The BCR V region has been implicated as a potential avenue of T cell help for autoreactive B cells in systemic lupus erythematosus. In principle, either germline-encoded or somatically generated sequences could function as targets of such help. Preceding studies have indicated that class II MHC-restricted T cells in normal mice attain a state tolerance to germline-encoded Ab diversity. In this study, we tested whether this tolerance is intact in systemic lupus erythematosus-prone (New Zealand Black × SWR)F1 mice (SNF1). Using a hybridoma sampling approach, we found that SNF1 T cells were tolerant to germline-encoded Ab sequences. Specifically, they were tolerant to germline-encoded sequences derived from a lupus anti-chromatin Ab that arose spontaneously in this strain. This was true both for diseased and prediseased mice. Thus, there does not appear to be a global defect in T cell tolerance to Ab V regions in this autoimmune-prone strain either before or during autoimmune disease.

A hallmark of systemic lupus erythematosus is the unregulated synthesis of autoreactive Abs with biologically significant affinities for self-Ags that include nuclear structures, such as chromatin, dsDNA, and ribonucleoprotein. These autoantibodies form immune complexes, which may deposit in the kidney and induce or exacerbate renal disease (1, 2, 3, 4). Spontaneous mouse models of lupus, such as the MRL/Mp-lpr/lpr strain, and F1 hybrids between New Zealand Black (NZB)3 and either New Zealand White (NZW) or SWR strains have been exploited in studies to address the pathological mechanism of autoantibody production. A considerable body of evidence obtained from these animal models supports the idea that some lupus autoantibodies are products of T cell-dependent immune responses (5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17). Autoantibody development can be precluded by experimental manipulations that inhibit T cell-B cell collaboration (7, 8, 9, 10, 17). Moreover, hybridoma sampling studies have shown that lupus autoantibodies often bind autoantigens with significant avidity; they are the result of oligoclonal B cell expansion; and they are products of class switch recombination and V region gene hypermutation (6, 11, 12, 13, 14, 15, 16).

Although there is good evidence for T cell help to autoreactive B cells in lupus, the antigenic specificity of this help has remained obscure. There is some evidence that T cell help in lupus is directed to ubiquitous self-Ag, such as chromatin or ribonuclear protein (18, 19, 20, 21). More subtle self-Ag, generated by posttranslational modifications are also potential targets (22, 23, 24). Finally, peptides from the BCR V region may provide an avenue of T cell help to autoreactive B cells. The BCR V region is promising in this regard because of the enormous diversity encompassed by V region peptides and because such peptides can be self-presented in class II MHC by activated B cells, which presumably are dependent upon T cell help (25, 26, 27, 28, 29, 30, 31, 32, 33, 34). We refer to this potential avenue of help to autoreactive B cells as the receptor presentation hypothesis (35).

Several groups have reported evidence in support of receptor presentation in autoimmunity (27, 30, 31, 35, 36, 37, 38, 39, 40). In a preceding study, we conducted a genealogical analysis of somatic mutations within an autoreactive B cell lineage obtained from a spontaneously diseased SNF1 mouse. The results of this work implicated somatic mutations within the BCR V region in creating an avenue of help to chromatin-reactive B cells (35). Other groups have reported more promiscuous reactions by T cells against autoantibody V regions, and in at least one case, the T cells were apparently reactive to a peptide specified by a germline-encoded stretch of DNA (41, 42, 43). This observation suggests that T cells in autoimmune-prone mice might lose, or fail to attain, tolerance to germline-encoded Ab sequences.

Distinguishing between T cell help via germline-encoded vs somatically generated Ab sequences is important because of concentration and temporal considerations. Compared with somatically generated sequences, germline-encoded sequences are more abundant, both during T cell development in the thymus and activation in the periphery. More importantly, somatic hypermutation of Ab V regions in mice and humans occurs primarily in the germinal center at a late stage of development in rare Ag-selected B cells (44, 45, 46, 47, 48, 49, 50). Although previous studies have indicated that T cells in normal mice are tolerant with respect to germline-encoded Ab diversity (26, 32, 33), this issue has not been explicitly addressed in lupus-prone mice. In this report, we provide evidence that lupus-prone SNF1 mice attain tolerance to germline-encoded Ab diversity and that this state is apparently maintained in diseased animals.

(SWR × NZB)F1 mice (SNF1) were bred in house at the Biological Resource Center using parental strains purchased from The Jackson Laboratory. A variant of BW5147 lacking the α- and β-chains of the TCR was used for T cell hybridoma formation (51). mAb SN5-18 is an anti-H2A/H2B/dsDNA mAb with two known VH somatic mutations that produce amino acid replacements. This Ab was derived from a spontaneously diseased female SNF1 mouse (12). SN5-18R is a germline-reverted version of mAb SN5-18, in which the two somatic mutations were converted to the germline sequence of the corresponding NZB VH gene (35). Twenty-nine overlapping 15-mer peptides encompassing the entire amino acid sequence of mAb SN5-18R VH, excluding the third CDR (CDR3), were synthesized by Mimotopes. Each peptide overlapped its neighbor by 12 aa. A mutated framework 1 (FR1) peptide spanning the residue 28 replacement mutation in mAb SN5-18 and three CDR3 peptides were also synthesized by the Molecular Resource Center (National Jewish Medical and Research Center). Their sequences and locations within the VH region are listed in Table I.

Table I.

