MHC class II-bound self peptides from autoimmune MRL/lpr mice reveal potential T cell epitopes for autoantibody production in murine systemic lupus erythematosus.

The systemic lupus erythematosus-like syndrome in MRL/lpr mice involves high-titered IgG autoantibodies, particularly antinuclear Abs that target histones, DNA, and RNA particles. Although T cell help is required for the generation of antinuclear Abs, the epitopes recognized by such helper T cells are unknown. To address this question, we isolated and sequenced self peptides bound by MHC class II molecules from MRL/lpr mice. We identified a number of peptides that are not seen in similar preparations from nonautoimmune C3H animals. The "abnormal" peptide donors include histone, a protein component of a small nuclear ribonucleoprotein, ribosomal proteins, and RNA processing enzymes. We postulate that the peptides from these donors are T cell epitopes required for the generation of the most frequent antinuclear Abs specificities seen in MRL/lpr mice.

Induction of autoimmunity in a transgenic model of B cell receptor peripheral tolerance: changes in coreceptors and B cell receptor-induced tyrosine-phosphoproteins.

Abrogation of peripheral tolerance in transgenic mice that express a uniform B-cell receptor may create a powerful tool to examine the molecular mechanisms that underlie the autoimmune response in B cells. Here we report that processes that induce a systemic lupus erythematosus-like syndrome in normal mice, namely chronic graft vs host reaction, trigger systemic autoimmunity in a well-established transgenic mice model of B cell receptor peripheral tolerance. The induction of graft vs host reaction in mice that carry both a rearranged B cell Ag receptors specific for hen egg lysozyme and expressing chronically circulating hen egg lysozyme Ag resulted in induction of high and sustained levels of circulating anti-hen egg lysozyme autoantibodies and glomerulonephritis with proteinuria. This was associated with marked changes in expression of cell-surface proteins, such as CD23 and complement receptor 2. B cells from the graft vs host-induced mice could proliferate in vitro in response to self-Ag, and upon stimulation with anti-IgD demonstrated rapid phosphotyrosine phosphorylation of specific proteins, which could not be induced in the anergic double transgenic B cells. Conversely, loss of tolerance was not associated with a higher induction in the level of Syk kinase phosphorylation following stimulation with anti-IgD. Taken collectively, these data establish that 1) processes that induce a systemic lupus erythematosus-like syndrome in normal mice can abrogate peripheral tolerance in transgenic mice expressing self-tolerized B cells, and that 2) loss of tolerance in this model is associated with marked changes in surface expression of B cell coreceptors as well as with selective changes in IgD-induced signaling by discrete tyrosine-phosphoproteins, but not Syk kinase.

Differential control of autoantibodies and lymphoproliferation by Fas ligand expression on CD4+ and CD8+ T cells in vivo.

We have previously shown that the gld autoimmune syndrome is suppressed in lethally irradiated gld mice reconstituted with a mixture of normal and gld bone marrow (BM). Furthermore, in vivo depletion of normal Thy-1+ cells restores lymphoproliferation and autoantibody production in such chimeras, suggesting that T cells bearing Fas ligand are responsible for correcting the gld defect. In this study, mixed-BM chimeras lacking either normal CD4+ (B6CD4KO-B6gld) or normal CD8+ T cells (B6CD8KO-B6gld) were generated to determine the contribution of the normal T cell subsets to disease suppression. Lymphoproliferation was completely suppressed in B6CD4KO-B6gld chimeras but only modestly in B6CD8KO-B6gld chimeras. On the other hand, both types of mixed-BM chimeras had incomplete effects on the suppression of serum autoantibodies when compared with B6gld reconstituted with isologous BM. These results suggest that both T cell subsets provide Fas ligand to suppress immune cells responsible for autoantibody production; however, CD8+ T cells are mainly responsible for preventing lymphoproliferation.

Diverse antibody recognition patterns of the multiple Sm-D antigen polypeptides.

