Identification of candidate genes that influence sex hormone-dependent disease phenotypes in mouse lupus.

Ninety percent of systemic lupus erythematosus patients are female, and gender differences in lupus susceptibility are also observed in (New Zealand Black x New Zealand White)F1 (BWF1) lupus-prone mice. We followed orchiectomized, intact male and female BWF1 mice for lupus-like disease for 1 year. A comparative gene expression analysis was then used to identify candidate genes potentially responsible for gender-dependent differences in lupus susceptibility. Seven genes encoded on the sex chromosomes and 77 probe sets, including 14 immunoglobulin genes, encoded on the autosomal chromosomes were identified as differentially expressed in male versus female BWF1 splenocytes prior to disease onset. Five genes were determined to be regulated by either estradiol or dihydrotestosterone in an in vivo system and most of them were preferentially expressed in antigen-presenting cells. Gender differences in the expression of Csf3-r, Histh1c, Serpinb2, Slc6a4 and Cd22 in BWF1 mice are the result of transcriptional modification by sex hormones and warrant further investigation. The identification of candidate genes and their expression patterns in splenocyte sub-populations provide new information regarding the mechanisms by which sex hormones influence the development of mouse lupus.

Type I interferon signaling is involved in the spontaneous development of lupus-like disease in B6.Nba2 and (B6.Nba2 x NZW)F(1) mice.

Several studies have described a role for type I interferons (IFNalphabeta) in the initiation and/or prolongation of autoimmune diseases. Most pronounced has been the association of disease activity with what is now known as 'the interferon signature' of gene expression in peripheral blood mononuclear cells from lupus patients. In correlation, studies have shown that inhibition of IFNalphabeta signaling abrogates disease in various mouse models of lupus. New Zealand black (NZB) and B6.Nba2 congenic mice spontaneously develop elevated levels of serum anti-nuclear autoantibodies (ANAs). Nevertheless, neither of these strains develop fatal renal disease. The female F1 offspring of NZB or B6.Nba2 crossed with New Zealand white (NZW) mice do, however, develop kidney disease. We have previously shown that increases in endogenous IFNalphabeta levels in (B6.Nba2 x NZW)F1 mice leads to accelerated development of renal disease in an IFNalphabeta-dependent manner. We now show that B6.Nba2 and (B6.Nba2 x NZW)F1 mice deficient for the IFNalphabeta-receptor fail to develop ANA and renal disease, although the mice have substantial immune complex deposition in the glomeruli. Thus, endogenous IFNalphabeta might influence disease by affecting B-cell activation and differentiation, as well as the kidneys' susceptibility to damage, the latter perhaps through induction of a local inflammatory milieu.

Genetic susceptibility to polyI:C-induced IFNalpha/beta-dependent accelerated disease in lupus-prone mice.

Systemic lupus erythematosus (SLE) is an autoimmune disease of unknown etiology. Associations between viral infections and the onset of SLE have been suggested, and recent studies have provided evidence that type I interferons (IFNalpha/beta) might play a role in the SLE disease process. Viruses and interferons have also been implicated in mouse models of SLE. We generated a model of accelerated proteinuria, in which lupus-prone mice were injected repeatedly with polyinosinic:polycytidylic acid (polyI:C), mimicking exposure to virus-derived double stranded RNA (dsRNA), leading to the production of IFNalpha/beta. PolyI:C-treated (B6.Nba2 x NZW)F1 and (B6 x NZW)F1 hybrid mice developed significantly increased levels of anti-dsDNA autoantibodies, characteristic of lupus. Most significantly, polyI:C-treated (B6.Nba2 x NZW)F1 mice, but not (B6 x NZW)F1 or parental strains, developed lupus-like nephritis in an accelerated fashion, which was dependent on IFNalpha/beta and associated with elevated deposition of total IgG, IgG2a and complement factor C3 in the glomerular capillary walls. These data suggest that reagents, which increase the levels of endogenous IFNalpha/beta (directly or indirectly), can accelerate the course of lupus-like nephritis, the development of which is dependent on the presence of both NZW- and Nba2-encoded genes.

Chronic beryllium disease: immune-mediated destruction with implications for organ-specific autoimmunity.