Synthetic peptides from VH region of mAb SN5′-18R

NameResidues in VH GeneSequence
p1 1–15 EVKLVESEGGLVQPG 
p2 4–18 LVESEGGLVQPGSSM 
p3 7–21 SEGGLVQPGSSMKLS 
p4 10–24 GLVQPGSSMKLSCTA 
p5 13–27 QPGSSMKLSCTASGF 
p6 16–30 SSMKLSCTASGFTFS 
p7 19–33 KLSCTASGFTFSDYY 
p8 22–36 CTASGFTFSDYYMAW 
p9 25–39 SGFTFSDYYMAWVRQ 
p10 28–42 TFSDYYMAWVRQVPE 
p11 31–45 DYYMAWVRQVPEKGL 
p12 34–48 MAWVRQVPEKGLEWV 
p13 37–51 VRQVPEKGLEWVANI 
p14 40–54 VPEKGLEWVANINYD 
p15 43–57 KGLEWVANINYDGSS 
p16 46–60 EWVANINYDGSSTYY 
p17 49–63 ANINYDGSSTYYLDS 
p18 52–66 NYDGSSTYYLDSLKG 
p19 55–69 GSSTYYLDSLKGRFI 
p20 58–72 TYYLDSLKGRFIISR 
p21 61–75 LDSLKGRFIISRDNA 
p22 64–78 LKGRFIISRDNAKNI 
p23 67–81 RFIISRDNAKNILYL 
p24 70–84 ISRDNAKNILYLQMS 
p25 73–87 DNAKNILYLQMSSLK 
p26 76–90 KNILYLQMSSLKSED 
p27 79–93 LYLQMSSLKSEDTAT 
p28 82–96 QMSSLKSEDTATYYC 
p29 85–99 SSLKSEDTATYYCAR 
Mutant FR1 17–39 MKLSCTASGFIFSDYYMAWVRQ 
Germline FR1 17–39 SMKLSCTASGFTFSDYYMAWVRQ 
VHCDR3–1 91–106 (V-D junction) TATYYCARPVGTTRAR 
VHCDR3–2 96–111 (D mostly) CARPVGTTRARGAWFA 
VHCDR3–3 100–116 (D-J junction) VGTTRARGAWFAYWGQ 
VκCDR3  TYYCQQYSKLPWTFGGGTKLEIK 
NameResidues in VH GeneSequence
p1 1–15 EVKLVESEGGLVQPG 
p2 4–18 LVESEGGLVQPGSSM 
p3 7–21 SEGGLVQPGSSMKLS 
p4 10–24 GLVQPGSSMKLSCTA 
p5 13–27 QPGSSMKLSCTASGF 
p6 16–30 SSMKLSCTASGFTFS 
p7 19–33 KLSCTASGFTFSDYY 
p8 22–36 CTASGFTFSDYYMAW 
p9 25–39 SGFTFSDYYMAWVRQ 
p10 28–42 TFSDYYMAWVRQVPE 
p11 31–45 DYYMAWVRQVPEKGL 
p12 34–48 MAWVRQVPEKGLEWV 
p13 37–51 VRQVPEKGLEWVANI 
p14 40–54 VPEKGLEWVANINYD 
p15 43–57 KGLEWVANINYDGSS 
p16 46–60 EWVANINYDGSSTYY 
p17 49–63 ANINYDGSSTYYLDS 
p18 52–66 NYDGSSTYYLDSLKG 
p19 55–69 GSSTYYLDSLKGRFI 
p20 58–72 TYYLDSLKGRFIISR 
p21 61–75 LDSLKGRFIISRDNA 
p22 64–78 LKGRFIISRDNAKNI 
p23 67–81 RFIISRDNAKNILYL 
p24 70–84 ISRDNAKNILYLQMS 
p25 73–87 DNAKNILYLQMSSLK 
p26 76–90 KNILYLQMSSLKSED 
p27 79–93 LYLQMSSLKSEDTAT 
p28 82–96 QMSSLKSEDTATYYC 
p29 85–99 SSLKSEDTATYYCAR 
Mutant FR1 17–39 MKLSCTASGFIFSDYYMAWVRQ 
Germline FR1 17–39 SMKLSCTASGFTFSDYYMAWVRQ 
VHCDR3–1 91–106 (V-D junction) TATYYCARPVGTTRAR 
VHCDR3–2 96–111 (D mostly) CARPVGTTRARGAWFA 
VHCDR3–3 100–116 (D-J junction) VGTTRARGAWFAYWGQ 
VκCDR3  TYYCQQYSKLPWTFGGGTKLEIK 

Abs were purified as described previously (35). Culture supernatants were passed through protein G columns, and mAb were eluted with a 0.5 M NaCl/0.1 M glycine (pH 2.5) solution and dialyzed against PBS. The crude mAb were treated with DNase (1 μg/ml; Worthington Biochemical) in the presence of 2 mM MgCl2 at 37°C for 90 min. This material was then passed through a Sepharose column conjugated with affinity-purified goat anti-mouse IgG (heavy and L chain specific; Zymed Laboratories). High salt solution (1.5 M NaCl in PBS (pH 7.2)) was then passed through the column to dissociate any histones associated with the bound mAb, which was subsequently eluted with 0.5 M NaCl/0.1 M glycine (pH 2.5) and dialyzed extensively against PBS. Ab purity was assessed by SDS-PAGE.

T cell hybridomas were produced as described previously (33). Briefly, SWR and SNF1 mice were immunized s.c. with 100 μl of whole mAb (100 μg) emulsified in CFA (Difco Laboratories). Mice were sacrificed 7 days following immunization, and draining lymph node cells were cultured 3 days with mAb (125 μg/ml) followed by a 3-day culture with IL-2 before polyethylene glycol-induced cell fusion to the BW5147 thymoma (TCRαβ) as described previously (33). T cell hybridomas were screened for IL-2 production in response to APC cultured with the mAb or peptide immunogens. In most assays, 105 T cells were cocultured overnight with 3 × 105 APC and Ag at a final concentration of 62.5 μg/ml for Ab or 2.5 μg/ml for peptide. A time-resolved fluoroimmunometric assay was used to measure IL-2 as described previously (29). For MHC restriction studies, NZB and SWR splenocytes were used as APC.

To determine whether SWR mice contain the germline VH gene encoding mAb SN5-18R, SWR and NZB genomic kidney DNA was digested separately with BamHI, BglII, and EcoRI, and analyzed in Southern blot with a 135-bp VH probe spanning codons 22–66 of the corresponding germline NZB VH gene (35). A 387-bp JH probe was used as a positive control for DNA integrity. Hybridizations were done at 42°C in 10 ml of a solution containing 8 × 107 cpm of PCR-labeled probe, 5× SSC, 10% dextran sulfate, 5× Denhardt’s solution, 500 μg/ml ssDNA, 1% SDS, and 50% formamide. Initial washes were performed in 50 ml of a solution containing 50% formamide, 0.4% SDS, and 5× SSC at 42°C for 1 h; and stringency washes were done at 47°C in 50 ml of a solution containing 2× SSC, 0.4% SDS, and 50% formamide (20 min). The membrane was wrapped and exposed for 4 h on phosphoimager film, then signals were read in Typhoon 9200 Variable Mode Imager (Amersham Biosciences).