Anti-Sm antibodies are intrinsically associated with systemic lupus erythematosus. The major targets are the so-called B and D polypeptides. The number of Sm targets increased upon the report that SDS-PAGE conditions could be manipulated to display not one, but three Sm-D polypeptides. To characterize the relative reactivities of Sm-D1, Sm-D2, and Sm-D3, both human and murine autoantibodies were screened. These sera displayed two distinct patterns of reactivity. The Sm-D1/D3 pattern was at least four times more common than Sm-D1/D2/D3 recognition. The predominant immunoreactive protein was Sm-D1. We screened over 40 monoclonal antibodies that were derived from MRL mice and were selected as anti-Sm positive. Of these, 27 reacted with at least one Sm-D polypeptide by protein blot, but in contrast to the MRL sera, none of these antibodies reacted with Sm-D2. Rather, there were two recognition patterns of approximately equal abundance, against Sm-D1/D3 or Sm-D1 alone. Last, we explored the immune response to isolated Sm-D (containing all three D antigens) from rabbit thymus. The autoantibody produced reacted only with Sm-D2. There is accumulating evidence that the anti-Sm response is at least partially antigen-driven. The details of the intragroup relationships within the Sm-D family of proteins provide further insight into the Sm autoimmune response.

The role of environmental antigens in the spontaneous development of autoimmunity in MRL-lpr mice.

It has been proposed that the "normal" stimulation of the immune system that occurs from interactions with environmental stimuli, whether infectious or dietary, is necessary for the initiation and/or continuation of autoimmunity. We tested this hypothesis by deriving a group of MRL-lpr mice into a germfree (GF) environment. At 5 mo of age, no differences between GF and conventional MRL-lpr mice were noted in lymphoproliferation, flow cytometric analysis of lymph node cells (LN), or histologic analysis of the kidneys. Autoantibody levels were comparably elevated in both groups. A second experiment tested the role of residual environmental stimuli by contrasting GF mice fed either a low m.w., ultrafiltered Ag-free (GF-AF) diet or an autoclaved natural ingredient diet (GF-NI). At 4 mo of age, both groups showed extensive lymphoproliferation and aberrant T cell formation, although the GF-AF mice had approximately 50% smaller LNs compared with sex-matched GF-NI controls. Autoantibody formation was present in both groups. Histologic analysis of the kidneys revealed that GF-AF mice had much lower levels of nephritis, while immunofluorescence analysis demonstrated no difference in Ig deposits but did reveal a paucity of C3 deposition in the kidneys of GF-AF mice. These data do not support a role for infectious agents in the induction of lymphoproliferation and B cell autoimmunity in MRL-lpr mice. Furthermore, they suggest that autoantibodies do not originate from B cells that were initially committed to exogenous Ags. They do suggest a possible contributory role for dietary exposure in the extent of lymphoproliferation and development of nephritis in this strain.

The role of host (endogenous) T cells in chronic graft-versus-host autoimmune disease.

Chronic graft-vs-host (cGVH) disease induced by the transfer of Ia-incompatible spleen cells from one normal mouse strain (such as B6.C-H2(bm12)/KhEg (bm12)) to another (such as C57BL/6) causes an autoimmune syndrome resembling systemic lupus erythematosus (SLE). The role of host-derived T cells in this response is not obvious. Previous reports suggested that host T cells might serve to down-regulate the autoimmune syndrome. To address this issue more definitively, we used CD4 knockout (KO) or CD8KO C57BL/6 (B6) mice as recipients in the bm12-->C57B6 cGVH model. CD4KO B6 mice injected with allogeneic bm12 spleen cells (bm12-->CD4KO group) showed no evidence of cGVH disease. They made no detectable autoantibodies, including anti-chromatin, anti-dsDNA, anti-ssDNA, and rheumatoid factor. They survived at least 20 wks after induction of cGVH disease; and they did not develop nephritis, based on the absence of detectable levels of proteinuria and normal renal histology at the time of sacrifice. By contrast, CD8KO B6 mice (bm12-->CD8KO group) and normal B6 mice (bm12-->B6 group) injected with bm12 spleen cells generally showed similar levels of mortality, nephritis, and autoantibodies, although the autoantibody titers declined somewhat after week 8 in the bm12-->CD8KO group. Control groups of recipients injected with B6 spleen cells showed no induction of autoantibodies. A surprising finding, however, was that the B6-->CD8KO group developed severe histologic glomerulonephritis in the absence of autoantibodies and with decreased immune deposits. These results indicate that endogenous (host) CD4+ T cells play an essential role in the cGVH autoimmune syndrome.

