Proc. Nati. Acad. Sci. USA Vol. 88, pp. 11271-11275, December 1991 Immunology
Junctional region sequences of T-cell receptor ,8-chain genes expressed by pathogenic anti-DNA autoantibody-inducing helper T cells from lupus mice: Possible selection by cationic autoantigens (systemic lupus erythematosus/nephritogenic helper T cells/T-ceil receptors)
SHARLENE ADAMS, PIERRE LEBLANC, AND SYAMAL K. DATTA* Department of Medicine, New England Medical Center and Sackler Immunology Program, Tufts University School of Medicine, Boston, MA 02111
Communicated by Leroy Hood, September 16, 1991 (received for review March 20, 1991)
subsets that are essential for inducing the production of these crucial pathogenic anti-DNA autoantibodies both in vivo and in vitro, appear only in the spleens of the SNF1 progeny just prior to the onset of lupus nephritis but not in young preautoimmune SNF1 mice or in the parental strains that do not develop nephritis (6-8). Although the antigenic specificities of such Th cells are unknown, we were able to clone them by means of their special functional property of inducing the production of pathogenic anti-DNA autoantibodies, and we report herein analysis of the T-cell receptor (TCR) 8 chains expressed by these pathogenic autoantibody-inducing Th cells.t
ABSTRACT We rescued from the spleens of 10 (SWR x NZB)F1 (SNF1) mice with lupus nephritis the T cells that were activated in vivo and cloned 268 T-cell lines and hybridomas. Only 12% of these T-cell clones had the functional ability to preferentially augment the production of pathogenic anti-DNA autoantibodies. Among these, 16 helper T-cell (Th-cell) clones that were mostly CD4+ and had the strongest autoantibodyinducing ability were analyzed for T-cell receptor (TCR) 18-chain gene usage. Seven of the 16 Th-cell clones expressed 13-chain variable region (Vp) Vp8 (8.2 or 8.3) genes and three expressed Vp4, whereas two clones each used a Vp31 or Vp2 or Vpl4 gene, suggesting some restriction in TCR gene usage. Although heterogeneous, the V-D-J junctional region sequences of TCR 13-chain genes used by these Tb-cell clones invariably encoded one or more negatively charged residues (aspartic or glutamic acid) that had been generated in most cases by unspecified nucleotide (N) additions. Representative pathogenic autoantibody-inducing Th-cell clones could rapidly induce the development of lupus nephritis when injected into young prenephritic SNF1 mice. The pathogenic autoantibodyinducing Tb cells expressing the anionic residues in their TCR 13-chain junctions (complementarity-determining region CDR3) were probably selected by some cationic autoantigenic peptide presented by the anti-DNA B cells they preferentially helped. These results offer a due regarding the nature of the primary autoantigen that may drive the pathogenic autoimmune response in lupus.
MATERIALS AND METHODS Mice and T-Cell Clones. NZB and SWR mice were obtained from The Jackson Laboratory, and their SNF1 progeny were bred by us. Female mice were used. Cloned T-cell lines from nephritic SNF1 mice were derived as described (7, 8). Herein we report also cloning T-cell hybridomas after fusing CD4enriched T cells (6-8) from the spleens of 6- to 7-mo-old nephritic SNF1 mice with the TCR a/,8-chain-negative variant of the BW5147 thymoma (9). To select for those T-cell lines and hybridomas that could preferentially augment the production of cationic, IgG-class anti-DNA autoantibodies, serial dilutions of T cells from each line or hybridoma were cultured with syngeneic B cells as described (6-8), except that the hybridoma T cells received 2200 rads (10) before coculture. Total polyclonal IgG (,ug/ml) or IgG-class autoantibodies (units/ml) to single-stranded DNA or doublestranded DNA produced in the cultures were quantitated by assaying separate aliquots from each culture supernatant as described (6-8). Cationic anti-DNA autoantibodies of IgG class were detected by affinity purification of culture supernatant aliquots on DNA-cellulose columns followed by isoelectric focusing (6-8). Pathogenicity of T-Cell Clones in Vivo. The SNF1 females begin to develop nephritis around 5 mo of age (2). To determine if the T-cell clones could accelerate nephritis development, 3- to 3.5-mo-old SNF1 females were injected i.v. twice, 7 days apart, with 107 or 2 x 107 T cells in 0.2 ml of Hanks' balanced salt solution. The T cells were first activated by stimulation with syngeneic, mitomycin C-treated antigen-presenting cells (APC) (7, 8) and 20 units of