Chronic beryllium disease (CBD) is caused by exposure to beryllium in the workplace and is characterized by an accumulation of beryllium-specific CD4+ T cells with granulomatous inflammation in the lung. Owing to its unique physical properties, beryllium is used in a variety of high-technology industries, and CBD continues to be an important public health concern. CBD develops in up to 16% of exposed workers, depending on genetic susceptibility and the nature of the exposure. Increased susceptibility has been associated with particular HLA-DP alleles, especially those possessing negatively charged residues at certain positions of the peptide-binding pocket. The mechanism for this disease association lies in the ability of certain HLA-DP molecules, with associated peptides, to bind and present beryllium to pathogenic CD4+ T cells. In patients with CBD, large numbers of effector memory CD4+ T cells are compartmentalized to the lung, and these cells are poised to release T helper 1-type cytokines upon beryllium recognition. In the same patients, however, beryllium-specific T cells are barely detectable in the circulation. As opposed to those present in blood, beryllium-specific cells in the lung no longer require the engagement of CD28 for optimal T-cell activation and in fact frequently lose the expression of CD28. These findings in CBD have important implications for studies in autoimmune diseases, including those in which the antigen is unknown and the target organ is inaccessible.

Evidence for an interferon-inducible gene, Ifi202, in the susceptibility to systemic lupus.

The Nba2 locus is a major genetic contribution to disease susceptibility in the (NZB x NZW)F(1) mouse model of systemic lupus. We generated C57BL/6 mice congenic for this NZB locus, and these mice produced antinuclear autoantibodies characteristic of lupus. F(1) offspring of congenic and NZW mice developed high autoantibody levels and severe lupus nephritis similar to (NZB x NZW)F(1) mice. Expression profiling with oligonucleotide microarrays revealed only two differentially expressed genes, interferon-inducible genes Ifi202 and Ifi203, in congenic versus control mice, and both were within the Nba2 interval. Quantitative PCR localized increased Ifi202 expression to splenic B cells and non-T/non-B cells. These results, together with analyses of promoter region polymorphisms, strain distribution of expression, and effects on cell proliferation and apoptosis, implicate Ifi202 as a candidate gene for lupus.

Autoimmune disease: why and where it occurs.

Autoimmune disease is controlled by genetic and environmental factors. Both of these affect susceptibility to autoimmunity at three levels: the overall reactivity of the immune system, the specific antigen and its presentation, and the target issue.

Characterization of CD4(+) T cell autoreactivity to self-MHC in New Zealand hybrid mice.

The New Zealand white (NZW) H2(z) locus is strongly associated with the development of autoimmune disease in (NZB x NZW)F(1) mice, a model of systemic lupus erythematosus. To better understand the role of H2(z) in autoimmunity, we generated CD4(+) T cell hybridomas from the spleen and lymph nodes of unimmunized (NZB x NZW)F(1) mice and characterized their specificity. We found that over 50% of the hybridomas responded to syngeneic (H2(d/z)) spleen cells in the absence of exogenous antigen. Many of these autoreactive hybridomas responded to spleen cells expressing H2(z) and used H2(z) class II (I-A(z) or I-E(z)) molecules for presentation. Interestingly, nearly one third of the H2(z)-reactive hybridomas could not respond to spleen cells expressing only H2(z) class II molecules. These studies characterize a frequent population of autoreactive CD4(+) T cells in lupus mice and indicate that major histocompatibility complex molecules in addition to class II may be important for this self-recognition.

Oligoclonal expansion of intraepidermal T cells in psoriasis skin lesions.

