Lines. The CD4+ CD8- T-cell clones specific for the their surface immunoglobulin and present it in context of idiotope of A2315 and restricted to I-Ed express an a/3 their MHC molecules (3). This proposal was based on the heterodimeric receptor and have been described extensively findings that T cells specific for an idiotype on the A2 light (7, 8). They recognize the FRN peptide (see above), thus chain from MOPC315 cells (A2315) recognized denatured localizing the idiotypic determinant of A2315 to amino acid forms of A2315 (4) and that this recognition was under H-2- positions between 91 and 108 (8). The B-lymphoma cell lines linked immune response (Ir) gene control (5). The hypothesis A20J, A20/10, and A20/46 (20) have been obtained from T. has been supported and extended by later work (6-8). Similar Leanderson and M. H. Julius. They were originally thought ideas have recently been proposed by others (9, 10), and to be independent, but Southern blot analysis of their MHC-restricted T cells specific for idiotypes (11, 12) and immunoglobulin genes suggested that they all might have allotypes (13) have been described. However, it is still unclear originated from A20 (data not shown), and two of them whether MHC-restricted T cells recognize processed forms of (originally called L10 and K46) were therefore renamed immunoglobulin exclusively and whether B cells can process A20/10 and A20/46. their own immunoglobulin. Gene Construction and Transfection. The plasmid pSV2A2 To test this directly we have used recently established was kindly provided by G. E. Wu (Basel Institute for Immu- T-cell clones specific for an idiotope ofA2315 in the context of nology). It consists of the 6.6-kilobase (kb) EcoRI fragment the I-Ed MHC antigen (7, 8). These clones, like the T cells containing the A2 gene of MOPC315 (15) inserted into the studied in vivo before (4, 5), recognize an idiotypic determi- EcoRI site ofthe expression vector pSV2neo (21). The mouse nant on A2315 around amino acid positions 94, 95, and 96 (7, immunoglobulin heavy chain enhancer (22, 23) contained in 8). At these three positions the A2 light chain of myeloma a 1-kb Xba I fragment of the Sp6 heavy chain gene (kindly protein M315 differs from the germ-line-encoded A2 sequence provided by A. Traunecker, Basel Institute for Immunology) due to somatic mutations (14, 15). For this reason, B cells was inserted into the unique Xba I site in the major intron of the A2 gene, as shown in Fig. la. This construct was The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: APC, antigen-presenting cell; MHC, major histocom- in accordance with 18 U.S.C. §1734 solely to indicate this fact. patibility complex. 282 Downloaded by guest on September 29, 2021 Immunology: Weiss and Bogen Proc. Natl. Acad. Sci. USA 86 (1989) 283 a EcoRI XbaI EcoRI VJX2 CA2 EcoRI XbaI EcoRI XbaI EcoFU VJA2 I Cn 2 H-chain enhaence b c .1 I 0 A20O10 1!I", A20110 oO ~~~~~4- I I 1 anti-jAd II, anti-Wd II I . IIII a I II i2 a II 1 II 0 8 II o p a I I I 0I~ ~ ~ 20 0 200 T.. I- .E j4f A FIO 'I FIO wlti4.Ad anti+Ed I IIII UI aI I 2] Ia, III % 10 26o0 ' 0 t0o 200 Log relative fluoescence FIG. 1. (a) Construct ofthe A2 gene ofMOPC315 containing the heavy chain enhancer. The 1-kb Xba I fragment of the enhancer was inserted into the A2 gene in the same orientation as it is found in the heavy chain. The modified A2 gene is integrated in the EcoRI site of the expression vector pSV2neo in the opposite transcriptional orientation with respect to the neo gene. (b) Electrophoretic analysis of 125I-iabeled surface proteins of A20/10 and the corresponding transfectant F10 precipitated with anti-mouse immunoglobulin and protein A-Sepharose. Similar results were obtained by biosynthetic labeling with [35S]methionine and binding of anti-K and anti-A antibodies to viable cells followed by lysis and immunoprecipitation (data not shown). (c) Cell surface analysis of A20/10 and the corresponding transfectant F10 clone in a fluorescence-activated cell sorter using fluorescein-conjugated monoclonal anti-I-Ad D3.137 (kindly provided by M. H. Julius) and anti-I-Ed 13/4 antibodies. A2 chain, I-Ad, and I-Ed expression was similar for the other cell lines. transfected into the B-lymphoma cells via protoplast fusion various amounts of supernatants. Rabbit afntiserum to mouse (24). Neomycin-resistant F9, F10, and F11 clones were immunoglobulin was used as second antibody and free A2315 obtained from A20/46, A20/10, and A20J, respectively. as standard. Fluorescence-Activated Cell Sorter (FACS) Analysis. B- Proliferation Assay. B-lymphoma cells or transfectants lymphoma cells and transfectants were stained with fluores- were treated with mitomycin C (7) or fixed with glutaral- cein-conjugated anti-I-Ed (13/4) and anti-I-Ad (D3.137) dehyde (25) as described. In assays, 3 x 104 B-lymphoma monoclonal antibodies for 30 min on ice, counterstained with cells and 4 x 104 T cells (taken 12 days after the last propidium iodide, and analyzed on a Becton Dickinson FACS stimulation with antigen) were cultured with or without 440 (analysis kindly performed by D. Thorpe). addition of antigen in flat-bottomed (mitomycin-C-treated Immunoprecipitation. Cells were surface iodinated by us- APCs) or round bottomed (fixed APCs) microtiter wells for ing glucose oxidase and lactoperoxidase, lysed with 0.5% 48 hr before a 12-hr pulse with 1 ,uCi (1 Ci = 37 GBq) of Nonidet P-40, and, after removal of nuclei, stored at -70°C. [methyl-3H]thymidine. For inhibitions, monoclonal anti-Ia Portions were precipitated with rabbit antiserum to mouse antibodies were added to APCs 15 min prior to addition of T immunoglobulin and protein A-Sepharose or with protein cells and antigen. Culture conditions have been described (7). A-Sepharose alone. Immunoprecipitates were analyzed on Growth Inhibition Assay. It has previously been shown that sodium dodecyl sulfate/1o polyacrylamide gels under re- the cell clones display A2315-specific and I-Ed-restricted ducing conditions.