Immunological and pathological consequences of mutations in both Fas and Fas ligand.

The lpr mutation in mice results in premature termination of transcription of the gene encoding the apoptosis-signaling receptor Fas. As a result, lpr mice develop severe lymphoproliferation and lupus-like autoantibodies. Growing evidence suggests that the lpr mutation is "leaky" and that low levels of Fas are expressed in lpr mice. To determine if Fas expressed in lpr mice is contributing to apoptosis we generated a novel strain of mice (B6/lprgld) which is homozygous for both the lpr mutation and the gld mutation which encodes nonfunctional Fas ligand (FasL) protein. If low levels of Fas in B6/lpr mice contribute to apoptosis and lessen the severity of disease, the B6/lprgld mice, which also lack functional FasL, would be expected to develop a more severe form of disease than B6/lpr mice. Our results revealed no significant increase in either lymphoproliferation or autoimmunity in B6/lprgld mice compared to B6/lpr or B6/gld mice. Additionally, no increase in surface expression of Fas was detected by flow cytometry on B6/lprgld lymphocytes compared to B6/lpr lymphocytes. However, histological examination of B6/lprgld liver revealed a marked increase in lymphocytic infiltration, compared to livers of B6/lpr and B6/gld mice. Our results suggest that, while low levels of Fas in lpr mice may not be contributing to amelioration of lymphoproliferation or autoimmunity, they may be partially protecting the liver from abnormalities which arise in the absence of Fas-mediated apoptosis.

Novel immunoregulatory B cell pathways revealed by lpr-+ mixed chimeras.

lpr, a murine mutation of the Fas apoptosis receptor, causes lymphadenopathy and autoantibody production, with lymphadenopathy primarily due to a population of CD4-CD8-B220+ T cells. Previous in vivo experiments, in which lpr and normal bone marrow cells were coinfused into lpr hosts, have demonstrated that only T cells of lpr origin accumulated abnormally and only B cells of lpr origin produced autoantibodies. Moreover, in these chimeras, B cells of normal origin were unable to respond to conventional, T cell-dependent exogenous Ag. To address the role of lpr B cells in regulation of lpr autoimmunity, we have prepared lpr-+ mixed chimeras and selectively eliminated lpr B cells using allele-specific, mAb treatment, thus allowing normal B cells to develop in an environment with lpr T cells. From these data, we arrived at four major conclusions: 1) Compared with control-treated chimeric mice, lpr B cell-depleted mice had greatly reduced total lymph node cell counts; 2) the T cells were derived equally from normal and lpr donors, and the percentage of lpr-derived CD4-CD8- T cells was greatly reduced; 3) despite the presence of the remaining lpr T cells, no autoantibodies were produced by the normal derived B cells; and 4) lpr T cells without lpr B cells were unable to prevent a normal B cell response to conventional Ag. These data demonstrate that B cells can play a critical and expansive regulatory role, not only for T cells, but for other B cells as well.

Up-regulation of Fas and the costimulatory molecules B7-1 and B7-2 on peripheral lymphocytes in autoimmune B6/gld mice.

C57BL/6-gld/gld (B6/gld) mice have a point mutation in the gene for Fas ligand (FasL) resulting in nonfunctional FasL protein. We hypothesized that the lack of normal Fas/FasL interactions in these mice might result in abnormalities of Fas expression. Thus, we compared spleen cells from B6/gld mice and normal B6 control mice. While B6 spleen cells consisted of two main populations, Fashigh (high Fas expression) and Faslow (low Fas expression), nearly all B6/gld spleen cells were Fashigh. Two-color immunofluorescence revealed that the Fashigh and Faslow populations in the B6 spleen were Thy-1.2+ (T cells) and IgM+ (B cells), respectively, whereas both T cells and B cells in the B6/gld spleen were Fashigh, indicating that Fas expression is increased on B cells in the B6/gld spleen. This phenomenon was age related and restricted to peripheral lymphocytes. In addition to Fas, B6/gld splenic B cells showed increased expression of the costimulatory molecule B7-2, while the related costimulatory molecule B7-1 was up-regulated on both B cells and T cells in the B6/gld spleen. In vitro, both B cells and T cells from the B6/gld spleen showed an increase in susceptibility to apoptosis mediated by soluble anti-Fas Ab. These results suggest that some lymphocytes in B6/gld mice are primed to undergo Fas-mediated apoptosis, but are unable to do so due to the absence of functional FasL. Further study of such abnormal lymphocytes in the B6/gld spleen may elucidate the nature of autoimmunity in these mice.