CD8(+) T cell infiltration into the epidermis is thought to be a key event in the pathogenesis of psoriasis. A quantitative competitive polymerase chain reaction method was developed to examine the expression of T cell receptor beta chain variable region 2, 3, 6.1-3, 8, and 13.1 genes in the epidermis of psoriatic lesions. Paired epidermal samples and peripheral blood samples from five psoriasis patients were studied. The results demonstrated the expansion of T cell receptor beta chain variable region 3 (two patients), 8 (two patients), and/or 2 (one patient). Contrary to previous reports, neither beta chain variable region 6.1-3 nor beta chain variable region 13.1 subgroups were expanded in any of the lesions. DNA sequence analysis revealed dominant T cell clones observed in all expanded beta chain variable region families and heterogeneous populations and/or small clones observed in non-expanded beta chain variable region families. Using CDR3 length analysis to examine the complete beta chain repertoire of the infiltrating T cells in the lesional epidermis, we found that approximately 50% of the T cell receptor beta chain variable region families in each patient's lesion demonstrated abnormal CDR3 DNA length distribution, indicating the presence of monoclonal or oligoclonal T cell expansion. Together, the results show that among different patients, T cell oligoclonality is not restricted to a limited number of T cell receptor beta chain variable region families. In an attempt to identify the pathogenic T cells among the many expanded T cell clones in the lesions, we compared T cell receptor expansion in the lesional epidermis with non-lesional epidermis. Particular T cell receptor were found to be preferentially expanded in lesional epidermis and these lesion-specific T cell clones may be most important in the pathogenesis and development of psoriatic lesions.

Beryllium presentation to CD4+ T cells underlies disease-susceptibility HLA-DP alleles in chronic beryllium disease.

Chronic beryllium disease results from beryllium exposure in the workplace and is characterized by CD4(+) T cell-mediated inflammation in the lung. Susceptibility to this disease is associated with particular HLA-DP alleles. We isolated beryllium-specific T cell lines from the lungs of affected patients. These CD4(+) T cell lines specifically responded to beryllium in culture in the presence of antigen-presenting cells that expressed class II MHC molecules HLA-DR, -DQ, and -DP. The response to beryllium was nearly completely and selectively blocked by mAb to HLA-DP. Additional studies showed that only certain HLA-DP alleles allowed presentation of beryllium. Overall, the DP alleles that presented beryllium to disease-specific T cell lines match those implicated in disease susceptibility, providing a mechanism for this association. Based on amino acid residues shared by these restricting and susceptibility DP alleles, our results provide insight into the residues of the DP beta-chain required for beryllium presentation.

Genetic control of glycoprotein 70 autoantigen production and its influence on immune complex levels and nephritis in murine lupus.

The F1 hybrids of New Zealand Black (NZB) and New Zealand White (NZW) mice spontaneously develop an autoimmune disease that serves as a model for human systemic lupus erythematosus. Autoimmunity in (NZB x NZW)F1 mice includes the production of autoantibodies to the endogenous retroviral envelope glycoprotein, gp70, and gp70-anti-gp70 immune complexes (gp70 IC) have been implicated in the development of lupus nephritis in these animals. We used backcross and intercross combinations of C57BL/6 (B6; low gp70 levels) and NZB mice (high gp70 levels) to examine the contribution of serum gp70 Ag levels to the development of gp70 IC and nephritis. Analysis of (B6.H2z x NZB)F1 x NZB backcross mice and (NZB x B6)F2 mice showed a much stronger association of gp70 IC with kidney disease compared with IgG anti-chromatin autoantibodies in both populations of mice. Serum levels of gp70 correlated with production of gp70 IC in mice producing autoantibodies, although the overall effect on nephritis appeared to be small. Genetic mapping revealed three NZB-derived regions on chromosomes 2, 4, and 13 that were strongly linked with increased gp70 levels, and together, accounted for over 80% of the variance for this trait. However, additional linkage analyses of these crosses showed that loci controlling autoantibody production rather than gp70 levels were most important in the development of nephritogenic immune complexes. Together, these studies characterize a set of lupus-susceptibility loci distinct from those that control autoantibody production and provide new insight into the components involved in the strong association of gp70 IC with murine lupus nephritis.

Enhanced susceptibility to lupus contributed from the nonautoimmune C57BL/10, but not C57BL/6, genome.

Genes from New Zealand Black and New Zealand White mice have been implicated in the development of a disease similar to human systemic lupus erythematosus. In an attempt to define the MHC class II genes involved in disease, we previously studied similarly designed backcrosses of New Zealand Black mice with C57BL/6 (B6) mice transgenic for Ez genes or with C57BL/10 (B10) mice transgenic for Az genes. Although the transgenes showed no effect on the development of autoantibody production or lupus nephritis in either backcross, surprisingly, there was greatly increased expression of these disease traits in the backcrosses involving B10 compared with B6 mice. These studies therefore implicated genetic contributions in B10 vs B6 backgrounds, despite their 98% identity. A genome-wide linkage analysis uncovered a B10 locus on mid-chromosome 13, which enhanced nephritis and was strongly linked with the production of pathogenic retroviral gp70-anti-gp70 immune complexes when contributed by B10, but not B6, mice. The subsequent identification of a single marker polymorphic between B10 and B6, along with the extreme genetic similarity between the two strains in this region, is likely to permit expedited identification of the lupus-susceptibility gene from this nonautoimmune strain.