The basic mechanism of systemic lupus erythematosus (SLE) remains elusive because the primary antigen(s) that drive the pathogenic autoimmune response in this disease is unknown. To address this complex problem, we have been analyzing the select populations of T and B cells that are responsible for the pathogenesis of lupus nephritis in the F1 progeny of crosses between autoimmune NZB (New Zealand Black) and the normal SWR strains of mice (reviewed in ref. 1). In marked contrast to the parental strains, the SNF1 mice uniformly die of an accelerated glomerulonephritis that resembles human lupus very closely in pathology and natural history (2). The nephritis-prone SNF1 mice produce a select population of pathogenic anti-DNA autoantibodies that are IgG in class and cationic in charge, and they share a recurrent cross-reactive idiotype as well as highly homologous heavy chain variable (VH) region sequences with numerous basic residues in the complementarity-determining regions (CDRs) (1-3). These cationic autoantibodies and immunocomplexes initiate lupus nephritis probably by binding to anionic sites (proteoglycans) in the renal glomeruli (4). The genes for these pathogenic autoantibodies are contributed to the SNF1 mice by the parental strains (3, 5). However, helper T-cell (Th-cell)
Abbreviations: SNF1, (SWR x NZB)F1 or (NZB x SWR)F1; Th cell, helper T cell; TCR, T-cell receptor; CDR, complementaritydetermining region; VH and Vp, heavy chain; and A-chain variable regions. *To whom reprint requests should be addressed at: Department of Medicine, Box 52, New England Medical Center, 750 Washington Street, Boston, MA 02111. tThe 16 T-cell receptor sequences reported in this paper have been deposited in the GenBank data base (accession nos. M80439M80454).
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 11271
11272
Immunology: Adams et al.
recombinant interleukin-2 (Cetus) per ml for 5 days and washed prior to injection. The mice were monitored weekly for proteinuria by albustix (2) and sacrificed at 4-4.5 mo of age (i.e., 3 weeks after the last injection with T cells). Sections from the kidneys were stained for detection of glomerulonephritis by light microscopy, and immunofluorescence of frozen sections to detect IgG-immunocomplex deposits was done as described (2) by fluorescein isothiocyanate-conjugated-goat anti-mouse IgG F(ab')2 fragment (Cappel Laboratories). The sections were coded and analyzed by two investigators. Sequencing TCR 3-Chain V Region Genes (Vp) Used by the T-Cell Clones. The TCR Va gene expressed by each clone was first determined by staining with a panel of anti-TCR monoclonal antibodies (mAbs) or by RNA blot-hybridization (Northern) analysis with Vp gene probes as described (8); in addition, we used the monoclonal antibody to V,14 (14-2, rat IgM; gift of D. Raulet, ref. 11). Total RNA was extracted from each T-cell line or hybridoma as described (8). Firststrand cDNA was synthesized by using 1-5 ttg of total RNA with oligo(dT) (Promega) and Moloney murine leukemia virus reverse transcriptase (BRL) in a final volume of 20 /Al as described (12). The reaction was stopped by heating at 940C for 5 min. The cDNA in each reaction was immediately amplified by the polymerase chain reaction (PCR) (12) using one of the Va primers (sense) together with the anti-sense 3-chain constant region (Ca) primer (described below) at a final concentration of 0.25 1LM. Amplifications were performed in 100 ul with 2.5 units of Taq polymerase (PerkinElmer/Cetus), 0.1 mM dNTPs, and 0.5 mM MgCl2 with a thermocycler (Perkin-Elmer/Cetus) under the following conditions: 94TC melting for 1 min, 500C or 550C annealing for 2 min, and 72TC extension for 2 min for 25 cycles followed by a 7-min extension at 720C. The amplified product containing the expected-size TCR gene fragment was digested with EcoRI, purified, and cloned into pUC19 for dideoxy chaintermination sequencing by standard techniques (12-14). Pos-