To determine whether SWR mice carry a germline Vκ gene matching the sequence of the Vκ10.2b of mAb SN5-18R, SWR genomic DNA was subjected to PCR amplification with primers corresponding to a sequence located 70 bases upstream of the leader exon and to the 3′ end of the Vκ10.2b gene expressed by mAb SN5-18R: 5′ primer, GCA TGC TCT CAC TTC CTA TCT TTG; and 3′ primer, GCT TAC TAT ACT GCT GAC AAT AG. Thirty PCR cycles were performed with high-fidelity DNA polymerase (Phusion; MJ Bioworks) as follows: 94°C, 30 s; 62°C, 1 min; and 72°C, 1 min. Following Taq-mediated addition of 3′ A bases, the PCR products were inserted into a TOPO TA Cloning vector (Invitrogen Life Technologies) for sequencing with vector primers, according the manufacturer’s protocol. DNA sequencing was performed by the National Jewish Molecular Resource Center.

Anti-chromatin Ab were detected in a fluoroimmunometric assay by coating 96-well microtiter plates with mouse chromatin (10 μg/ml). After incubating with a blocking buffer (2% BSA, and 1% gelatin in PBS), dilutions of SNF1 sera were added to the plates for 1 h. IgG anti-chromatin Ab were detected with a biotin-labeled rat anti-mouse IgG (Southern Biotechnology Associates) followed by a streptavidin-Eu3+ conjugate. Europium fluorescence was measured at 615 nm on a Wallac Victor (PerkinElmer Wallac) as described previously (29). Anti-Smith Ag (Sm) Ab and anti-cardiolipin Ab were identified similarly, except that plates were coated with 10 μg/ml calf thymus Sm (ImmunoVision) or bovine cardiolipin (10 μg/ml; Sigma-Aldrich).

For immunohistology, one of the kidneys was removed and embedded in Cyromolds (Miles) filled with Optimal Cutting Temperature compound (Sakura Finetek). Serial sections (5–7 μm) were cut at −16°C and placed on microscope slides. Tissues were fixed by submerging slides in acetone (5 min), air-dried, and stored at −80°C. Tissue samples were then thawed at room temperature (RT) and incubated at RT for 15 min with 5% normal goat serum in PBS. Fifty microliters of diluted flurochrome-labeled Abs (goat anti-mouse IgG and goat anti-mouse IgM; BD Pharmingen) were added and incubated for 30 min at RT followed by three washes with PBS. Coverslips were placed on the samples with mounting media (Biomedia) for fluorescence and sealed with nail polish. Another of the kidneys was paraffin-embedded, sliced, and stained with H&E. H&E staining was performed by the Histology Facility (National Jewish Medical and Research Center).

Prior results from our laboratory indicated that tolerance to germline-encoded V region peptides is attained by CD4+ T cells in nonautoimmune-prone mice (32). In this study, we tested whether autoimmune-prone SNF1 mice were self-tolerant to a germline-encoded autoantibody, specifically one reactive with chromatin (H2A/H2B/DNA). As in our preceding study, tolerance/responsiveness was assessed using a T cell hybridoma readout (32). This approach offers the advantage of enhanced definition that comes from an ability to repeatedly test hybridomas for peptide specificity, MHC-restriction, dose response, and cross-reactions. In addition, the CFA immunization and stimulations in vitro with Ag and IL-2 before fusion elicit clonal expansion of even rare, Ag-specific T cells.

Six young (8 wk old), prediseased SNF1 mice were immunized with a germline version of a spontaneous anti-chromatin Ab (mAb SN5-18R). The original mAb (SN5-18), containing two replacement somatic mutations in the VH7183 region, was derived from an autoimmune SNF1 female mouse (12). In mAb SN5-18R, these mutations were reverted to germline sequence, and the V gene was expressed in the context of a BALB/c γ2b constant gene (35, 52). The L chain of mAb SN5-18R (Vκ10.2b-Jκ1) contains no somatic mutations (12).

From these cell fusions, we screened 261 T hybridomas for those that produced IL-2 in response to mAb SN5-18R in the context of SNF1 splenic APCs. Twenty-six hybridomas, some for each mouse, reacted with the IgG2b constant region, which contains an allotypic T cell epitope encoded by the BALB/c γ2b gene. Other T cell hybridomas responded spontaneously to SNF1 APC without deliberately added Ag. Representative data are shown in Fig. 1,A. In all, only seven hybridomas responded to the VH/D/JH region of mAb SN5-18R. However, in additional tests, we found that all seven of these reacted to a pair of CDR3 peptides spanning the VH-D boundary (Fig. 1,B). No T cell hybridoma reacted to strictly germline-specified peptides. Table II summarizes the results of the T cell hybridoma analysis. Collectively, these results indicate that the germline-encoded sequences within the anti-chromatin Ab were not immunogenic in SNF1 mice.

FIGURE 1.

Representative response patterns of T cell hybridomas from immunized SNF1 mice. A, D1.33 is a nonresponder; D1.23 reacts to APC (SNF1 splenocytes) without added Ag; D1.41 reacts to a γ2b polymorphism in the H chain constant region; and D5.32 reacts to the VH/D/JH region of mAb SN5-18R. B, VHCDR3 specificities of all T cell hybridomas that respond to the VH/D/JH of mAb SN5-18R. Control peptide is Germline FR1 as shown in Table I. Error bars indicate SDs.

FIGURE 1.

Representative response patterns of T cell hybridomas from immunized SNF1 mice. A, D1.33 is a nonresponder; D1.23 reacts to APC (SNF1 splenocytes) without added Ag; D1.41 reacts to a γ2b polymorphism in the H chain constant region; and D5.32 reacts to the VH/D/JH region of mAb SN5-18R. B, VHCDR3 specificities of all T cell hybridomas that respond to the VH/D/JH of mAb SN5-18R. Control peptide is Germline FR1 as shown in Table I. Error bars indicate SDs.

Close modal
Table II.