Functional subsets within clonally expanded CD8(+) memory T cells in elderly humans.

With advancing age, healthy humans frequently demonstrate large clonal expansions of CD8(+) T cells in the peripheral blood, which persist for long periods of time and appear to be maintained as a population of memory cells. We studied nine large T cell clones in five elderly individuals. We noted that in most cases the expanded clones were dominated by cells that did not express CD28, a pivotal molecule in T cell activation, and these clones proliferated poorly in culture. However, nearly all of the clonal expansions had CD28(+) fractions and some of these cells appeared to lose CD28 gene expression with stimulation in culture. CD28(+) cells demonstrated greater proliferation in both bulk and limiting dilution cultures compared to CD28(-) cells bearing the same TCR, whereas CD28(-) cells showed increased perforin expression. Together, these data suggest that loss of CD28 expression marks functional differentiation to cytotoxic memory cells within these clonal expansions and likely within CD8(+) memory populations in general.

Use of soluble peptide-DR4 tetramers to detect synovial T cells specific for cartilage antigens in patients with rheumatoid arthritis.

Considerable evidence indicates that CD4(+) T cells are important in the pathogenesis of rheumatoid arthritis (RA), but the antigens recognized by these T cells in the joints of patients remain unclear. Previous studies have suggested that type II collagen (CII) and human cartilage gp39 (HCgp39) are among the most likely synovial antigens to be involved in T cell stimulation in RA. Furthermore, experiments have defined dominant peptide determinants of these antigens when presented by HLA-DR4, the most important RA-associated HLA type. We used fluorescent, soluble peptide-DR4 complexes (tetramers) to detect synovial CD4(+) T cells reactive with CII and HCgp39 in DR4(+) patients. The CII-DR4 complex bound in a specific manner to CII peptide-reactive T cell hybridomas, but did not stain a detectable fraction of synovial CD4(+) cells. A background percentage of positive cells (<0.2%) was not greater in DR4 (DRB1*0401) patients compared with those without this disease-associated allele. Similar results were obtained with the gp39-DR4 complex for nearly all RA patients. In a small subset of DR4(+) patients, however, the percentage of synovial CD4(+) cells binding this complex was above background and could not be attributed to nonspecific binding. These studies demonstrate the potential for peptide-MHC class II tetramers to be used to track antigen-specific T cells in human autoimmune diseases. Together, the results also suggest that the major oligoclonal CD4(+) T cell expansions present in RA joints are not specific for the dominant CII and HCgp39 determinants.

Immune reconstitution in the first year of potent antiretroviral therapy and its relationship to virologic response.

The effects of 1 year of zidovudine, lamivudine, and ritonavir treatment on immune reconstitution were evaluated in 34 human immunodeficiency virus (HIV)-infected individuals. After 48 weeks of therapy, 20 (59%) subjects had <100 copies HIV RNA/mL. CD4+ T cells increased from a median of 192/mm3 at baseline to 362/mm3 at week 48. Lymphocyte proliferative responses to Candida normalized within 12 weeks, but responses to HIV and tetanus remained depressed throughout therapy. Alloantigen responses increased within 12 weeks and then declined to baseline levels. Recovery of delayed-type hypersensitivity responses occurred after 12 weeks for Candida and after 48 weeks for mumps. The magnitude of virologic suppression was correlated with numeric increases in CD4+ T cells, but not with measures of functional immune reconstitution. Plasma virus suppression <100 copies/mL was not significantly correlated with increases in CD4+ T cells or functional immune reconstitution.

Abnormal thymic expression of epithelial cell adhesion molecule (EP-CAM) in New Zealand Black (NZB) mice.