Proc. Nati. Acad. Sci. USA 88 (1991)
itive clones were sequenced with the Cp primer or the universal primer with the 17Sequencing kit according to the manufacturer (Pharmacia-LKB). Two independent cDNA clones from each of two separate PCR products obtained from each T-cell line or hybridoma RNA were sequenced. The oligonucleotide primers for PCR were synthesized according to published sequences (13-16) or, in case of the VP2 gene, were designed from its CDR2 region sequence (14), and an EcoRI restriction site sequence was added to the 5' end of each primer for cloning the amplified TCR Va cDNA product in pUC19. The amplification primers were as follows: Vn1, 5'-dGGAATTCAGCGCTGAGAAGCCGCCAG-3'; V,92, 5'-
dGGAATTCGCTGATTACCTGGCCACACGGG-3'; V,4, 5'-dGGAATTCATGTTTTCCTACAGCTATC-3'; V,38, 5'dGGAATTCAACACATGGAGGCTGCAGTC-3'; VP14, 5'dGGAATTCCAGGTAGAGTCGGTGGTGCA-3', and Cp, 5'-dGGAATTCGCCATTCACCCACCAGCTCAG-3'. Sequences were analyzed with Wisconsin Genetics Computer Group software (17).
RESULTS Functional Properties of T-Cell Clones. The pathogenic anti-DNA autoantibody inducing Th cells are activated in vivo in SNF1 mice with lupus nephritis (6-8). Since activated T cells express interleukin 2 receptors, we could isolate and clone those T cells by limiting-dilution cultures with low doses of recombinant interleukin 2 and irradiated syngeneic spleen cells as feeders (7, 8). Only 25 (17%) of 149 T-cell lines that were derived from the spleens of eight SNF1 mice with lupus nephritis were capable of preferentially augmenting the production of IgG anti-DNA autoantibodies when cocultured with a polyclonal population of syngeneic B cells (8). Among these, 10 CD4' Th-cell lines were the strongest inducers of the cationic or pathogenic variety of autoantibodies (7, 8). Eight of these particular Th-cell lines whose TCR have been sequenced here, are designated by the prefix L- in Table 1.
Table 1. Functional properties and TCR Vp gene usage of Th-cell clones derived from nephritic SNF1 mice Fold help for IgG antibody productiont T-cell TCR Vp clone* IgG anti-ssDNA Total IgG IgG anti-dsDNA usage L-1A 20.7 28.0 7.4 8.2 L-9w.7 26.2 7.0 4.9 8.2 L-9.2 8.0 2.8 4.6 8.3 L-8w.45 6.4 2.9 2.7 8.3 H-16.B11 202.1 7.1 20.6 8.2 41.8 H-12.C4 2.6 10.0 8.3 H-12.E4 6.0 3.3 3.2 8.3 L-3A 6.0 12.7 4.1 4 L-9.10 6.6 2.2 4.9 4 6.7 L-9.37 4.2 5.0 4 H-15.G9 44.5 3.6 20.3 14 H-15.E3 16.3 2.4 14 6.2 L-9.7 11.0 2.9 5.7 2 H-16.B6 22.4 6.6 3.8 2 H-10.B5 12.5 2.0 9.9 1 18.0 H-16.G10 15.3 12.9 1 *Cloned Th-cell lines are designated by the initial letter L, and the hybridomas, by H. Th-cell clones that augmented the production of cationic IgG anti-DNA autoantibodies when cocultured with syngeneic B cells are shown. The first number identifying the cloned T-cell lines is the number of the nephritic SNF1 animal from which the line was derived. The hybridoma clones were from two other nephritic mice, H-14, H-15, and H-16 series from one and H-10 and H-12 from another. tThe values represent increase in folds in production of the IgG antibodies when the T cells were cocultured with syngeneic Bh cells as compared with B cells cultured alone in the same assay. Serial dilutions of cells from the Th-cell clones were tested several times, and the optimum help provided is shown. B cells cultured by themselves produced 0.015 0.005 unit and 0.006 0.002 unit of IgG autoantibodies per ml to ssDNA and dsDNA, respectively, and 0.38 0.07 ,ug of total polyclonal IgG ±
±
per ml.
