In-person versus virtual administration of the American College of Rheumatology gold standard cognitive battery in systemic lupus erythematosus: Are they interchangeable?

OBJECTIVE: During the COVID-19 pandemic, many research studies were adapted, including our longitudinal study examining cognitive impairment (CI) in systemic lupus erythematosus (SLE). Cognitive testing was switched from in-person to virtual. This analysis aimed to determine if the administration method (in-person vs. virtual) of the ACR-neuropsychological battery (ACR-NB) affected participant cognitive performance and classification. METHODS: Data from our multi-visit, SLE CI study included demographic, clinical, and psychiatric characteristics, and the modified ACR-NB. Three analyses were undertaken for cognitive performance: (1) all visits, (2) non-CI group visits only and (3) intra-individual comparisons. A retrospective preferences questionnaire was given to participants who completed the ACR-NB both in-person and virtually. RESULTS: We analysed 328 SLE participants who had 801 visits (696 in-person and 105 virtual). Demographic, clinical, and psychiatric characteristics were comparable except for ethnicity, anxiety and disease-related damage. Across all three comparisons, six tests were consistently statistically significantly different. CI classification changed in 11/71 (15%) participants. 45% of participants preferred the virtual administration method and 33% preferred in-person. CONCLUSIONS: Of the 19 tests in the ACR-NB, we identified one or more problems with eight (42%) tests when moving from in-person to virtual administration. As the use of virtual cognitive testing will likely increase, these issues need to be addressed - potentially by validating a virtual version of the ACR-NB. Until then, caution must be taken when directly comparing virtual to in-person test results. If future studies use a mixed administration approach, this should be accounted for during analysis.

Suppression of autoimmunity by CD5(+) IL-10-producing B cells in lupus-prone mice.

Systemic lupus erythematosus is a complex autoimmune disorder characterized by the production of pathogenic anti-nuclear antibodies. Previous work from our laboratory has shown that the introgression of a New Zealand Black-derived chromosome 4 interval onto a lupus-prone background suppresses the disease. Interestingly, the same genetic interval promoted the expansion of both Natural Killer T- and CD5(+) B cells in suppressed mice. In this study, we show that ablation of NKT cells with a CD1d knockout had no impact on either the suppression of lupus or the expansion of CD5(+) B cells. On the other hand, suppressed mice had an expanded population of IL-10-producing B cells that predominantly localized to the CD5(+)CD1d(low) compartment. The expansion of CD5(+) B cells negatively correlated with the frequency of pro-inflammatory IL-17 A-producing T-cells and kidney damage. Adoptive transfer with a single injection of total B cells with an enriched CD5(+) compartment reduced the frequency of memory/activated, IFNγ-producing, and IL-17 A-producing CD4 T-cells but did not significantly reduce autoantibody levels. Taken together, these data suggest that the expansion of CD5(+) IL-10-producing B cells and not NKT cells protects against lupus in these mice, by limiting the expansion of pro-inflammatory IL-17 A- and IFNγ-producing CD4 T-cells.

Identification of a lupus-susceptibility locus leading to impaired clearance of apoptotic debris on New Zealand Black chromosome 13.

Systemic lupus erythematosus is a chronic multi-organ autoimmune disease marked mainly by the production of anti-nuclear antibodies. Nuclear antigens become accessible to the immune system following apoptosis and defective clearance of apoptotic debris has been shown in several knockout mouse models to promote lupus. However, genetic loci associated with defective clearance are not well defined in spontaneously arising lupus models. We previously showed that introgression of the chromosome 13 interval from lupus-prone New Zealand Black (NZB) mice onto a non-autoimmune B6 genetic background (B6.NZBc13) recapitulated many of the NZB autoimmune phenotypes. Here, we show that B6.NZBc13 mice have impaired clearance of apoptotic debris by peritoneal and tingible-body macrophages and have narrowed down the chromosomal interval of this defect using subcongenic mice with truncated NZB chromosome 13 intervals. This chromosomal region (81-94 Mb) is sufficient to produce polyclonal B- and T-cell activation, and expansion of dendritic cells. To fully recapitulate the autoimmune phenotypes seen in B6.NZBc13 mice, at least one additional locus located in the centromeric portion of the interval is required. Thus, we have identified a novel lupus susceptibility locus on NZB chromosome 13 that is associated with impaired clearance of apoptotic debris.