New Zealand Black (NZB) mice have been well documented to have a variety of thymic epithelial cell microenvironmental abnormalities, including disruption of corticoepithelial cell networks and medullary cell clusters. These abnormalities of the thymic stromal network are particularly important because similar observations have been noted in other models of murine lupus. Thymic epithelial cells, a key component of the microenvironment, play an important role in selection of the mature T cell receptor repertoire. Recently, a homotypic calcium-independent human and murine epithelial cell adhesion molecule, Ep-CAM, has been described which is located at the thymocyto-cortical cell junction. The function of Ep-CAM is still unclear but its unique location within the thymus suggests that it is critical in the process of providing maturation signals. Consequently, we examined the thymic expression of Ep-CAM in a series of autoimmune prone mice by thymic distribution of Ep-CAM in NZB, NZW, NZB/W, BXSB-Yaa, MRL- lpr/lpr, C3H- gld/gld and the control strains BALB/c, C57BL6, C3H and MRL(+/+), by immunohistology and flow cytometry. Interestingly, NZB mice are similar to control mice from day 4 to 2 weeks of age, having a very low expression of Ep-CAM at the thymocyto-cortical junction. In control strains, there is a marked increased in expression of Ep-CAM beginning at 5 weeks of age. In contrast, NZB mice fail to show significant expression of Ep-CAM even well into adulthood. This abnormality of NZB mice was also noted in NZB/W F1 and BXSB mice, but not MRL- lpr/lpr or C3H- gld/gld mice. Given the potential importance of Ep-CAM in thymic selection, this study provides important evidence that a defective stromal microenvironment is likely to be of etiological significance in the susceptibility of NZB to autoimmune disease.

Control of separate pathogenic autoantibody responses marks MHC gene contributions to murine lupus.

Previous studies have suggested that MHC and non-MHC genes contribute to the development of autoimmune disease in F1 hybrids of New Zealand black (NZB) and white (NZW) mice. We conducted a genome-wide screen of 148 female (NZB x NZW)F1 x NZB backcross mice to map dominant NZW genetic loci linked with lupus disease traits. In this backcross analysis, inheritance of the NZW MHC (H2(d/z) vs. H2(d/d)) was strongly linked with the development of lupus nephritis (P approximately 1 x 10(-16)), increasing the risk of disease by over 30-fold. H2(d/z) was also linked with elevated serum levels of IgG autoantibodies to single-stranded DNA, double-stranded DNA, histones, and chromatin but not with anti-gp70 autoantibodies, measured as circulating gp70-anti-gp70 immune complexes. Non-MHC contributions from NZW seemed weak in comparison to MHC, although NZW loci on chromosomes 7 and 16 were noted to be suggestively linked with autoantibody production. Strikingly, H2(d/z) (compared with H2(d/d)) enhanced antinuclear antibodies in a coordinate fashion but did not affect anti-gp70 production in the current backcross. However, the opposite influence was noted for H2(d/z) (compared with H2(z/z)) when (NZB x NZW)F1 x NZW backcross mice were analyzed. These results suggest that H2(z) and H2(d) haplotypes differentially regulate two different sets of nephritogenic autoantibody responses. This study confirms a critical role for H2(z) compared with other dominant NZW loci in (NZB x NZW)F1 mice and provides an explanation as to why H2(d/z) heterozygosity is required for full expression of disease in this model.

Identification of pathogenic T cells in patients with beryllium-induced lung disease.

Chronic beryllium disease (CBD) is caused by beryllium exposure and is characterized by granulomatous inflammation with accumulation of CD4+ T cells in the lung. We analyzed TCR beta-chain and alpha-chain genes expressed by these CD4+ T cells. In the lungs of individual patients, as well as among four of five CBD patients studied, different oligoclonal expansions within the Vbeta3 subset were found to express homologous or even identical CDR3 amino acid sequences. These related expansions were specific for CBD patients, were compartmentalized to lung, and persisted at high frequency in patients with active disease. Limiting dilution cloning and analysis of coexpressed TCR alpha-chain genes confirmed that these TCRs were selectively expanded by a common Ag involving beryllium. Overall, homologous TCR beta- and alpha-chains showed identical V regions and invariant charged residues within the CDR3 but considerable variability in TCRJ usage. Remarkably, CBD patients expressing nearly identical TCRs did not share common HLA-DRB1 or DQ alleles. These results implicate particular CD4+ cells in the pathogenesis of CBD and provide insight into how beryllium is recognized in human disease.