Immunology: Adams et al.
Proc. Natl. Acad. Sci. USA 88 (1991)
Since the cloned Th-celi lines had to be grown for 4-6 weeks before they could be tested (7, 8), in vitro selection factors could have influenced their establishment in culture. Therefore, in this study we also derived 119 T-cell hybridomas from two more nephritic SNF1 mice by fusing their activated CD4' T cells with the TCR-loss variant fusion partner; only the T cells that were activated in vivo would fuse in this procedure. Merely 8% of the 119 hybridomas could induce the production ofpathogenic autoantibodies. They are designated by the prefix H- in Table 1. All of the Th-cell hybridomas were CD4+ except H-12.E4 and 16.B11 (not shown). T-cell hybridomas can loose expression of CD4 molecules. All of the Th-cell clones (L or H) in Table 1, could augment the production of IgG anti-ssDNA autoantibodies more than total polyclonal IgG, and some Th-cell clones were more potent, increasing autoantibody production by 202-fold. This marked increase in the IgG autoantibodies, measured in units/ml, was not reflected by an increase in total polyclonal IgG (ug/ml) because the former constituted only a small component of the total IgG produced in the cultures. All of these Tb clones also augmented production of anti-dsDNA autoantibodies, but some were more potent (Table 1). The Th-cell clones were also capable of helping syngeneic B cells to produce IgG anti-DNA autoantibodies that were cationic in charge on isoelectric focusing (not shown, but similar to results in refs. 1, 6, and 7). Seven ofthe 16 pathogenic autoantibody-inducer Th-cell clones were TCR V,8+ (8.2 or 8.3). However, Vp4, V,914, Vp2, or V1l1 TCR genes were also used recurrently by the other Th-cell clones (Table 1). TCR 3-Chain Junction Sequences. The TCR (-chain gene segments used by the SNF1-derived T-cell clones (Table 1) were amplified and sequenced. Seven of the 16 cationic autoantibody-inducing Th clones expressed Vp8 genes (8.2 or
8.3). The V,8 primer used here allowed us to sequence the entire V,8 gene segment (13, 14), and we did not detect any polymorphisms in the SNF1 V,98 genes (not shown) that would have rendered them reactive to Mis self-antigens (18). The TCR (3 V-D-Jjunctional region (CDR3) sequences of the T-cell clones were heterogeneous (Fig. 1; D, diversity, J, joining). However, the TCRs expressed by all of the cationic autoantibody-inducing Th-cell clones had one or more negatively charged amino acid residues (aspartic or glutamic acid) in their CDR3 between positions 99 and 105 (highlighted in Fig. 1). The TCRs of 6 pathogenic autoantibody-inducing Th-cell clones had an aspartic acid residue at position 99 and another 5 had an aspartic or glutamic acid residue at position 100. In the case of 10 of these 16 cationic autoantibodyinducing Th-cell clones, one of the negative residues was formed by unspecified nucleotide (N) additions. Moreover, in 8 Th-cell clones, the glutamic acid residue encoded by J4 was positioned at 104 or 105 because of the nature of the joining of their TCR gene segments. And in 5 Th-cell clones, because of the nature of the recombination process, an aspartic acid residue was encoded by Dp sequences, which in the germ-line reading frame does not code for aspartic acid (ref. 14; see footnote to Fig. 1). Comparison with Other TCR Sequences. The frequency of anionic residues in a total of 181 junctional region sequences of TCR (3 chains expressed by mature T cells of mice was analyzed. These TCRs originated from four groups: (i), 16 pathogenic anti-DNA autoantibody-inducing Th-cell clones described here; (ii), 57 T-cell clones specific for a known cationic peptide derived from cytochrome c or myelin basic protein (MBP) or sperm whale myoglobin or phage A repressor antigen (19-25); (iii), 29 TCR (-chain cDNA sequences obtained from the T cells in the enlarged lymph nodes of