The lupus phenotype in B6.NZBc1 congenic mice reflects interactions between multiple susceptibility loci and a suppressor locus.

Lupus susceptibility loci on chromosome 1 have an important role in the development of autoimmunity in the New Zealand Black (NZB) mouse. We have previously shown that C57BL/6 congenic mice with an introgressed homozygous NZB chromosome 1 interval extending from ∼35 to 106 cM develop anti-nuclear antibodies and mild glomerulonephritis. In this study, we produced subcongenic mouse strains to localize the susceptibility loci in this interval and investigate how they promote autoimmunity. Our results indicate at least four susceptibility alleles and a suppressor allele. One allele is located in the 96-100 cM region and is sufficient to breach tolerance to chromatin. Addition of a second locus in the 88-96 cM interval enhances anti-dsDNA antibody production and promotes renal disease, which together with a third susceptibility allele in the 70-88 interval results in significant mortality. We further demonstrate the presence of a suppressor locus in the 35-70 or 100-102 cM interval that abrogates these phenotypes and an additional susceptibility allele in the 102-106 cM interval that restores a milder autoimmune phenotype. Several of these loci alter T-cell function. Thus, there is substantial genetic complexity in the NZB 35-106 cM interval, with disease reflecting a balance between susceptibility and suppressor loci.

A targeted association study in systemic lupus erythematosus identifies multiple susceptibility alleles.

Systemic lupus erythematosus (SLE) is a multisystem autoimmune disease. Multiple genetic and environmental factors contribute to the pathogenesis of this disease. Recent genome-wide association studies have added substantially to the number of genes associated with SLE. To replicate some of these susceptibility loci, single-nucleotide polymorphisms reported to be associated to SLE were evaluated in a cohort of 245 well-phenotyped Canadian SLE trios. Our results replicate previously reported associations to alleles of interferon regulatory factor 5 (IRF5), major histocompatibility complex (MHC), tumor necrosis factor (ligand) superfamily member 4 (TNFSF4), Kell blood group complex subunit-related family member 6 (XKR6), B-cell scaffold protein with ankyrin repeats 1 (BANK1), protein tyrosine phosphatase non-receptor type 22 (PTPN22), ubiquitin-conjugating enzyme E2L 3 (UBE2L3) and islet cell autoantigen 1 (ICA1). We also identify putative associations to cytotoxic T-lymphocyte-associated protein 4 (CTLA4), a gene associated with several autoimmune disorders, and ERBB3, a locus on 12q13 that was previously reported to be associated with type 1 diabetes. This study confirms the existence of multiple genetic risk factors for SLE, and supports the notion that some risk factors for SLE are shared with other inflammatory disorders.

Lack of association between the interferon-alpha signature and longitudinal changes in disease activity in systemic lupus erythematosus.

OBJECTIVE: To study the longitudinal expression of interferon (IFN)-inducible genes in systemic lupus erythematosus (SLE) and determine their suitability as disease biomarkers. METHODS: RNA was isolated from the peripheral blood of 94 patients with SLE and 11 controls and reverse transcribed into cDNA. The expression levels of five IFN-responsive genes (LY6E, OAS1, IFIT1, ISG15 and MX1) were determined by quantitative PCR, normalised to GAPDH and summed to generate a global IFN score. Patients were followed longitudinally for a period of 3-12 months, and the association between disease activity, as measured by the SLE disease activity index (SLEDAI-2K), and other clinical and laboratory variables was examined. RESULTS: The expression of all five IFN-responsive genes was significantly higher in patients with SLE than in controls. The expression of LY6E, OAS1, IFIT1 and the global IFN score was associated with high disease activity. The global IFN score was also associated with active renal disease, a decreased C3, and the presence of anti-dsDNA or anti-RNA binding protein antibodies at a single point in time. However, there was a poor correlation between changes in this score and changes in disease activity, C3 or anti-dsDNA antibody levels in patients followed longitudinally. In most patients the levels of IFN-induced gene expression remained relatively stable over 3-12 months despite marked changes in disease activity. Nevertheless, in patients with low/moderate disease activity, those with high IFN scores had a more recent history of sustained high disease activity. CONCLUSION: The findings indicate that IFN-induced gene expression has limited clinical utility as a biomarker of acute changes in disease activity.