B cell genotype determines the fine specificity of autoantibody in lpr mice.

Anti-Sm Abs are specific markers of human systemic lupus erythematosus (SLE) and of murine models of this disease. In humans, anti-Sm Abs are mostly IgG1, and in MRL/lpr mice, IgG2a; both are T-dependent isotypes. Other lpr strains, such as B6/lpr, do not produce anti-Sm Ab spontaneously. The present study was aimed at identifying the cellular expression of background genes responsible for generation of the anti-Sm Ab response in MRL/lpr mice. We used double chimeric mice made by transferring MRL/lpr and B6/lpr bone marrows into irradiated allotype heterozygous F1 mice. Five mo after reconstitution, FACS analysis of lymph node (LN) and spleen cells revealed that both MRL/lpr and B6/lpr cells coexisted in roughly equal numbers. Ab produced by each donor could be distinguished by allotype-specific assays. IgG2a anti-Sm was made only by MRL-derived B cells despite the presence of T cells that might potentially provide help to the B6/lpr B cells. The frequency of anti-Sm Ab-producing individuals was similar to that of unmanipulated MRL/lpr mice (about 25%). IgG2a anti-chromatin and total IgG2a was mostly dominated by the MRL-derived B cells. B6-derived B cells produced more rheumatoid factor (RF) against their own IgG2b(b), while RF against IgG2a was dominated by MRL-derived B cells. This suggests that the control of the production of particular autoantibody specificities, such as anti-Sm, is determined by the expression of MRL or B6 background genes in B cells.

Distinctive immune response patterns of human and murine autoimmune sera to U1 small nuclear ribonucleoprotein C protein.

The Ul small nuclear ribonucleoprotein (snRNP), a complex of nine proteins with Ul RNA, is a frequent target of autoantibodies in human and murine systemic lupus erythematosus (SLE). Anti-Sm antibodies recognizing the B'/B, D, E, F, and G proteins of Ul snRNPs are highly specific for SLE, and are nearly always accompanied by anti-nRNP antibodies recognizing the Ul snRNP-specific 70K, A, and/or C proteins. Previous studies suggest that human anti-nRNP antibodies recognize primarily the U1-70K and Ul-A proteins, whereas recognition of Ul-C is less frequent. We report here that autoantibodies to U1-C are more common in human autoimmune sera than believed previously. Using a novel immunoprecipitation technique to detect autoantibodies to native Ul-C, 75/78 human sera with anti-nRNP/ Sm antibodies were anti-Ul-C (+). In striking contrast, only 1/65 anti-nRNP/Sm (+) MRL mouse sera of various Igh allotypes was positive. Two of ten anti-nRNP/Sm (+) sera from BALB/c mice with a lupus-like syndrome induced by pristane recognized Ul-C. Thus, lupus in MRL mice was characterized by a markedly lower frequency of anti-U1-C antibodies than seen in human SLE or pristane-induced lupus. The results may indicate different pathways of intermolecular-intrastructural diversification of autoantibody responses to the components of Ul snRNPs in human and murine lupus, possibly mediated by alterations in antigen processing induced by the autoantibodies themselves.

Both Sm and DNA are selecting antigens in the anti-Sm B cell response in autoimmune MRL/lpr mice.