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Junctional region sequences of T-cell receptor ,8-chain genes expressed by pathogenic anti-DNA autoantibody-inducing helper T cells from lupus mice: Possible selection by cationic autoantigens (systemic lupus erythematosus/nephritogenic helper T cells/T-ceil receptors)
SHARLENE ADAMS, PIERRE LEBLANC, AND SYAMAL K. DATTA* Department of Medicine, New England Medical Center and Sackler Immunology Program, Tufts University School of Medicine, Boston, MA 02111
Communicated by Leroy Hood, September 16, 1991 (received for review March 20, 1991)
subsets that are essential for inducing the production of these crucial pathogenic anti-DNA autoantibodies both in vivo and in vitro, appear only in the spleens of the SNF1 progeny just prior to the onset of lupus nephritis but not in young preautoimmune SNF1 mice or in the parental strains that do not develop nephritis (6-8). Although the antigenic specificities of such Th cells are unknown, we were able to clone them by means of their special functional property of inducing the production of pathogenic anti-DNA autoantibodies, and we report herein analysis of the T-cell receptor (TCR) 8 chains expressed by these pathogenic autoantibody-inducing Th cells.t
ABSTRACT We rescued from the spleens of 10 (SWR x NZB)F1 (SNF1) mice with lupus nephritis the T cells that were activated in vivo and cloned 268 T-cell lines and hybridomas. Only 12% of these T-cell clones had the functional ability to preferentially augment the production of pathogenic anti-DNA autoantibodies. Among these, 16 helper T-cell (Th-cell) clones that were mostly CD4+ and had the strongest autoantibodyinducing ability were analyzed for T-cell receptor (TCR) 18-chain gene usage. Seven of the 16 Th-cell clones expressed 13-chain variable region (Vp) Vp8 (8.2 or 8.3) genes and three expressed Vp4, whereas two clones each used a Vp31 or Vp2 or Vpl4 gene, suggesting some restriction in TCR gene usage. Although heterogeneous, the V-D-J junctional region sequences of TCR 13-chain genes used by these Tb-cell clones invariably encoded one or more negatively charged residues (aspartic or glutamic acid) that had been generated in most cases by unspecified nucleotide (N) additions. Representative pathogenic autoantibody-inducing Th-cell clones could rapidly induce the development of lupus nephritis when injected into young prenephritic SNF1 mice. The pathogenic autoantibodyinducing Tb cells expressing the anionic residues in their TCR 13-chain junctions (complementarity-determining region CDR3) were probably selected by some cationic autoantigenic peptide presented by the anti-DNA B cells they preferentially helped. These results offer a due regarding the nature of the primary autoantigen that may drive the pathogenic autoimmune response in lupus.
MATERIALS AND METHODS Mice and T-Cell Clones. NZB and SWR mice were obtained from The Jackson Laboratory, and their SNF1 progeny were bred by us. Female mice were used. Cloned T-cell lines from nephritic SNF1 mice were derived as described (7, 8). Herein we report also cloning T-cell hybridomas after fusing CD4enriched T cells (6-8) from the spleens of 6- to 7-mo-old nephritic SNF1 mice with the TCR a/,8-chain-negative variant of the BW5147 thymoma (9). To select for those T-cell lines and hybridomas that could preferentially augment the production of cationic, IgG-class anti-DNA autoantibodies, serial dilutions of T cells from each line or hybridoma were cultured with syngeneic B cells as described (6-8), except that the hybridoma T cells received 2200 rads (10) before coculture. Total polyclonal IgG (,ug/ml) or IgG-class autoantibodies (units/ml) to single-stranded DNA or doublestranded DNA produced in the cultures were quantitated by assaying separate aliquots from each culture supernatant as described (6-8). Cationic anti-DNA autoantibodies of IgG class were detected by affinity purification of culture supernatant aliquots on DNA-cellulose columns followed by isoelectric focusing (6-8). Pathogenicity of T-Cell Clones in Vivo. The SNF1 females begin to develop nephritis around 5 mo of age (2). To determine if the T-cell clones could accelerate nephritis development, 3- to 3.5-mo-old SNF1 females were injected i.v. twice, 7 days apart, with 107 or 2 x 107 T cells in 0.2 ml of Hanks' balanced salt solution. The T cells were first activated by stimulation with syngeneic, mitomycin C-treated antigen-presenting cells (APC) (7, 8) and 20 units of