Association of LY9 in UK and Canadian SLE families.

Systemic lupus erythematosus (SLE) is a complex disease trait of unknown aetiology. Genome-wide linkage studies in human SLE identified several linkage regions, including one at 1q23, which contains multiple susceptibility genes, including the members of the signalling lymphocyte activation molecule (SLAM) locus. In mice there is a syntenic linkage region, Sle1. The SLAM genes are functionally related cell-surface receptors, which regulate signal transduction of cells in the immune system. Family-based association study in UK and Canadian SLE families identified variants in the promoter and coding region of SLAMF7 and LY9 contributing to SLE disease susceptibility. The strongest association was from rs509749, in exon 8 of LY9 (P=0.00209). rs509749 encodes a Val/Met nonsynonymous change in amino acid 602 in the cytoplasmic domain of LY9. In the parents and affected individuals from the Canadian SLE families, the risk allele of rs509049 skews the T-cell population by increasing the number of CD8+ memory T cells, while decreasing the proportion of CD4+ naïve T cells and activated T cells. Since rs509749 lies within the consensus binding site for SAP/SH2D1a, which influences downstream signalling events from LY9, the mechanism for increased CD8+ memory T cells may include differential binding SAP/SH2D1a to the cytoplasmic domain of LY9.

T-cell tolerance induction is normal in the (NZB x NZW)F1 murine model of systemic lupus erythematosus.

The (New Zealand black (NZB) x New Zealand white (NZW))F1 (NZB/W) mouse strain spontaneously develops an autoimmune disease characterized by anti-dsDNA antibody production and glomerulonephritis. Although evidence suggests that production of pathogenic autoantibodies is T-cell dependent, the immunological defects that lead to activation of these autoreactive T cells are unknown. In particular, it has not been resolved whether autoreactive T cells become activated in these mice because of a generalized defect in T-cell tolerance induction. Previous work has demonstrated that thymic and peripheral tolerance to strongly deleting antigens are intact in NZB/W mice. In this study we investigate whether these mice possess a more subtle T-cell tolerance defect. To this end, we have produced NZB/W mice carrying a transgene encoding beef insulin (BI) which is expressed at levels close to the threshold for T-cell tolerance induction. In BALB/c mice this transgene produces a profound but incomplete state of BI-specific T-cell tolerance, mediated predominantly by clonal anergy. Comparison of BI-specific tolerance in NZB/W, major histocompatibility complex (MHC)-matched (BALB/c x NZW)F1, and BALB/c BI-transgenic mice clearly demonstrates that T-cell tolerance induction is normal in NZB/W mice. The data suggest that the loss of T-cell tolerance that ultimately supports nephritogenic autoantibody production in NZB/W mice does not result from a generalized defect in T-cell tolerance, and by extension likely results from aberrant activation of specific autoreactive T cells.

Genetic dissection of B cell traits in New Zealand black mice. The expanded population of B cells expressing up-regulated costimulatory molecules shows linkage to Nba2.

B cell abnormalities are a prominent feature of the immunologic derangement in NZB and NZB / W mice. We recently demonstrated that these mice have an increased proportion of splenic B cells expressing B7.1 and elevated levels of B7.2 and ICAM-1 that possess the characteristics of marginal zone B cells (CD23(low / -) CD5(-) CD44(hi) CD24(hi) IgD(- / low) IgM(hi)) and are found as early as 4 - 6 weeks of age. These findings suggest that activated B cells in NZB and NZB / W mice could serve a costimulatory function leading to activation of autoreactive T cells. However, it remains unclear whether there is any association between B abnormalities and nephritis in these mice. Here we have used genetic mapping techniques to address this issue. We show that increases in the proportion of B cells expressing costimulatory molecules, serum IgM levels, the number of IgM ELISpots, and IgG anti-single-stranded (ss) DNA antibody production, are significantly associated with a chromosomal region that overlaps with Nba2, a genetic locus previously linked to nephritis. Based on these findings we propose that immune mechanisms leading to polyclonal B cell activation and up-regulation of costimulatory molecules in these mice play a central role in the loss of tolerance that leads to production of pathogenic autoantibodies.

