Lisa K. Peterson

SLAP Deficiency Enhances Number and Function of Regulatory T Cells Preventing Chronic Autoimmune Arthritis in SKG Mice

To test if manipulating TCR complex-mediated signaling (TCR signaling) could treat autoimmune disease, we generated the double SKG Src-like adapter protein (SLAP) knockout (DSSKO) mouse model. The SKG mutation in ZAP70 and SLAP have opposing functions on the regulation of TCR signaling. The combination of these two mutations alters TCR signaling in the context of a defined genetic background, uniform environmental conditions, and a well-characterized signaling disruption. In contrast to SKG mice, DSSKO mice do not develop zymosan-induced chronic autoimmune arthritis. This arthritis prevention is not due to significant alterations in thymocyte development or repertoire selection but instead enhanced numbers of regulatory T cells (Tregs) and decreased numbers of Th17 cells skewing the ratio of Tregs to autoreactive effector T cells. Treg depletion and/or functional blockade led to the development of arthritis in DSSKO mice. In vitro suppression of effector T cell proliferation was also enhanced, demonstrating that DSSKO mice have increased numbers of Tregs with increased function. Understanding how TCR signals influence development, expansion, and function of Tregs in DSSKO mice could advance our ability to manipulate Treg biology to treat ultimately autoimmune disease.

Role of CD5+ B-1 cells in EAE pathogenesis

Hybridoma cell lines producing natural autoantibodies (NAA), generated from A.SW mice with progressive experimental autoimmune encephalomyelitis (P-EAE), have been shown to cause demyelination and renal pathology when injected into naïve mice. To investigate the relative contribution of these antibodies to disease pathogenesis, B-1 cells, the major producers of NAA, were depleted by hypotonic shock. Depletion of B-1 cells during the effector phase of EAE significantly decreased the severity of demyelination and overall pathology in the brain. There was also a decreased incidence of P-EAE and a decrease in clinical score. Depletion during the induction phase of the disease resulted in an increase in the incidence of P-EAE and in the clinical score. Overall, B-1 cells were found to modulate EAE pathogenesis.

SLAP deficiency increases TCR avidity leading to altered repertoire and negative selection of cognate antigen-specific CD8+ T cells.

How T cell receptor (TCR) avidity influences CD8+ T cell development and repertoire selection is not yet fully understood. To fill this gap, we utilized Src-like adaptor protein (SLAP)-deficient mice as a tool to increase TCR avidity on double positive (DP) thymocytes. We generated SLAP−/− mice with the transgenic MHC class I-restricted TCR (OT-1) and SLAP−/− Vβ5 mice, expressing only the β-chain of the TCR OT-1 transgene, to examine the effects of increased TCR surface levels on CD8+ T cell development and repertoire selection. In comparing SLAP−/− OT-1 and Vβ5 mice with wild-type controls, we performed compositional analysis and assessed thymocyte signaling by measuring CD5 levels. In addition, we performed tetramer and compositional staining to measure affinity for the cognate antigen, ovalbumin (OVA) peptide, presented by MHC. Furthermore, we quantified differences in α-chain repertoire in SLAP−/− Vβ5 mice. We have found that SLAP−/− OT-1 mice have fewer CD8+ thymocytes but have increased CD5 expression. SLAP−/− OT-1 mice have fewer DP thymocytes expressing Vα2, signifying increased endogenous α-chain rearrangement, and more non-OVA-specific CD8+ splenocytes upon tetramer staining. Our data demonstrate that SLAP−/− Vβ5 mice also have fewer OVA-specific cells and increased Vα2 usage in the peripheral Vβ5 CD8+ T cells that were non-OVA-specific, demonstrating differences in α-chain repertoire. These studies provide direct evidence that increased TCR avidity in DP thymocytes enhances CD8+ T cell negative selection deleting thymocytes with specificity for cognate antigen, an antigen the mature T cells may never encounter. Collectively, these studies provide new insights into how TCR avidity during CD8+ T cell development influences repertoire selection.