Reactivities of T cell hybridomas from prediseased SNF1 mice immunized with germline Ab SN5–18R

Mouse (gender)Hybridomas ScreenedTest Ag
mAb SN5–18RaControl IgG2bbGermline peptides (p1–p29)cCDR3 peptides
SNF1D1 (male) 42 
SNF1D2 (male) 19 
SNF1D3 (female) 21 
SNF1D4 (female) 32 
SNF1D5 (male) 94 10 
SNF1D6 (male) 53 12 
Total hybridomas 261 33 26 
Mouse (gender)Hybridomas ScreenedTest Ag
mAb SN5–18RaControl IgG2bbGermline peptides (p1–p29)cCDR3 peptides
SNF1D1 (male) 42 
SNF1D2 (male) 19 
SNF1D3 (female) 21 
SNF1D4 (female) 32 
SNF1D5 (male) 94 10 
SNF1D6 (male) 53 12 
Total hybridomas 261 33 26 
a

Codons 28 and 57 were reverted back to germline sequence from original mAb SN5–18.

b

Allotypic response to constant region of the BALB/c γ2b.

c

p < 0.0003 that none of the SNF1 mice would yield hybridomas by chance alone given the frequencies of such hybridomas obtained from SWR-positive control mice (see Table III).

A lack of immunogenicity could be due to intrinsic properties of mAb SN5-18R. It was possible, for example, that no peptides from the VH region of this Ab could be appropriately processed and presented in the context of class II MHC. To determine whether the absence of a T cell response to mAb SN5-18R in SNF1 mice was due to tolerance, we assessed its immunogenicity in parental SWR mice. Although we knew that the VH gene encoding this Ab was derived from the NZB parental strain, it was important in tests of tolerance to demonstrate that the SWR strain lacked this VH gene (12). To this end, we performed a Southern blot using a DNA probe derived from the VH gene of mAb SN5-18R. In this assay, genomic DNA from NZB and SWR mice was digested separately with three restriction enzymes. With each enzyme, a single hybridization signal was detected in lanes containing NZB DNA. The signals identified DNA fragments containing a previously defined NZB VH7183 gene used by the anti-chromatin Ab (35). No signal was seen in any of the lanes containing SWR DNA that was hybridized with the VH probe. In contrast, single signals were seen in all lanes hybridized with a JH probe, thus confirming integrity of the digested and transferred SWR DNA. In additional tests, we were never able to amplify the corresponding VH gene from SWR genomic DNA by PCR. These results indicate that the SWR parental strain lacks the germline VH7183 gene for mAb SN5-18R (Fig. 2).

FIGURE 2.

Southern blot defines NZB VH gene for mAbSN5-18R that is absent from the SWR genome. Genomic kidney DNA from NZB and SWR strains was digested with either BglII, BamHI, or EcoRI and subjected to a Southern hybridization procedures using a 135-bp VH probe (codons 22–66) derived from VH gene of SN5-18R (left) and a 387-bp JH probe (right). Single signals observed with NZB DNA indicates that the 135-bp probe detects a single germline VH gene present in the NZB genome but absent from the SWR genome.

FIGURE 2.

Southern blot defines NZB VH gene for mAbSN5-18R that is absent from the SWR genome. Genomic kidney DNA from NZB and SWR strains was digested with either BglII, BamHI, or EcoRI and subjected to a Southern hybridization procedures using a 135-bp VH probe (codons 22–66) derived from VH gene of SN5-18R (left) and a 387-bp JH probe (right). Single signals observed with NZB DNA indicates that the 135-bp probe detects a single germline VH gene present in the NZB genome but absent from the SWR genome.

Close modal

To determine whether mAb SN5-18R was immunogenic in SWR mice, which lack the corresponding VH gene, we generated and screened T cell hybridomas from SWR mice immunized with mAb SN5-18R as described above. This time, 52 of 291 hybridomas from four SWR mice (8 wk old) responded to the V region of the mAb (Table III). Of 29 hybridomas that were successfully recloned, 13 responded to VH CDR3 peptides, and one responded to a VκCDR-3 peptide. However, the remaining 15 responded to germline-encoded VH peptides, one of which was located in FR1 (p7 and p8), and two of which were located in CDR2 (p16 and p17 or p19). Because p16 and p19 overlap by only six residues, which are insufficient to confer binding to class II MHC, we conclude that two distinct MHC binding epitopes are present within CDR2. All of the hybridomas reacted to Ag in the context of SNF1 or SWR APC, but not NZB APC, indicating their restriction to I-Aq (data not shown). Responses to germline-specified peptides are shown in Fig. 3.

Table III.

Reactivities of T cell hybridomas from SWR mice immunized with germline Ab SN5–18R

Mouse No. (gender)Hybridomas ScreenedTest Ag
mAb SN5–18RControl IgG2bV region specificGermline peptides (p1–p29)aCDR3 peptides
SWR 13 (male) 77 19 11 
SWR 14 (male) 118 31 24 
SWR 15 (female) 42 
SWR 16 (female) 54 11 10 
Total hybridomas 291 67 16 52 15 14 
Mouse No. (gender)Hybridomas ScreenedTest Ag
mAb SN5–18RControl IgG2bV region specificGermline peptides (p1–p29)aCDR3 peptides
SWR 13 (male) 77 19 11 
SWR 14 (male) 118 31 24 
SWR 15 (female) 42 
SWR 16 (female) 54 11 10 
Total hybridomas 291 67 16 52 15 14 
a

Twenty-nine of 52 V region-specific T hybriodomas were tested for responses to V region peptides.

FIGURE 3.

Responses of SWR T cell hybridomas to germline-encoded VH peptides of mAb SN5-18R. p6, p7, and p8 are FR1 peptides, and p16, p17, p18, and p19 are CDR2 peptides, as specified in Table I. A, T cell hybridomas respond to a FR1 epitope (p7 and p8). B, T cell hybridomas respond to two CDR2 epitopes (p16 and p17 or p19). Error bars indicate SDs.

FIGURE 3.

Responses of SWR T cell hybridomas to germline-encoded VH peptides of mAb SN5-18R. p6, p7, and p8 are FR1 peptides, and p16, p17, p18, and p19 are CDR2 peptides, as specified in Table I. A, T cell hybridomas respond to a FR1 epitope (p7 and p8). B, T cell hybridomas respond to two CDR2 epitopes (p16 and p17 or p19). Error bars indicate SDs.