Activated B cells express increased levels of costimulatory molecules in young autoimmune NZB and (NZB x NZW)F(1) mice.

Polyclonal B cell activation is a hallmark of autoimmune disease in NZB and (NZB x NZW)F(1) (NZB/W) mice. However, the mechanism by which this activated cell subset facilitates disease development is unknown. We recently showed that resting B cells from these mice demonstrate enhanced expression of costimulatory molecules in response to CD40 crosslinking (Jongstra-Bilen et al., J. Immunol. 159,5810-5820, 1997). This led us to question whether activated B cells expressed costimulatory molecules in vivo. Using flow cytometry we found that NZB and NZB/W mice have an increased proportion of splenic B cells expressing B7.1 and elevated levels of B7.2 and ICAM-1. These B cells isolate within the low-density activated population and possess the phenotypic characteristics of marginal zone B cells. The levels of B7.1 on the activated B cell population are similar to those induced by CD40 stimulation raising the possibility that activated B cells in NZB and NZB/W mice provide costimulatory signals to self-reactive T cells leading to loss of tolerance.

The orientation and nature of the interaction between beef insulin-specific TCRs and the insulin/class II MHC complex.

Recent crystallographic studies suggest that TCR interact with peptide/class I MHC complexes in a single preferred orientation. Although similar studies have not been performed for class II-restricted TCR, it has been proposed that T cell recognition of peptide/class II complexes has similar orientational restrictions. This study represents a functional approach to systematic analysis of this question. Twenty-one mutant A beta(d) molecules were produced by alanine scanning mutagenesis and assessed for their ability to present species variants of insulin to a panel of beef insulin-specific T cell hybridomas with limited TCR alpha- and/or beta-chain sequence differences. We demonstrate that all beef insulin-specific TCR have the same orientation on the insulin/Ad complex, such that the alpha-chain interacts with the carboxyl-terminal region of the A beta(d) alpha-helix, and the beta-chain complementarity-determining region 3 interacts with the carboxyl-terminal portion of the peptide, consistent with that observed for crystallized TCR-peptide/class I complexes. Despite this structural constraint, even TCR that share structural similarity show remarkable heterogeneity in their responses to the panel of MHC mutants. This variability appears to result from conformational changes induced by binding of the TCR to the complex and the exquisite sensitivity of the threshold for T cell activation.

Autoimmunity develops in lupus-prone NZB mice despite normal T cell tolerance.

NZB mice spontaneously develop an autoimmune disease characterized by production of anti-RBC, -lymphocyte, and -ssDNA Abs. Evidence suggests that the NZB mouse strain has all of the immunologic defects required to produce lupus nephritis but lacks an MHC locus that allows pathogenic anti-dsDNA Ab production. The capacity to produce diverse autoantibodies in these mice raises the possibility that they possess a generalized defect in self-tolerance. To determine whether this defect is found within the T cell subset, we backcrossed a transgene encoding bovine insulin (BI) onto the NZB background. In nonautoimmune BALB/c mice, the BI transgene induces a profound but incomplete state of T cell tolerance mediated predominantly by clonal anergy. Comparison of tolerance in NZB and BALB/c BI-transgenic mice clearly demonstrated that NZB T cells were at least as tolerant to BI as BALB/c T cells. NZB BI-transgenic mice did not spontaneously produce anti-BI Abs, and following antigenic challenge, BI-specific Ab production was comparably reduced in both BI-transgenic NZB and BALB/c mice. Further, in vitro BI-specific T cell proliferation and cytokine secretion were appropriately decreased for primed lymph node and splenic T cells derived from NZB BI-transgenic relative to their nontransgenic counterparts. These data indicate that a generalized T cell tolerance defect does not underlie the autoimmune disease in NZB mice. Instead, we propose that the T cell-dependent production of pathogenic IgG autoantibodies in these mice arises from abnormal activation of T cells in the setting of normal but incomplete tolerance.