Monoclonal MOG-reactive autoantibody from progressive EAE has the characteristics of a natural antibody

A.SW mice sensitized with myelin oligodendrocyte glycoprotein (MOG)92-106 is an animal model for progressive multiple sclerosis (MS). We isolated MOG-reactive monoclonal antibodies that were immunoglobulin (Ig)M and polyreactive, similar to natural autoantibodies. Upon analysis of the variable (V) light chains and the diversity (D) and joining (J) regions of V heavy chains, we found they were identical to germ line Vkappa19/28, Jkappa5, DFL16.1e and JH4, respectively. The sequence of the VH region had 99.7% and 100% identity at the nucleotide and amino acid levels, respectively, compared with the germ line encoded antibody, P3, of the Q52 family. Although A strain mice have been reported to have an insertion in BAFF-R, the receptor for BAFF (B cell activation factor from the tumor necrosis factor family), which could explain our results, A.SW mice have no mutations in BAFF-R.

Novel method for quantitative ANA measurement using near-infrared imaging.

Antinuclear antibodies (ANA) have been detected in patients with systemic rheumatic diseases and are used in the screening and/or diagnosis of autoimmunity in patients as well as mouse models of systemic autoimmunity. Indirect immunofluorescence (IIF) on HEp-2 cells is the gold standard for ANA screening. However, its usefulness is limited in diagnosis, prognosis and monitoring of disease activity due to the lack of standardization in performing the technique, subjectivity in interpreting the results and the fact that it is only semi-quantitative. Various immunological techniques have been developed in an attempt to improve upon the method to quantify ANA, including enzyme-linked immunosorbent assays (ELISAs), line immunoassays (LIAs), multiplexed bead immunoassays and IIF on substrates other than HEp-2 cells. Yet IIF on HEp-2 cells remains the most common screening method for ANA. In this study, we describe a simple quantitative method to detect ANA which combines IIF on HEp-2 coated slides with analysis using a near-infrared imaging (NII) system. Using NII to determine ANA titer, 86.5% (32 of 37) of the titers for human patient samples were within 2 dilutions of those determined by IIF, which is the acceptable range for proficiency testing. Combining an initial screening for nuclear staining using microscopy with titration by NII resulted in 97.3% (36 of 37) of the titers detected to be within two dilutions of those determined by IIF. The NII method for quantitative ANA measurements using serum from both patients and mice with autoimmunity provides a fast, relatively simple, objective, sensitive and reproducible assay, which could easily be standardized for comparison between laboratories.

Cross-reactive myelin antibody induces renal pathology

Experimental autoimmune encephalomyelitis (EAE) is an autoimmune model for multiple sclerosis (MS). Previously, we reported renal immunoglobulin (Ig) deposition in mice with myelin oligodendrocyte glycoprotein (MOG92–106)-induced progressive EAE and naïve mice injected with MOG92–106 hybridoma cells producing antibody that cross-reacts with various autoantigens including double-stranded DNA. To assess whether MOG92–106 antibodies actually induce kidney changes, the extent of renal Ig deposition and changes in glomerular histology and filtration were investigated. Mice with progressive EAE exhibited Ig deposition, glomerular hypercellularity and proteinuria indicating kidney dysfunction. MOG92–106 hybridoma cell injected mice also had Ig deposition and proteinuria. Therefore, sensitization with MOG92–106 and transfer of MOG92–106 antibodies can induce both central nervous system and renal pathology. The renal involvement reported in MS is believed to occur as a side effect of nephrotoxic drugs or neurogenic bladder. Our results demonstrate that an autoimmune response against myelin could induce pathologic changes in the kidney and may help explain renal changes reported in patients with progressive MS.

Ethnic Differences in Autoantibody Diversity and Hierarchy: More Clues from a US Cohort of Patients with Systemic Sclerosis

