Abatacept increases T cell exhaustion in early RA individuals who carry HLA risk alleles.

Exhausted CD8 T cells (TEX) are associated with worse outcome in cancer yet better outcome in autoimmunity. Building on our past findings of increased TIGIT+KLRG1+ TEX with teplizumab therapy in type 1 diabetes (T1D), in the absence of treatment we found that the frequency of TIGIT+KLRG1+ TEX is stable within an individual but differs across individuals in both T1D and healthy control (HC) cohorts. This TIGIT+KLRG1+ CD8 TEX population shares an exhaustion-associated EOMES gene signature in HC, T1D, rheumatoid arthritis (RA), and cancer subjects, expresses multiple inhibitory receptors, and is hyporesponsive in vitro, together suggesting co-expression of TIGIT and KLRG1 may broadly define human peripheral exhausted cells. In HC and RA subjects, lower levels of EOMES transcriptional modules and frequency of TIGIT+KLRG1+ TEX were associated with RA HLA risk alleles (DR0401, 0404, 0405, 0408, 1001) even when considering disease status and cytomegalovirus (CMV) seropositivity. Moreover, the frequency of TIGIT+KLRG1+ TEX was significantly increased in RA HLA risk but not non-risk subjects treated with abatacept (CTLA4Ig). The DR4 association and selective modulation with abatacept suggests that therapeutic modulation of TEX may be more effective in DR4 subjects and TEX may be indirectly influenced by cellular interactions that are blocked by abatacept.

Abatacept and non-melanoma skin cancer in patients with rheumatoid arthritis: a comprehensive evaluation of randomised controlled trials and observational studies.

OBJECTIVES: This study aims to evaluate non-melanoma skin cancer (NMSC) risk associated with abatacept treatment for rheumatoid arthritis (RA). METHODS: This evaluation included 16 abatacept RA clinical trials and 6 observational studies. NMSC incidence rates (IRs)/1000 patient-years (p-y) of exposure were compared between patients treated with abatacept versus placebo, conventional synthetic (cs) disease-modifying antirheumatic drugs (DMARDs) and other biological/targeted synthetic (b/ts)DMARDs. For observational studies, a random-effects model was used to pool rate ratios (RRs). RESULTS: ~49 000 patients receiving abatacept were analysed from clinical trials (~7000) and observational studies (~42 000). In randomised trials (n=4138; median abatacept exposure, 12 (range 2-30) months), NMSC IRs (95% CIs) were not significantly different for abatacept (6.0 (3.3 to 10.0)) and placebo (4.0 (1.3 to 9.3)) and remained stable throughout the long-term, open-label period (median cumulative exposure, 28 (range 2-130 months); 21 335 p-y of exposure (7044 patients over 3 years)). For registry databases, NMSC IRs/1000 p-y were 5-12 (abatacept), 1.6-10 (csDMARDs) and 3-8 (other b/tsDMARDs). Claims database IRs were 19-22 (abatacept), 15-18 (csDMARDs) and 14-17 (other b/tsDMARDs). Pooled RRs (95% CIs) from observational studies for NMSC in patients receiving abatacept were 1.84 (1.00 to 3.37) vs csDMARDs and 1.11 (0.98 to 1.26) vs other b/tsDMARDs. CONCLUSIONS: Consistent with the warnings and precautions of the abatacept label, this analysis suggests a potential increase in NMSC risk with abatacept use compared with csDMARDs. No significant increase was observed compared with b/tsDMARDs, but the lower limit of the 95% CI was close to unity.

Abatacept, Cenicriviroc, or Infliximab for Treatment of Adults Hospitalized With COVID-19 Pneumonia: A Randomized Clinical Trial.