Close modal

The consistent positive response to germline-specified peptides by SWR mice, which lack the SN5-18R VH gene, in contrast to the lack of a response by SNF1 mice, which carry this VH gene (p < 0.0003) (Table III), supports the hypothesis that T cells in autoimmune-prone SNF1 mice attain a state of tolerance to germline-encoded Ab sequences. Specifically, tolerance was attained to three distinct epitopes in the VH region of a lupus autoantibody.

The absence of an SWR T cell response to the germline SN5-18R Vκ region stood in striking contrast to a consistent VH-specific response. This could be explained if the SWR strain contained a germline Vκ10.1b gene identical in sequence to that encoding the L chain V region of mAb SN5-18R. If this were true, the T cell repertoire in SWR (normal) mice would be expected to attain a state of tolerance to SN5-18R Vk peptide sequences. To test this, we amplified and sequenced the Vκ10.2b gene from SWR genomic DNA using leader and 3′-most primers and found that the sequence of the SWR Vκ gene exactly matched that of SN5-18R Vκ. Thus, it is likely that T cells in both SWR and SNF1 mice attain a state of tolerance to germline Vκ10.2b sequences.

To determine whether tolerance was maintained in spontaneously autoimmune mice, we conducted immunogenicity tests as before, but this time in SNF1 mice with manifestations of lupus-like disease. Seven 6-mo-old female mice with evidence of proteinuria (100 mg/dl) were used in this experiment. All of these mice had high titers of serum Abs directed against chromatin and Sm, and six of them had Abs against cardiolipin (or β2-glycoprotein 1) before immunization. Moreover, at the conclusion of the experiment, four of four mice examined exhibited diffuse glomerulonephritis with deposition of IgM and IgG, evident by immunohistology (data not shown).

The diseased mice were assessed for tolerance to mAb SN5-18-R as described above. In all, 274 T cell hybridomas were generated and tested. Fourteen of these reacted against the γ2b constant region, and 19 reacted against the VH/D/JH region. All 19 of these latter hybridomas were specific for VHCDR3 sequences. These data, summarized in Table IV, support the idea that tolerance to germline-encoded Ab sequences is maintained, even in SNF1 mice with active disease.

Table IV.

Specificities of T cell hybridomas from diseased SNF1 mice immunized with germline Ab SN5–18R

Mouse (gender)Hybridomas ScreenedTest Ag
mAbSN5–18RControl IgG2b (allotype)Germline peptides (p1–p29)aVHCDR3
SNF1G1 (female) 18 
SNF1G2 (female) 
SNF1G3 (female) 16 
SNF1G4 (female) 48 
SNF1H1 (female) 79 11 
SNF1H2 (female) 51 
SNF1H3 (female) 53 
Total hybridomas 274 33 14 19 
Mouse (gender)Hybridomas ScreenedTest Ag
mAbSN5–18RControl IgG2b (allotype)Germline peptides (p1–p29)aVHCDR3
SNF1G1 (female) 18 
SNF1G2 (female) 
SNF1G3 (female) 16 
SNF1G4 (female) 48 
SNF1H1 (female) 79 11 
SNF1H2 (female) 51 
SNF1H3 (female) 53 
Total hybridomas 274 33 14 19 
a

p < 0.0002 that none of the mice would yield germline peptide-specific hybridomas by chance alone given the frequencies of such hybridomas obtained from the SWR-positive control group (Table III).

As a final positive control for immune responsiveness in SNF1 mice and a quality control for consistency in our results, we assessed immunogenicity of the original somatically mutated SN5-18 Ab. Our previous studies had demonstrated that a threonine to isoleucine mutation at residue 28 in VHFR1 of this Ab produced a T cell epitope restricted by I-Aq. This mutation was found in all members of an autoreactive lineage represented by seven B cell hybridomas, and provided the foundation of the receptor presentation hypothesis (35). From four SNF1 mice (8 wk old) immunized with mAbSN5-18, we screened 100 T cell hybridomas. Two hybridomas responded to the γ2b allotypic determinant; seven responded to VHCDR3 sequences, and six responded to the mutant FR1 sequence in a dose-dependent manner (Table V and Fig. 4). Importantly, the latter six hybridomas all required the Thr to Ile mutation for their responses, and they were restricted by SWR APC (I-Aq).

Table V.

Reactivities of T cell hybridomas from prediseased SNF1 mice immunized with the mutated mAb SN5–18

Mouse No. (gender)Hybridomas ScreenedTest Ag
mAb SN5–18 (mutant)mAbSN5–18R (germline)Control IgG2b (allotype)Mutant FR1 PeptideGermline FR1 Peptide and p1-p29VHCDR3 Peptides
SNF1E1 (male) 30 
SNF1E2 (male) 18 
SNF1E3 (female) 23 
SNF1E4 (female) 29 
Total hybridomas 100 15 6a 
Mouse No. (gender)Hybridomas ScreenedTest Ag
mAb SN5–18 (mutant)mAbSN5–18R (germline)Control IgG2b (allotype)Mutant FR1 PeptideGermline FR1 Peptide and p1-p29VHCDR3 Peptides
SNF1E1 (male) 30 
SNF1E2 (male) 18 
SNF1E3 (female) 23 
SNF1E4 (female) 29 
Total hybridomas 100 15 6a 
a

Hybridomas responding to mutant FR1 were restricted by SWR APC.

FIGURE 4.

A representative T cell hybridoma reactive against the mutated FR1 of mAb SN5-18 (E4.13). This hybridoma was generated in an SNF1 mouse immunized with mAb SN5-18. The mutant peptide contains an Ile residue at codon 28, whereas the germline peptide contains a Thr. Error bars indicate SDs.

FIGURE 4.

A representative T cell hybridoma reactive against the mutated FR1 of mAb SN5-18 (E4.13). This hybridoma was generated in an SNF1 mouse immunized with mAb SN5-18. The mutant peptide contains an Ile residue at codon 28, whereas the germline peptide contains a Thr. Error bars indicate SDs.