More than half of the anti-Sm hybridomas isolated from MRL/Mp-lpr/lpr (MRL/lpr) mice produce Abs that also bind ssDNA, and half of these bind dsDNA. Intraclonal comparisons indicate that DNA is a selecting Ag for at least some dual-binding clones. To determine whether Sm itself is a selecting Ag for anti-Sm, we have identified the somatic mutations within the expressed VH and V kappa genes of eight anti-Sm hybridomas, six of which do not bind DNA. We find these V genes have between 0 and 12 somatic mutations each, and that four hybridomas possess a higher number of heavy or light chain CDR replacement (R) mutations than expected by chance, suggesting that these anti-Sm-producing B cells have undergone Ag selection. To demonstrate directly the effect of somatic mutation on Sm binding, we have engineered the unmutated counterpart of Ab 2-12, an Sm-specific hybridoma Ab with a nonrandom distribution of V kappa CDR R mutations, and compared its ability to bind Sm and ssDNA with that of the originally isolated 2-12 Ab. We find that the unmutated Ab has a much lower avidity for Sm than the mutant, but, unlike the mutant, it binds ssDNA. We conclude that Sm can drive clonal expansion in the anti-Sm response, and that Sm-only binding B cells can arise from Sm/DNA dual-binding B cell clonal precursors. These data also suggest that dual binding is not necessary to sustain clonal expansion. Thus, this response is unique in that it can be driven by either of two Ags.

MHC genes modify systemic autoimmune disease. The role of the I-E locus.

The MHC exerts an important influence on systemic autoimmune disease. In C57BL/6-lpr/lpr (B6/lpr) mice, substitution of the H-2d instead of the H-2b MHC haplotype results in a global reduction in autoantibody levels. Since H-2d expresses both I-A and I-E, while H-2b expresses only I-A, general down-regulation of autoimmunity in the d haplotype might be due to I-E expression. This was tested with I-E alpha d transgenic B6/lpr mice, which expressed a functional surface I-E molecule. Five-month-old transgene-positive B6/lpr mice had much lower total IgG, IgG anti-chromatin, anti-DNA, and IgM rheumatoid factor directed against IgG1 and against IgG2b than transgene-negative littermates (p < or = 0.002), as well as significantly lower spleen and lymph node weights (p < or = 0.002). Decreases in autoantibody levels in the transgenic lpr mice were not due to a nonspecific effect of the I-E alpha d transgene, since transgene-positive B6/lpr.H-2d mice had levels of autoantibodies comparable with transgene-negative B6/lpr.H-2d mice. To determine whether autoantibody was preferentially made by I-E-negative B cells, irradiated (B6/lpr.Igha x B6/lpr.I-E alpha d)F1 mice were reconstituted with equal amounts of B6/lpr.Igha and B6/lpr.I-E alpha d bone marrow. Allotype-specific ELISA showed that most autoantibody was produced by the I-E negative B cells (range 97% to 84%). The results show that a functional I-E molecule in lpr mice leads to generalized reduction in autoantibody levels through a direct effect on the B cell. The molecular mechanism of this effect remains to be determined.

Phenotypic abnormalities of splenic and bone marrow B cells in lpr and gld mice.

Mice homozygous for the mutant Fas gene lpr develop generalized lymphoproliferation and produce autoantibodies resembling those found in human SLE. We have previously shown that these autoantibodies are produced by B2 cells rather than B1 cells and that the autoantibody- producing B cells are intrinsically abnormal. We investigated further the lpr B cell with a large panel of antibodies to B-cell surface markers to identify phenotypic abnormalities. B cells from spleen and bone marrow of age-matched congenic mice differing only at the lpr locus were examined by flow cytometry. Two consistent phenotypic differences were identified. First, spleen cells from older lpr mice had an increase in the number and percentage of IgM+ B cells expressing low levels of CD23. Second, lpr bone marrow had decreased numbers of B220hiIgM+-syndecan-1+CD23+ B cells. All other markers tested, except the previously identified modest increase of Ia on lpr spleen cells, showed no consistent differences. B cells from gld mice showed the same phenotypic abnormalities as those from lpr. Compared to T cells, the relative paucity of cell surface marker differences between lpr and +/+ B cells suggests that B cells may have fewer regulatory mechanisms to silence autoreactive specificities. The phenotypic differences identified may provide clues to the mechanism of autoantibody production in lpr mice, while the overwhelming phenotypic similarity between lpr and +/+ B cells suggests that the major abnormality of lpr B cells may lie in their specificity, that is, in their inability to delete autoreactive subsets.