The B-cell transmembrane protein CD72 binds to and is an in vivo substrate of the protein tyrosine phosphatase SHP-1.

BACKGROUND: Signals from the B-cell antigen receptor (BCR) help to determine B-cell fate, directing either proliferation, differentiation, or growth arrest/apoptosis. The protein tyrosine phosphatase SHP-1 is known to regulate the strength of BCR signaling. Although the B-cell co-receptor CD22 binds SHP-1, B cells in CD22-deficient mice are much less severely affected than those in SHP-1-deficient mice, suggesting that SHP-1 may also regulate B-cell signaling by affecting other signaling molecules. Moreover, direct substrates of SHP-1 have not been identified in any B-cell signaling pathway. RESULTS: We identified the B-cell transmembrane protein CD72 as a new SHP-1 binding protein and as an in vivo substrate of SHP-1 in B cells. We also defined the binding sites for SHP-1 and the adaptor protein Grb2 on CD72. Tyrosine phosphorylation of CD72 correlated strongly with BCR-induced growth arrest/apoptosis in B-cell lines and in primary B cells. Preligation of CD72 attenuated BCR-induced growth arrest/death signals in immature and mature B cells or B-cell lines, whereas preligation of CD22 enhanced BCR-induced growth arrest/apoptosis. CONCLUSIONS: We have identified CD72 as the first clear in vivo substrate of SHP-1 in B cells. Our results suggest that tyrosine-phosphorylated CD72 may transmit signals for BCR-induced apoptosis. By dephosphorylation CD72. SHP-1 may have a positive role in B-cell signaling. These results have potentially important implications for the involvement of CD72 and SHP-1 in B-cell development and autoimmunity.

Coexistence of Fabry's disease and systemic lupus erythematosus.

This report describes a case of a female with systemic lupus erythematosus, who was subsequently diagnosed with Fabry's disease. Due to similarities in the organs involved by these two multisystem disorders, difficulties were encountered in establishing a prompt diagnosis of Fabry's disease. That and subsequent management of this patient are discussed. A literature review of the coexistence of the two disorders along with the potential pathogenic mechanisms explaining this association are explored.

Resting B cells from autoimmune lupus-prone New Zealand Black and (New Zealand Black x New Zealand White)F1 mice are hyper-responsive to T cell-derived stimuli.

To determine whether B cells from New Zealand Black (NZB) and (New Zealand Black x New Zealand White)F1 (NZB/W) mice possess intrinsic defects that lead to altered immune responsiveness, we purified resting B cells from these mice and compared their surface phenotype and function with those of resting B cells isolated from BALB/c and DBA/2 nonautoimmune mouse strains. Flow cytometric analysis of freshly isolated resting B cells revealed that NZB and NZB/W resting B cells are conventional B2-type cells similar to their nonautoimmune counterparts. Despite this, resting B cells from young NZB and NZB/W mice express lower levels of CD23 on their surface and aberrant levels of intracellular IgM. Upon stimulation, resting B cells from young NZB and NZB/W mice demonstrate increased proliferation, IgM secretion, or enhanced expression of costimulatory molecules in response to a variety of different T cell-derived stimuli, including cytokines and signals generated through CD40. Therefore, B cell hyper-responsiveness to T cell stimuli is immunodominant or codominant in NZB/W mice. Taken together, our results suggest that intrinsic B cell hyper-responsiveness may play a role in the pathogenesis of autoimmune disease in NZB and NZB/W mice. The increased clonal expansion of these B cells together with increased Ig production and enhanced costimulatory capacity serve to amplify the immune response. In the context of normal but incomplete T cell tolerance, B cell hyperresponsiveness to the limited signals provided by partially tolerant T cells may be sufficient to yield an autoantibody response.

Tolerance is overcome in beef insulin-transgenic mice by activation of low-affinity autoreactive T cells.

