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The Journal of Immunology, 2005, 175: 2184-2190.
Copyright © 2005 by The American Association of Immunologists

T Cell Tolerance to Germline-Encoded Antibody Sequences in a Lupus-Prone Mouse1

Wenzhong Guo*, Diana Smith*, Amanda Guth{dagger}, Katja Aviszus* and Lawrence J. Wysocki2,*

* Integrated Department of Immunology, National Jewish Medical and Research Center, and University of Colorado Health Science Center, Denver, CO 80206; and {dagger} Department of Clinical Sciences, Colorado State University, Fort Collins, CO 80523


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
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 x 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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
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.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Animals, cell lines, Abs, and peptides

(SWR x 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 {alpha}- and {beta}-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.


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  		Table I. Synthetic peptides from VH region of mAb SN5'-18R

 
Purification of Abs

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.

Production of T cell hybridomas

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 (TCR{alpha}–{beta}–) 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 x 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.

Southern blot

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 x 107 cpm of PCR-labeled probe, 5x SSC, 10% dextran sulfate, 5x 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 5x SSC at 42°C for 1 h; and stringency washes were done at 47°C in 50 ml of a solution containing 2x 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).

Sequencing the SWR V{kappa}10.2b gene

To determine whether SWR mice carry a germline V{kappa} gene matching the sequence of the V{kappa}10.2b 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 V{kappa}10.2b 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.

Binding assays of serum autoantibodies

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).

Histology

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).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
No detectable T cell response to germline-encoded VH segment of an anti-chromatin Ab

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 {gamma}2b constant gene (35, 52). The L chain of mAb SN5-18R (V{kappa}10.2b-J{kappa}1) 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 {gamma}2b gene. Other T cell hybridomas responded spontaneously to SNF1 APC without deliberately added Ag. Representative data are shown in Fig. 1A. 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. 1B). 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.



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  		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 {gamma}2b 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.

 

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

 
T cell response in parental SWR mice to germline-encoded VH sequences of an anti-chromatin Ab

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).



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  		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.

 
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 V{kappa}CDR-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.


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

 


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  		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.

 
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 V{kappa} region stood in striking contrast to a consistent VH-specific response. This could be explained if the SWR strain contained a germline V{kappa}10.1b 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 V{kappa}10.2b gene from SWR genomic DNA using leader and 3'-most primers and found that the sequence of the SWR V{kappa} gene exactly matched that of SN5-18R V{kappa}. Thus, it is likely that T cells in both SWR and SNF1 mice attain a state of tolerance to germline V{kappa}10.2b sequences.

Maintenance of T cell tolerance to germline-encoded portion of an anti-chromatin Ab in diseased SNF1 mice

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 {beta}2-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 {gamma}2b 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.


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

 
T cell reactions to a somatically mutated portion of an anti-chromatin Ab in SNF1 mice

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 {gamma}2b 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).


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

 


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  		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.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
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 x NZW)F1 and (NZB x 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 x 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).


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


   Disclosures
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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. Back

2 Address correspondence and reprint requests to Dr. Lawrence J. Wysocki, Department of Immunology, K902, National Jewish Medical and Research Center, 1400 Jackson Street, Denver, CO 80206. E-mail address: WysockiL{at}njc.org Back

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

Received for publication September 30, 2004. Accepted for publication May 31, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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