Suppression and reversal of gld disease by parabiosis with normal mice.

The disruption of the Fas receptor or Fas ligand by the lpr or gld mutations, respectively, results in severe autoimmune and lymphoproliferative disease due to the failure of Fas-mediated deletion of self-reactive lymphocytes. Recently, we have shown in mixed chimeras that gld-induced autoimmunity could be corrected by normal bone marrow, in particular by normal T cells. In contrast, lpr-mediated autoimmunity could not be influenced by normal bone marrow-derived cells. In the present report, we have studied the role of normal lymphocytes in suppressing or reversing gld-induced autoimmunity by parabiosis with normal mice. Our results show a suppression of lymphadenopathy, fewer CD4-CD8- T cells, and lower levels of autoantibody production in gld mice parabiosed with normal mice at 4-6 weeks of age. The gld mice parabiosed with normal mice at 4 months of age also exhibited a substantial reduction of both total and CD4-CD8- T cells in the periphery 2 months after surgery. However, they showed little reduction of autoantibodies compared to gld mice parabiosed with gld mice. In contrast, older lpr mice did not exhibit any reduction in lymphadenopathy or autoantibody production after parabiosis with normal mice. The prevention or reversal of lymphadenopathy in parabiosed gld mice suggests that ongoing Fas-mediated deletion in the periphery may play an important role in maintaining self-tolerance. The relative irreversibility of autoantibody synthesis in older parabiosed gld mice suggests that autoantibody-producing B cells or their committed precursors are long lived and do not express functional Fas receptor.

bcl-2 transgenic Lpr mice show profound enhancement of lymphadenopathy.

The lpr gene encodes a defective form of the fas gene that mediates apoptosis, and its expression results in autoantibodies and massive lymphadenopathy. bcl-2, another gene locus that affects programmed cell death, acts to inhibit apoptosis. Since multiple mechanisms controlling programmed cell death may contribute to systemic autoimmunity, the effect of the bcl-2 transgene on the lpr model was examined by crossing bcl-2 transgenic and C57BL/6-lpr mice. Compared with bcl-2-/lpr mice, bcl-2+/lpr showed dramatic increases in lymphadenopathy and T cell accumulation, but not in autoantibodies or B cell numbers. Short term transfer studies demonstrated that double negative T cells normally have a limited lifespan, and their survival is enhanced by the bcl-2 transgene. Thus, defects in separate apoptosis mechanisms may combine to produce enhanced pathologic effects.

The Yaa gene-mediated acceleration of murine lupus: Yaa- T cells from non-autoimmune mice collaborate with Yaa+ B cells to produce lupus autoantibodies in vivo.

The BXSB Y chromosome-linked mutant gene, Yaa, promotes autoimmune responses in mice predisposed to a lupus-like autoimmune disease. We have previously shown that a cognate interaction of T cells with B cells expressing the Yaa gene appears to be responsible for the accelerated production of autoantibodies. To investigate whether T cells that provide help for autoantibody production by Yaa+ B cells need to express the Yaa gene, we have made radiation bone marrow chimeras containing two sets of T and B cells from mice with or without the Yaa gene and differing by the Thy-1 and Igh allotypes. We then determined autoantibody production following the selective elimination of T cells of Yaa+ origin by treating mice with allele-specific anti-Thy-1 monoclonal antibody. Our results demonstrated that the selective production of autoantibodies by Yaa+ B cells in Yaa(+)-Yaa- double bone marrow chimeras can be mediated as efficiently by T cells from non-autoimmune mice lacking the Yaa gene as by T cells from autoimmune mice bearing the Yaa gene. This indicates that T cells from non-autoimmune Yaa- mice are capable of providing help for autoimmune responses by collaborating with Yaa+ B cells. These data thus strongly suggest that the Yaa gene defect is not functionally expressed in T cells, but only in B cells, and contrast with parallel experiments in the lpr model, in which defects of the Fas antigen in both T and B cells are crucial for the lpr gene-mediated promotion of autoantibody production.

Modulation of disease activity in murine systemic lupus erythematosus by cytokine gene delivery.