The basic mechanism of systemic lupus erythematosus (SLE) remains elusive because the primary antigen(s) that drive the pathogenic autoimmune response in this disease is unknown. To address this complex problem, we have been analyzing the select populations of T and B cells that are responsible for the pathogenesis of lupus nephritis in the F1 progeny of crosses between autoimmune NZB (New Zealand Black) and the normal SWR strains of mice (reviewed in ref. 1). In marked contrast to the parental strains, the SNF1 mice uniformly die of an accelerated glomerulonephritis that resembles human lupus very closely in pathology and natural history (2). The nephritis-prone SNF1 mice produce a select population of pathogenic anti-DNA autoantibodies that are IgG in class and cationic in charge, and they share a recurrent cross-reactive idiotype as well as highly homologous heavy chain variable (VH) region sequences with numerous basic residues in the complementarity-determining regions (CDRs) (1-3). These cationic autoantibodies and immunocomplexes initiate lupus nephritis probably by binding to anionic sites (proteoglycans) in the renal glomeruli (4). The genes for these pathogenic autoantibodies are contributed to the SNF1 mice by the parental strains (3, 5). However, helper T-cell (Th-cell)
Abbreviations: SNF1, (SWR x NZB)F1 or (NZB x SWR)F1; Th cell, helper T cell; TCR, T-cell receptor; CDR, complementaritydetermining region; VH and Vp, heavy chain; and A-chain variable regions. *To whom reprint requests should be addressed at: Department of Medicine, Box 52, New England Medical Center, 750 Washington Street, Boston, MA 02111. tThe 16 T-cell receptor sequences reported in this paper have been deposited in the GenBank data base (accession nos. M80439M80454).
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 11271
11272
Immunology: Adams et al.
recombinant interleukin-2 (Cetus) per ml for 5 days and washed prior to injection. The mice were monitored weekly for proteinuria by albustix (2) and sacrificed at 4-4.5 mo of age (i.e., 3 weeks after the last injection with T cells). Sections from the kidneys were stained for detection of glomerulonephritis by light microscopy, and immunofluorescence of frozen sections to detect IgG-immunocomplex deposits was done as described (2) by fluorescein isothiocyanate-conjugated-goat anti-mouse IgG F(ab')2 fragment (Cappel Laboratories). The sections were coded and analyzed by two investigators. Sequencing TCR 3-Chain V Region Genes (Vp) Used by the T-Cell Clones. The TCR Va gene expressed by each clone was first determined by staining with a panel of anti-TCR monoclonal antibodies (mAbs) or by RNA blot-hybridization (Northern) analysis with Vp gene probes as described (8); in addition, we used the monoclonal antibody to V,14 (14-2, rat IgM; gift of D. Raulet, ref. 11). Total RNA was extracted from each T-cell line or hybridoma as described (8). Firststrand cDNA was synthesized by using 1-5 ttg of total RNA with oligo(dT) (Promega) and Moloney murine leukemia virus reverse transcriptase (BRL) in a final volume of 20 /Al as described (12). The reaction was stopped by heating at 940C for 5 min. The cDNA in each reaction was immediately amplified by the polymerase chain reaction (PCR) (12) using one of the Va primers (sense) together with the anti-sense 3-chain constant region (Ca) primer (described below) at a final concentration of 0.25 1LM. Amplifications were performed in 100 ul with 2.5 units of Taq polymerase (PerkinElmer/Cetus), 0.1 mM dNTPs, and 0.5 mM MgCl2 with a thermocycler (Perkin-Elmer/Cetus) under the following conditions: 94TC melting for 1 min, 500C or 550C annealing for 2 min, and 72TC extension for 2 min for 25 cycles followed by a 7-min extension at 720C. The amplified product containing the expected-size TCR gene fragment was digested with EcoRI, purified, and cloned into pUC19 for dideoxy chaintermination sequencing by standard techniques (12-14). Pos-