Close modal

There is a growing body of evidence that peptides from the BCR V region may provide an avenue of CD4+ T cell help for autoreactive B cells (27, 28, 30, 31, 35, 38, 39, 40, 41, 42). Much of the evidence for this receptor presentation idea comes from studies performed in (NZB × NZW)F1 and (NZB × SWR)F1 mice (SNF1), which develop an age-dependent systemic autoimmune disease, with many of the hallmarks of human lupus (27, 31, 35). Testing the receptor presentation idea is complex, in part because of the diversity within Ab V regions. The diversity arises from both germline sequences and two fundamentally distinct somatic processes that occur at different points in B cell development. Results of previous studies have indicated that normal mice attain a state of tolerance to germline-encoded Ab diversity (25, 26, 32, 33). In this study, we tested whether lupus-prone SNF1 mice exhibit a global deficiency in such tolerance. Our results support the idea that tolerance to germline-encoded Ab sequences is intact in SNF1 mice and apparently maintained, even in diseased animals.

We tested for tolerance by performing immunizations with an intact unmutated Ab, generating T cell hybridomas and testing these for reactivity to the Ab V regions and corresponding peptides. The test Ab was specific for chromatin and represented a germline version of an original mutated isolate derived from an autoimmune SNF1 mouse. No T cell hybridoma reactive to germline-encoded sequences of the anti-chromatin Ab was obtained from syngeneic SNF1 mice that were immunized either before or after developing manifestations of lupus. In contrast, they were readily obtained from parental SWR mice, which lacked the corresponding VH gene encoding this Ab. SNF1 mice did produce hybridomas that reacted to somatically generated VHCDR3 sequences and to a somatically mutated VHFR1 sequence when immunized with the corresponding mutant Ab.

Although T cells appear to be tolerant to the SN5-18R lupus autoantibody, we cannot formally exclude the possibility that some V genes in the repertoire are expressed at inordinately low levels and that self-tolerance to these might not be efficiently attained by T cells in the repertoire. Moreover, the T cell hybridoma approach does not permit us to determine how T cell tolerance is attained. V region reactive CD4 T cells might be deleted in the thymus (53), or they might be suppressed or incapable of full differentiation in the periphery for some other reason. It is even conceivable that V region-specific T cells survive longer in autoimmune-prone SNF1 and provide limited functions without being endowed a capacity to fully develop to a state that can be immortalized by fusion. The T cell hybridoma approach is also labor intensive. Nevertheless, we consider it a rigorous test for the presence of fully competent Ag-specific T cells. CFA was used in the immunizations, and both IL-2 and Ag stimulations in vitro preceded cell fusions. Moreover, with hybridomas, it is possible to conduct repeated tests to confirm and further define response patterns, and to identify TCR gene use and construct corresponding transgenic animals (53). In contrast, we have repeatedly found that whereas lymph node proliferation assays yield excellent results for conventional foreign proteins, they are not reliable for assessing tolerance to Ig. Even in positive control tests, specific proliferation was inconsistent and weak in the best cases. We do not know why this is so. Perhaps, tolerance to the multiple members of V region families leaves only a very low precursor frequency of T cells reactive against any specific V region.

A major reason for conducting this study derives from our previous observation that the clonal expansion of a spontaneous autoreactive B cell lineage from an SNF1 mouse was correlated with a pair of VH somatic mutations. Although neither mutation affected the affinity of the Ab product for chromatin, one of them, a Thr to Ile mutation at residue 28, produced a class II MHC-restricted T cell epitope (35). This mutation was shared by all seven members of the lineage, thus marking a point of massive clonal expansion. Complementary studies of T cell proliferation in lupus-prone (NZB × NZW)F1 mice from other laboratories have also provided evidence for the receptor presentation concept (28, 30, 38, 54). However, results of some of these studies suggest that T cells may respond more promiscuously to autoreactive Ab V regions (41, 42). In particular, determinant spreading to multiple parts of the V region was reported to occur in aged mice with disease (42). And at least one response pattern to a germline-specified VH peptide was noted (43).

Although our results do not formally rule out the possibility of T cell responses to germline V sequences in murine lupus, we can conclude that if this occurs, it is not due to global defect in tolerance to germline sequences in the SNF1 mouse. Logically, somatically mutated sequences are the most attractive candidates for avenues of T cell help to autoreactive B cells because they arise in rare B cells at a late stage of differentiation when the animal has a full repertoire of “educated” T cells. As such, somatic mutations can create neoantigens precisely within the B cell seeking T cell help and in the realm of an Ag-processing pathway that makes this possible. The idea that T cells attain a state of tolerance to germline-encoded Ab sequences has important implications to the design of idiopeptide-specific immunotherapies for autoimmunity and cancers of the lymphoid lineage (55, 56, 57).

We thank Thiago Detanico for his critical reading of the manuscript.

The authors have no financial conflict of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This work was supported by National Institutes of Health Grant AI48108.

3

Abbreviations used in this paper: NZB, New Zealand Black; FR1, framework 1; Sm, Smith Ag; RT, room temperature.