Importance: Immune dysregulation contributes to poorer outcomes in COVID-19. Objective: To investigate whether abatacept, cenicriviroc, or infliximab provides benefit when added to standard care for COVID-19 pneumonia. Design, Setting, and Participants: Randomized, double-masked, placebo-controlled clinical trial using a master protocol to investigate immunomodulators added to standard care for treatment of participants hospitalized with COVID-19 pneumonia. The results of 3 substudies are reported from 95 hospitals at 85 clinical research sites in the US and Latin America. Hospitalized patients 18 years or older with confirmed SARS-CoV-2 infection within 14 days and evidence of pulmonary involvement underwent randomization between October 2020 and December 2021. Interventions: Single infusion of abatacept (10 mg/kg; maximum dose, 1000 mg) or infliximab (5 mg/kg) or a 28-day oral course of cenicriviroc (300-mg loading dose followed by 150 mg twice per day). Main Outcomes and Measures: The primary outcome was time to recovery by day 28 evaluated using an 8-point ordinal scale (higher scores indicate better health). Recovery was defined as the first day the participant scored at least 6 on the ordinal scale. Results: Of the 1971 participants randomized across the 3 substudies, the mean (SD) age was 54.8 (14.6) years and 1218 (61.8%) were men. The primary end point of time to recovery from COVID-19 pneumonia was not significantly different for abatacept (recovery rate ratio [RRR], 1.12 [95% CI, 0.98-1.28]; P = .09), cenicriviroc (RRR, 1.01 [95% CI, 0.86-1.18]; P = .94), or infliximab (RRR, 1.12 [95% CI, 0.99-1.28]; P = .08) compared with placebo. All-cause 28-day mortality was 11.0% for abatacept vs 15.1% for placebo (odds ratio [OR], 0.62 [95% CI, 0.41-0.94]), 13.8% for cenicriviroc vs 11.9% for placebo (OR, 1.18 [95% CI 0.72-1.94]), and 10.1% for infliximab vs 14.5% for placebo (OR, 0.59 [95% CI, 0.39-0.90]). Safety outcomes were comparable between active treatment and placebo, including secondary infections, in all 3 substudies. Conclusions and Relevance: Time to recovery from COVID-19 pneumonia among hospitalized participants was not significantly different for abatacept, cenicriviroc, or infliximab vs placebo. Trial Registration: ClinicalTrials.gov Identifier: NCT04593940.

Abatacept for Treatment of Adults Hospitalized with Moderate or Severe Covid-19.

Background: We investigated whether abatacept, a selective costimulation modulator, provides additional benefit when added to standard-of-care for patients hospitalized with Covid-19. Methods: We conducted a master protocol to investigate immunomodulators for potential benefit treating patients hospitalized with Covid-19 and report results for abatacept. Intravenous abatacept (one-time dose 10 mg/kg, maximum dose 1000 mg) plus standard of care (SOC) was compared with shared placebo plus SOC. Primary outcome was time-to-recovery by day 28. Key secondary endpoints included 28-day mortality. Results: Between October 16, 2020 and December 31, 2021, a total of 1019 participants received study treatment (509 abatacept; 510 shared placebo), constituting the modified intention-to-treat cohort. Participants had a mean age 54.8 (SD 14.6) years, 60.5% were male, 44.2% Hispanic/Latino and 13.7% Black. No statistically significant difference for the primary endpoint of time-to-recovery was found with a recovery-rate-ratio of 1.14 (95% CI 1.00-1.29; p=0.057) compared with placebo. We observed a substantial improvement in 28-day all-cause mortality with abatacept versus placebo (11.0% vs. 15.1%; odds ratio [OR] 0.62 [95% CI 0.41- 0.94]), leading to 38% lower odds of dying. Improvement in mortality occurred for participants requiring oxygen/noninvasive ventilation at randomization. Subgroup analysis identified the strongest effect in those with baseline C-reactive protein >75mg/L. We found no statistically significant differences in adverse events, with safety composite index slightly favoring abatacept. Rates of secondary infections were similar (16.1% for abatacept; 14.3% for placebo). Conclusions: Addition of single-dose intravenous abatacept to standard-of-care demonstrated no statistically significant change in time-to-recovery, but improved 28-day mortality. Trial registration: ClinicalTrials.gov ( NCT04593940 ).