Proc. Nati. Acad. Sci. USA 88 (1991)
itive clones were sequenced with the Cp primer or the universal primer with the 17Sequencing kit according to the manufacturer (Pharmacia-LKB). Two independent cDNA clones from each of two separate PCR products obtained from each T-cell line or hybridoma RNA were sequenced. The oligonucleotide primers for PCR were synthesized according to published sequences (13-16) or, in case of the VP2 gene, were designed from its CDR2 region sequence (14), and an EcoRI restriction site sequence was added to the 5' end of each primer for cloning the amplified TCR Va cDNA product in pUC19. The amplification primers were as follows: Vn1, 5'-dGGAATTCAGCGCTGAGAAGCCGCCAG-3'; V,92, 5'-
dGGAATTCGCTGATTACCTGGCCACACGGG-3'; V,4, 5'-dGGAATTCATGTTTTCCTACAGCTATC-3'; V,38, 5'dGGAATTCAACACATGGAGGCTGCAGTC-3'; VP14, 5'dGGAATTCCAGGTAGAGTCGGTGGTGCA-3', and Cp, 5'-dGGAATTCGCCATTCACCCACCAGCTCAG-3'. Sequences were analyzed with Wisconsin Genetics Computer Group software (17).
RESULTS Functional Properties of T-Cell Clones. The pathogenic anti-DNA autoantibody inducing Th cells are activated in vivo in SNF1 mice with lupus nephritis (6-8). Since activated T cells express interleukin 2 receptors, we could isolate and clone those T cells by limiting-dilution cultures with low doses of recombinant interleukin 2 and irradiated syngeneic spleen cells as feeders (7, 8). Only 25 (17%) of 149 T-cell lines that were derived from the spleens of eight SNF1 mice with lupus nephritis were capable of preferentially augmenting the production of IgG anti-DNA autoantibodies when cocultured with a polyclonal population of syngeneic B cells (8). Among these, 10 CD4' Th-cell lines were the strongest inducers of the cationic or pathogenic variety of autoantibodies (7, 8). Eight of these particular Th-cell lines whose TCR have been sequenced here, are designated by the prefix L- in Table 1.
Table 1. Functional properties and TCR Vp gene usage of Th-cell clones derived from nephritic SNF1 mice Fold help for IgG antibody productiont T-cell TCR Vp clone* IgG anti-ssDNA Total IgG IgG anti-dsDNA usage L-1A 20.7 28.0 7.4 8.2 L-9w.7 26.2 7.0 4.9 8.2 L-9.2 8.0 2.8 4.6 8.3 L-8w.45 6.4 2.9 2.7 8.3 H-16.B11 202.1 7.1 20.6 8.2 41.8 H-12.C4 2.6 10.0 8.3 H-12.E4 6.0 3.3 3.2 8.3 L-3A 6.0 12.7 4.1 4 L-9.10 6.6 2.2 4.9 4 6.7 L-9.37 4.2 5.0 4 H-15.G9 44.5 3.6 20.3 14 H-15.E3 16.3 2.4 14 6.2 L-9.7 11.0 2.9 5.7 2 H-16.B6 22.4 6.6 3.8 2 H-10.B5 12.5 2.0 9.9 1 18.0 H-16.G10 15.3 12.9 1 *Cloned Th-cell lines are designated by the initial letter L, and the hybridomas, by H. Th-cell clones that augmented the production of cationic IgG anti-DNA autoantibodies when cocultured with syngeneic B cells are shown. The first number identifying the cloned T-cell lines is the number of the nephritic SNF1 animal from which the line was derived. The hybridoma clones were from two other nephritic mice, H-14, H-15, and H-16 series from one and H-10 and H-12 from another. tThe values represent increase in folds in production of the IgG antibodies when the T cells were cocultured with syngeneic Bh cells as compared with B cells cultured alone in the same assay. Serial dilutions of cells from the Th-cell clones were tested several times, and the optimum help provided is shown. B cells cultured by themselves produced 0.015 0.005 unit and 0.006 0.002 unit of IgG autoantibodies per ml to ssDNA and dsDNA, respectively, and 0.38 0.07 ,ug of total polyclonal IgG ±
±
per ml.