To gain insight into the factors controlling the maintenance or loss of T cell self tolerance we produced beef insulin (BI)-transgenic BALB/c mice. Transgenic mice express BI under control of the human insulin promoter and secrete physiological amounts of beef insulin. Although these mice are tolerant to BI, as evidenced by the lack of insulin-specific IgG antibody production following intraperitoneal immunization, tolerance is not complete. Footpad immunization results in a weak antigen-specific T cell proliferative response, indicating the presence of self-reactive BI-specific T cell in the periphery. These T cells are functional in vivo, providing support for IgG1, IgG2a, and IgG2b BI-specific antibody production, but require higher higher concentrations of antigen than nontransgenic T cells (both in vivo and following recall responses in vitro) to become activated. In vitro, BI-specific T cell proliferation in BI-transgenic mice can be largely restored by addition of interleukin-2, indicating that a significant component of T cell tolerance is mediated by anergy. To characterize the autoreactive T cells that become activated when tolerance is broken, BI-specific T cell hybridomas were generated from transgenic mice and compared to a panel of hybridomas previously derived from nontransgenic BALB/c mice. The majority of BI-transgenic hybridomas recognized the immunodominant A1-14 beef insulin peptide but with lower avidity than BALB/c hybridomas. Consistent with this, none of the dominant T cell receptor rearrangements found in the BALB/c BI-specific T cell receptor repertoire were found in the transgenic hybridomas. These results indicate that, despite evidence for clonal inactivation of many BI-specific T cells in BI-transgenic mice, loss of tolerance results from activation of low-affinity antigen-specific T cells that appear to have escaped this process.

Both MHC and background gene heterozygosity alter T cell receptor repertoire selection in an antigen-specific response.

Many autoimmune diseases are associated with specific class II MHC alleles; however, this association is not complete. One explanation for the variable expression of disease in susceptible individuals is that variability in the TCR repertoire may alter the potential to generate pathogenic autoreactive T cells. The current study was undertaken to examine the possibility that MHC and background heterozygosity, which is the norm in the outbred human population, alters the expressed TCR repertoire and, if so, whether this has an impact on peptide recognition and antigenic specificity. We, therefore, systematically analysed the beef insulin-specific TCR repertoire in inbred BALB/c mice before and after introduction of MHC heterozygosity (BALB/c x BALB.K)F1 mice, or MHC and background gene heterozygosity (BALB/c x A/J)F1 mice. We show that T cells from all three repertoires are predominantly Ad-restricted and recognize the same immunodominant peptide. Despite this, the beef insulin-specific TCR repertoires in F1 mice differ from those seen in BALB/c mice with the most dramatic changes seen in (BALB/c x A/J)F1 mice. These changes are accompanied by subtle differences in the antigenic specificity of the T cells. The results demonstrate that both MHC and background gene heterozygosity affect TCR repertoire selection, suggesting that the variable expression of autoimmune disease in individuals with a susceptible MHC allele may result, in part, from variability in the TCR repertoire introduced by this heterozygosity.

Clinical utility and specificity of anticardiolipin antibodies.

Established solid phase assays for anticardiolipin antibodies (aCL) are often characterized by high levels of nonspecific binding. As a result, only very high levels of aCL have been reported to be associated with a variety of clinical conditions including systemic lupus erythematosus (SLE), recurrent intravascular thrombosis and unexplained recurrent fetal loss. We have developed an ELISA replacing direct evaporation of soluble cardiolipin with cardiolipin micelles in physiological saline as the antigen binding step in the assay. Levels of IgG aCL were detected in various sera at dilutions of 1/100 to 1/3200, showing improved assay sensitivity. Assay specificity was determined using double stranded DNA and ovalbumin as irrelevant binding antigens and no crossreactivity was found. The controversial use of Tween 20 in the assay was investigated and results showed it decreases nonspecific binding without interfering in antibody detection. This assay has enabled us to identify differences in the prevalence and level of aCL antibodies in sera from healthy nonpregnant controls (0/25 positive), healthy pregnant controls (5/47 positive for IgG and 8/47 positive for IgM) and from women with unexplained recurrent fetal loss (16/62 and 14/62 positive, respectively). We support the observation that aCL are not normally distributed, and therefore nonparametric methods of statistical analysis are necessary to determine population prevalence. We confirm that aCL IgM are a relatively nonspecific finding, and extreme caution must be used in basing any clinical decisions on the presence of this antibody alone.