Objective. To determine the autoantibody repertoire and clinical associations in a multiethnic cohort of American patients with systemic sclerosis (SSc). Methods. There were 1000 patients with SSc (196 Hispanic, 228 African American, 555 white, and 21 other) who were screened for antinuclear antibodies (ANA), including anticentromere antibodies (ACA) by indirect immunofluorescence assay, antitopoisomerase-1 (topo-1/Scl-70) by immunodiffusion, and anti-RNA polymerase III (RNAP III) by ELISA. Sera from 160 patients with mainly nucleolar and/or speckled ANA pattern, but negative for ACA, Scl-70, and RNAP III, were further characterized by immunoprecipitation for SSc-specific antibodies. Results. The prevalence of antibodies against RNAP III, Th/To, and PM/Scl did not differ significantly among the ethnic groups. The frequency of anti-Scl-70 was lowest in whites (18.0%) compared with 24.0% and 26.8% in Hispanics and African Americans (p = 0.01), respectively. Compared with African American patients, Hispanic and white subjects had a higher frequency of ACA (p < 0.0001) and lower frequency of U3-RNP (p < 0.0001). U3-RNP antibodies were uniquely higher in African American patients, independent of clinical subset, while Th/To autoantibodies were associated with limited cutaneous SSc in white subjects. Overall, Hispanic and African American patients had an earlier age of onset and a predominance of diffuse cutaneous SSc compared with their white counterparts. Conclusion. SSc-specific antibodies may predict disease subset; however, the hierarchy of their prevalence differs across ethnic groups. This study provides the most extensive analysis to date on the relevance of autoantibodies in the diagnosis and clinical manifestations of SSc in Hispanic American patients.

SLAP deficiency decreases dsDNA autoantibody production.

Src-like adaptor protein (SLAP) adapts c-Cbl, an E3 ubiquitin ligase, to activated components of the BCR signaling complex regulating BCR levels and signaling in developing B cells. Based on this function, we asked whether SLAP deficiency could decrease the threshold for tolerance and eliminate development of autoreactive B cells in two models of autoantibody production. First, we sensitized mice with a dsDNA mimetope that causes an anti-dsDNA response. Despite equivalent production of anti-peptide antibodies compared to BALB/c controls, SLAP(-/-) mice did not produce anti-dsDNA. Second, we used the 56R tolerance model. SLAP(-/-) 56R mice had decreased levels of dsDNA-reactive antibodies compared to 56R mice due to skewed light chain usage. Thus, SLAP is a critical regulator of B-cell development and function and its deficiency leads to decreased autoreactive B cells that are otherwise maintained by inefficient receptor editing or failed negative selection.

TCR signal strength controls thymic differentiation of iNKT cell subsets

During development in the thymus, invariant natural killer T (iNKT) cells commit to one of three major functionally different subsets, iNKT1, iNKT2, and iNKT17. Here, we show that T cell antigen receptor (TCR) signal strength governs the development of iNKT cell subsets, with strong signaling promoting iNKT2 and iNKT17 development. Altering TCR diversity or signaling diminishes iNKT2 and iNKT17 cell subset development in a cell-intrinsic manner. Decreased TCR signaling affects the persistence of Egr2 expression and the upregulation of PLZF. By genome-wide comparison of chromatin accessibility, we identify a subset of iNKT2-specific regulatory elements containing NFAT and Egr binding motifs that is less accessible in iNKT2 cells that develop from reduced TCR signaling. These data suggest that variable TCR signaling modulates regulatory element activity at NFAT and Egr binding sites exerting a determinative influence on the dynamics of gene enhancer accessibility and the developmental fate of iNKT cells.Invariant natural killer T (iNKT) cells can be subsetted by their cytokine profiles, but how they develop in the thymus is unclear. Here the authors show, by analysing mice carrying mutant Zap70 genes, that T cell receptor signaling strength induces epigenetic changes of genes to modulate iNKT lineages.

ROLE OF B:T CELL RATIO IN SUPPRESSION OF CLINICAL SIGNS: A MODEL FOR SILENT MS

B10.S mice have been considered resistant to experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis. However, sensitization with a myelin oligodendrocyte glycoprotein (MOG) peptide, MOG(92-106), induced clinical signs in 30% of mice and central nervous system (CNS) pathology in 93% of mice. Symptomatic mice had more demyelination, inflammation, perivascular cuffing and axonal damage in the CNS compared to asymptomatic mice, but no strong correlations between CNS pathology and clinical score were found. Interestingly, the ratio of B cells to T cells in cellular infiltrates correlated with clinical score. This suggests that the balance between B and T cells contributes to expression of clinical signs.