Immunology: Adams et al.
Proc. Natl. Acad. Sci. USA 88 (1991)
Since the cloned Th-celi lines had to be grown for 4-6 weeks before they could be tested (7, 8), in vitro selection factors could have influenced their establishment in culture. Therefore, in this study we also derived 119 T-cell hybridomas from two more nephritic SNF1 mice by fusing their activated CD4' T cells with the TCR-loss variant fusion partner; only the T cells that were activated in vivo would fuse in this procedure. Merely 8% of the 119 hybridomas could induce the production ofpathogenic autoantibodies. They are designated by the prefix H- in Table 1. All of the Th-cell hybridomas were CD4+ except H-12.E4 and 16.B11 (not shown). T-cell hybridomas can loose expression of CD4 molecules. All of the Th-cell clones (L or H) in Table 1, could augment the production of IgG anti-ssDNA autoantibodies more than total polyclonal IgG, and some Th-cell clones were more potent, increasing autoantibody production by 202-fold. This marked increase in the IgG autoantibodies, measured in units/ml, was not reflected by an increase in total polyclonal IgG (ug/ml) because the former constituted only a small component of the total IgG produced in the cultures. All of these Tb clones also augmented production of anti-dsDNA autoantibodies, but some were more potent (Table 1). The Th-cell clones were also capable of helping syngeneic B cells to produce IgG anti-DNA autoantibodies that were cationic in charge on isoelectric focusing (not shown, but similar to results in refs. 1, 6, and 7). Seven ofthe 16 pathogenic autoantibody-inducer Th-cell clones were TCR V,8+ (8.2 or 8.3). However, Vp4, V,914, Vp2, or V1l1 TCR genes were also used recurrently by the other Th-cell clones (Table 1). TCR 3-Chain Junction Sequences. The TCR (-chain gene segments used by the SNF1-derived T-cell clones (Table 1) were amplified and sequenced. Seven of the 16 cationic autoantibody-inducing Th clones expressed Vp8 genes (8.2 or
8.3). The V,8 primer used here allowed us to sequence the entire V,8 gene segment (13, 14), and we did not detect any polymorphisms in the SNF1 V,98 genes (not shown) that would have rendered them reactive to Mis self-antigens (18). The TCR (3 V-D-Jjunctional region (CDR3) sequences of the T-cell clones were heterogeneous (Fig. 1; D, diversity, J, joining). However, the TCRs expressed by all of the cationic autoantibody-inducing Th-cell clones had one or more negatively charged amino acid residues (aspartic or glutamic acid) in their CDR3 between positions 99 and 105 (highlighted in Fig. 1). The TCRs of 6 pathogenic autoantibody-inducing Th-cell clones had an aspartic acid residue at position 99 and another 5 had an aspartic or glutamic acid residue at position 100. In the case of 10 of these 16 cationic autoantibodyinducing Th-cell clones, one of the negative residues was formed by unspecified nucleotide (N) additions. Moreover, in 8 Th-cell clones, the glutamic acid residue encoded by J4 was positioned at 104 or 105 because of the nature of the joining of their TCR gene segments. And in 5 Th-cell clones, because of the nature of the recombination process, an aspartic acid residue was encoded by Dp sequences, which in the germ-line reading frame does not code for aspartic acid (ref. 14; see footnote to Fig. 1). Comparison with Other TCR Sequences. The frequency of anionic residues in a total of 181 junctional region sequences of TCR (3 chains expressed by mature T cells of mice was analyzed. These TCRs originated from four groups: (i), 16 pathogenic anti-DNA autoantibody-inducing Th-cell clones described here; (ii), 57 T-cell clones specific for a known cationic peptide derived from cytochrome c or myelin basic protein (MBP) or sperm whale myoglobin or phage A repressor antigen (19-25); (iii), 29 TCR (-chain cDNA sequences obtained from the T cells in the enlarged lymph nodes of
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