1
Pisetsky, D. S..
1986
. Systemic lupus erythematosus.
Med. Clin. N. Am.
70
:
337
-353.
2
Tan, E. M..
1989
. Antinuclear antibodies: diagnostic markers for autoimmune diseases and probes for cell biology.
Adv. Immunol.
44
:
93
-151.
3
Datta, S. K..
1988
. Murine lupus.
Methods Enzymol.
162
:
385
-412.
4
Liang, Z., C. Xie, C. Chen, D. Kreska, K. Hsu, L. Li, X. J. Zhou, C. Mohan.
2004
. Pathogenic profiles and molecular signatures of antinuclear autoantibodies rescued from NZM2410 lupus mice.
J. Exp. Med.
199
:
381
-398.
5
Sobel, E. S., V. N. Kakkanaiah, M. Kakkanaiah, R. L. Cheek, P. L. Cohen, R. A. Eisenberg.
1994
. T-B collaboration for autoantibody production in lpr mice is cognate and MHC-restricted.
J. Immunol.
152
:
6011
-6016.
6
Bloom, D. D., E. W. St. Clair, D. S. Pisetsky, S. H. Clarke.
1994
. The anti-La response of a single MRL/Mp-lpr/lpr mouse: specificity for DNA and VH gene usage.
Eur. J. Immunol.
24
:
1332
-1338.
7
Datta, S. K..
1998
. Production of pathogenic antibodies: cognate interactions between autoimmune T and B cells.
Lupus
7
:
591
-596.
8
Finck, B. K., P. S. Linsley, D. Wofsy.
1994
. Treatment of murine lupus with CTLA4Ig.
Science
265
:
1225
-1227.
9
Jevnikar, A. M., M. J. Grusby, L. H. Glimcher.
1994
. Prevention of nephritis in major histocompatibility complex class II-deficient MRL-lpr mice.
J. Exp. Med.
179
:
1137
-1143.
10
Kalled, S. L., A. H. Cutler, S. K. Datta, D. W. Thomas.
1998
. Anti-CD40 ligand antibody treatment of SNF1 mice with established nephritis: preservation of kidney function.
J. Immunol.
160
:
2158
-2165.
11
Marion, T. N., D. M. Tillman, N. T. Jou.
1990
. Interclonal and intraclonal diversity among anti-DNA antibodies from an (NZB × NZW)F1 mouse.
J. Immunol.
145
:
2322
-2332.
12
Portanova, J. P., G. Creadon, X. Zhang, D. S. Smith, B. L. Kotzin, L. J. Wysocki, J. Ellenberger.
1995
. An early post-mutational selection event directs expansion of autoreactive B cells in murine lupus.
Mol. Immunol.
32
:
117
-135.
13
Shlomchik, M., M. Mascelli, H. Shan, M. Z. Radic, D. Pisetsky, A. Marshak-Rothstein, M. Weigert.
1990
. Anti-DNA antibodies from autoimmune mice arise by clonal expansion and somatic mutation.
J. Exp. Med.
171
:
265
-292.
14
Shlomchik, M. J., A. H. Aucoin, D. S. Pisetsky, M. G. Weigert.
1987
. Structure and function of anti-DNA autoantibodies derived from a single autoimmune mouse.
Proc. Natl. Acad. Sci. USA
84
:
9150
-9154.
15
Shlomchik, M. J., A. Marshak-Rothstein, C. B. Wolfowicz, T. L. Rothstein, M. G. Weigert.
1987
. The role of clonal selection and somatic mutation in autoimmunity.
Nature
328
:
805
-811.
16
Winkler, T. H., H. Fehr, J. R. Kalden.
1992
. Analysis of immunoglobulin variable region genes from human IgG anti-DNA hybridomas.
Eur. J. Immunol.
22
:
1719
-1728.
17
Wofsy, D., J. A. Ledbetter, P. L. Hendler, W. E. Seaman.
1985
. Treatment of murine lupus with monoclonal anti-T cell antibody.
J. Immunol.
134
:
852
-857.
18
Bell, D. A., B. Morrison, P. VandenBygaart.
1990
. Immunogenic DNA-related factors: nucleosomes spontaneously released from normal murine lymphoid cells stimulate proliferation and immunoglobulin synthesis of normal mouse lymphocytes.
J. Clin. Invest.
85
:
1487
-1496.
19
Mohan, C., S. Adams, V. Stanik, S. K. Datta.
1993
. Nucleosome: a major immunogen for pathogenic autoantibody-inducing T cells of lupus.
J. Exp. Med.
177
:
1367
-1381.
20
Kaliyaperumal, A., C. Mohan, W. Wu, S. K. Datta.
1996
. Nucleosomal peptide epitopes for nephritis-inducing T helper cells of murine lupus.
J. Exp. Med.
183
:
2459
-2469.
21
Datta, S. K., A. Kaliyaperumal.
1997
. Nucleosome-driven autoimmune response in lupus: pathogenic T helper cell epitopes and costimulatory signals.
Ann. NY Acad. Sci.
815
:
155
-170.
22
Cooke, M. S., N. Mistry, C. Wood, K. E. Herbert, J. Lunec.
1997
. Immunogenicity of DNA damaged by reactive oxygen species: implications for anti-DNA antibodies in lupus.
Free Radical Biol. Med.
22
:
151
-159.
23
Hottelet, M., W. J. van Venrooij, P. Anderson, P. J. Utz.
1998
. Proteins phosphorylated during stress-induced apoptosis are common targets for autoantibody production in patients with systemic lupus erythematosus.
J. Exp. Med.
187
:
547
-560.
24
Rubin, B., G. Sønderstrup.
2004
. Citrullination of self-proteins and autoimmunity.
Scand. J. Immunol.
60
:
112
-120.
25
Jorgensen, T., B. Bogen, K. Hannestad.
1983
. T helper cells recognize an idiotope located on peptide 88–114/117 of the light chain variable domain of an isologous myeloma protein (315).
J. Exp. Med.
158
:
2183
-2188.
26
Bogen, B., T. Jorgensen, K. Hannestad.
1985
. T helper cell recognition of idiotopes on λ2 light chains of M315 and T952: evidence for dependence on somatic mutations in the third hypervariable region.
Eur. J. Immunol.
15
:
278
-281.
27
Singh, R. R., V. Kumar, F. M. Ebling, S. Southwood, A. Sette, E. E. Sercarz, B. H. Hahn.
1995
. T cell determinants from autoantibodies to DNA can upregulate autoimmunity in murine systemic lupus erythematosus.
J. Exp. Med.
181
:
2017
-2027.
28
Singh, R. R., F. M. Ebling, E. E. Sercarz, B. H. Hahn.
1995
. Immune tolerance to autoantibody-derived peptides delays development of autoimmunity in murine lupus.
J. Clin. Invest.
96
:
2990
-2996.
29
Snyder, C. M., X. Zhang, L. J. Wysocki.
2002
. Negligible class II MHC presentation of B cell receptor-derived peptides by high density resting B cells.
J. Immunol.
168
:
3865
-3873.
30
Hahn, B. H., R. R. Singh, B. P. Tsao, F. M. Ebling.
1997