Somatic gene therapy is a novel approach with the potential to achieve prolonged increases in circulating levels of peptide hormones and cytokines. The present study evaluates the effects of monthly, intramuscular injections of cDNA expression vectors encoding for transforming growth factor beta (TGF beta) or interleukin 2 (IL-2) on disease activity in the MRL/lpr/lpr murine model of systemic lupus erythematosus (SLE). Monthly injections of plasmids cDNA between 6 and 26 weeks significantly elevated the serum levels of TGF beta (P < 0.005) and IL-2 (P < 0.05) compared with a control plasmid without insert. TGF beta encoding plasmid had beneficial effects in murine SLE with a prolonged survival of 70% at 26 weeks compared with 40% in the control group, decreased anti-chromatin and rheumatoid factor antibodies and a 50% decrease in total IgG production. Renal function was improved with reduced BUN levels and kidney inflammation as estimated by an histologic score. Those beneficial effects occurred in the apparent absence of local or systemic side-effects. In contrast, IL-2 cDNA injections appeared harmful with a decreased survival to 20% at 26 weeks, enhanced total IgG synthesis and autoantibodies production with a 4.5-fold increase in antichromatin antibodies. These results indicate that somatic gene therapy may provide a simple, inexpensive and effective mechanism for the long-term control of autoimmune diseases.

Sm and DNA binding by dual reactive B cells requires distinct VH, V kappa, and VH CDR3 structures.

We have previously demonstrated an overlap of the anti-Sm and anti-DNA responses in MRL/Mp-lpr/lpr mice. The Ab produced by many anti-Sm hybridomas bind DNA and are encoded by Ig V genes used by anti-DNA hybridomas. In addition, some anti-Sm Ab that bind DNA have acquired mutations that improve DNA binding, indicating that DNA is a selecting Ag in the anti-Sm response. To gain insight into the basis for the dual binding ability of these Ab, we coexpressed the H chain from the anti-Sm hybridoma 2-12 with nine different L chains. Hybridoma 2-12 binds Sm but not DNA, yet expresses the same J558 VH gene as three anti-Sm hybridomas that bind ssDNA and at least one anti-DNA hybridoma that does not bind Sm. We found that most of the transfectoma Ab bind Sm, but their avidities vary over more than 3 orders of magnitude. Five of the nine transfectoma Ab bind ssDNA, and none bind dsDNA. In general, the ability to bind each Ag follows the binding ability of the hybridoma from which the L chain is derived. H Chain swapping experiments indicate that the H chain, VH CDR3 in particular, contributes to the binding of both Sm and DNA. We conclude that Sm and DNA select for distinct features of VH, V kappa, and VH CDR3, suggesting selection by both Ag in the anti-Sm response.

The abnormal lpr double-negative T cell fails to proliferate in vivo.

Mice homozygous for the autosomal recessive gene lpr develop marked lymphadenopathy and a systemic autoimmune disease resembling human systemic lupus erythematosus. The enlarged nodes are dominated by T cells with an unusual surface phenotype: dull Thy-1+, dull CD3+, CD4-, CD8-, B220+ (double-negative T cells or DNTs). Despite their massive accumulation in vivo, these cells fail to proliferate in response to conventional T-cell mitogens in vitro. The identification of the lpr mutation as a defect in the Fas apoptosis receptor gene suggests that DNT accumulation may result from abnormal persistence rather than overproliferation. To test in vivo whether DNTs persist abnormally or have a capacity to differentiate into single-positive T cells, we have performed cell transfer experiments between congenic strains of lpr and +/+ mice differentially marked by expression of the Ly-1 or Thy-1 alleles. Although transferred lpr lymph node cells were mostly DNTs at the time of injection, most recovered cells of donor origin were single positive, particularly CD8+, at all time points after transfer. Furthermore, transfer of purified DNTs resulted in recovery of relatively few cells of donor origin. Transfer of lpr T cells enriched for CD8 expression confirmed the preferential survival of this subset. Thus, DNTs are a surprisingly transient population and have little capacity for transformation to single positives. This would suggest that DNTs are constantly being renewed, perhaps from CD4+ and CD8+ precursors.