. Peptides from Vh regions of antibodies to DNA activate T cell help to upregulate autoantibody synthesis.
Lupus
6
:
330
-332.
31
Hahn, B. H., R. R. Singh, F. M. Ebling.
1998
. Self Ig peptides that help anti-DNA antibody production: importance of charged residues.
Lupus
7
:
307
-313.
32
Eyerman, M. C., X. Zhang, L. J. Wysocki.
1996
. T cell recognition and tolerance of antibody diversity.
J. Immunol.
157
:
1037
-1046.
33
Eyerman, M. C., L. Wysocki.
1994
. T cell recognition of somatically-generated Ab diversity.
J. Immunol.
152
:
1569
-1577.
34
Bartnes, K., K. Hannestad.
1997
. Engagement of the B lymphocyte antigen receptor induces presentation of intrinsic immunoglobulin peptides on major histocompatibility complex class II molecules.
Eur. J. Immunol.
27
:
1124
-1130.
35
Zhang, X., D. S. Smith, A. Guth, L. J. Wysocki.
2001
. A receptor presentation hypothesis for T cell help that recruits autoreactive B cells.
J. Immunol.
166
:
1562
-1571.
36
Brosh, N., M. Dayan, M. Fridkin, E. Mozes.
2000
. A peptide based on the CDR3 of an anti-DNA antibody of experimental SLE origin is also a dominant T-cell epitope in (NZB × NZW)F1 lupus-prone mice.
Immunol. Lett.
72
:
61
-68.
37
Brosh, N., E. Eilat, H. Zinger, E. Mozes.
2000
. Characterization and role in experimental systemic lupus erythematosus of T-cell lines specific to peptides based on complementarity-determining region-1 and complementarity-determining region-3 of a pathogenic anti-DNA monoclonal antibody.
Immunology
99
:
257
-265.
38
Ebling, F. M., B. P. Tsao, R. R. Singh, E. Sercarz, B. H. Hahn.
1993
. A peptide derived from an autoantibody can stimulate T cells in the (NZB × NZW)F1 mouse model of systemic lupus erythematosus.
Arthritis Rheum.
36
:
355
-364.
39
Holmoy, T., B. Vandvik, F. Vartdal.
2003
. T cells from multiple sclerosis patients recognize immunoglobulin G from cerebrospinal fluid.
Mult. Scler.
9
:
228
-234.
40
Williams, W. M., N. A. Staines, S. Muller, D. A. Isenberg.
1995
. Human T cell responses to autoantibody variable region peptides.
Lupus
4
:
464
-471.
41
Singh, R. R., B. H. Hahn, B. P. Tsao, F. M. Ebling.
1998
. Evidence for multiple mechanisms of polyclonal T cell activation in murine lupus.
J. Clin. Invest.
102
:
1841
-1849.
42
Singh, R. R., B. H. Hahn.
1998
. Reciprocal T-B determinant spreading develops spontaneously in murine lupus: implications for pathogenesis.
Immunol. Rev.
164
:
201
-208.
43
Kalsi, J. K., B. H. Hahn.
2002
. The role of VH determinants in systemic lupus erythematosus.
Lupus
11
:
878
-884.
44
Liu, A. H., P. K. Jena, L. J. Wysocki.
1996
. Tracing the development of single memory-lineage B cells in a highly defined immune response.
J. Exp. Med.
183
:
2053
-2063.
45
Pascual, V., Y. J. Liu, A. Magalski, O. de Bouteiller, J. Banchereau, J. D. Capra.
1994
. Analysis of somatic mutation in five B cell subsets of human tonsil.
J. Exp. Med.
180
:
329
-339.
46
Jacob, J., R. Kassir, G. Kelsoe.
1991
. In situ studies of the primary immune response to (4-hydroxy-3-nitrophenyl)acetyl. I. The architecture and dynamics of responding cell populations.
J. Exp. Med.
173
:
1165
-1175.
47
Jacob, J., G. Kelsoe.
1992
. In situ studies of the primary immune response to (4-hydroxy-3-nitrophenyl)acetyl. II. A common clonal origin for periarteriolar lymphoid sheath-associated foci and germinal centers.
J. Exp. Med.
176
:
679
-687.
48
Jacob, J., J. Przylepa, C. Miller, G. Kelsoe.
1993
. In situ studies of the primary immune response to (4-hydroxy-3-nitrophenyl)acetyl. III. The kinetics of V region mutation and selection in germinal center B cells.
J. Exp. Med.
178
:
1293
-1307.
49
Berek, C., G. M. Griffiths, C. Milstein.
1985
. Molecular events during maturation of the immune response to oxazolone.
Nature
316
:
412
-418.
50
Vora, K. A., K. Tumas-Brundage, T. Manser.
1999
. Contrasting the in situ behavior of a memory B cell clone during primary and secondary immune responses.
J. Immunol.
163
:
4315
-4327.
51
White, J., M. Blackman, J. Bill, J. Kappler, P. Marrack, D. P. Gold, W. Born.
1989
. Two better cell lines for making hybridomas expressing specific T cell receptors.
J. Immunol.
143
:
1822
-1825.
52
Sompuram, S. R., J. Sharon.
1993
. Verification of a model F(ab) complex with phenylarsonate by olionucleotide-directed mutagenesis.
J. Immunol.
150
:
1822
-1828.
53
Snyder, C. M., K. Aviszus, R. A. Heiser, D. R. Tonkin, A. M. Guth, L. J. Wysocki.
2004
. Activation and tolerance in CD4+ T cells reactive to an immunoglobulin variable region.
J. Exp. Med.
200
:
1
-11.
54
Singh, R. R., F. M. Ebling, D. A. Albuquerque, V. Saxena, V. Kumar, E. H. Giannini, T. N. Marion, F. D. Finkelman, B. H. Hahn.
2002
. Induction of autoantibody production is limited in nonautoimmune mice.
J. Immunol.
169
:
587
-594.
55
Lou, Q., R. J. Kelleher, Jr, A. Sette, J. Loyall, S. Southwood, R. B. Bankert, S. H. Bernstein.
2004
. Germ line tumor-associated immunoglobulin VH region peptides provoke a tumor-specific immune response without altering the response potential of normal B cells.
Blood
104
:
752
-759.
56
Bendandi, M., C. D. Gocke, C. B. Kobrin, F. A. Benko, L. A. Sternas, R. Pennington, T. M. Watson, C. W. Reynolds, B. L. Gause, P. L. Duffey, et al
1999
. Complete molecular remissions induced by patient-specific vaccination plus granulocyte-monocyte colony-stimulating factor against lymphoma.
Nat. Med.
5
:
1171
-1177.
57
Baskar, S., C. B. Kobrin, L. W. Kwak.
2004
. Autologous lymphoma vaccines induce human T cell responses against multiple, unique epitopes.
J. Clin. Invest.
113
:
1498
-1510.