ACPA-negative and ACPA-positive RA patients achieving disease resolution demonstrate distinct patterns of MRI-detected joint-inflammation.

OBJECTIVES: Although sustained DMARD-free remission (SDFR; sustained absence of clinical-synovitis after DMARD-discontinuation) is increasingly achievable in RA, prevalence differs between ACPA-negative (40%) and ACPA-positive RA (5-10%). Additionally, early DAS remission (DAS4months<1.6) is associated with achieving SDFR in ACPA-negative, but not in ACPA-positive RA. Based on these differences, we hypothesized that longitudinal patterns of local tissue inflammation (synovitis/tenosynovitis/osteitis) also differ between ACPA-negative and ACPA-positive RA patients achieving SDFR. With the ultimate aim being to increase understanding of disease resolution in RA, we studied MRI-detected joint inflammation over time in relation to SDFR development in ACPA-positive RA and ACPA-negative RA. METHODS: A total of 198 RA patients (94 ACPA-negative, 104 ACPA-positive) underwent repeated MRIs (0/4/12/24 months) and were followed on SDFR development. The course of MRI-detected total inflammation, and synovitis/tenosynovitis/osteitis individually were compared between RA patients who did and did not achieve SDFR, using Poisson mixed models. In total, 174 ACPA-positive RA patients from the AVERT-1 were studied as ACPA-positive validation population. RESULTS: In ACPA-negative RA, baseline MRI-detected inflammation levels of patients achieving SDFR were similar to patients without SDFR but declined 2.0 times stronger in the first year of DMARD treatment [IRR 0.50 (95% CI; 0.32, 0.77); P < 0.01]. This stronger decline was seen in tenosynovitis/synovitis/osteitis. In contrast, ACPA-positive RA-patients achieving SDFR, had already lower inflammation levels (especially synovitis/osteitis) at disease presentation [IRR 0.45 (95% CI; 0.24, 0.86); P = 0.02] compared with patients without SDFR, and remained lower during subsequent follow-up (P = 0.02). Similar results were found in the ACPA-positive validation population. CONCLUSION: Compared with RA patients without disease resolution, ACPA-positive RA patients achieving SDFR have less severe joint inflammation from diagnosis onwards, while ACPA-negative RA patients present with similar inflammation levels but demonstrate a stronger decline in the first year of DMARD therapy. These different trajectories suggest different mechanisms underlying resolution of RA chronicity in both RA subsets.

Differential Changes in ACPA Fine Specificity and Gene Expression in a Randomized Trial of Abatacept and Adalimumab in Rheumatoid Arthritis.

INTRODUCTION: The biologics abatacept and adalimumab have different mechanisms of action (MoAs). We analyzed data from patients with rheumatoid arthritis treated in AMPLE (NCT00929864) to explore the pharmacodynamic effects of abatacept or adalimumab on anti-citrullinated protein antibodies (ACPAs) and gene expression. METHODS: AMPLE was a phase IIIb, 2-year, randomized, head-to-head trial of abatacept versus adalimumab. Post hoc analyses of baseline anti-cyclic citrullinated peptide-2 (anti-CCP2, an ACPA surrogate) positive (+) status and ACPA fine-specificity profiles over time, as well as transcriptional profiling (peripheral whole blood), were performed. RESULTS: Of 646 patients treated (abatacept, n = 318; adalimumab, n = 328), ACPA and gene expression data were available from 508 and 566 patients, respectively. In anti-CCP2+ patients (n = 388), baseline fine specificities for most ACPAs were highly correlated; over 2 years, levels decreased with abatacept but not adalimumab. By year 2, expression of genes associated with T cell co-stimulation and antibody production was lower for abatacept versus adalimumab; expression of genes associated with proinflammatory signaling was lower for adalimumab versus abatacept. Treatment modulated the expression of T- and B-cell gene signatures, with differences in CD8+ T cells, activated T cells, plasma cells, B cells, natural killer cells (all lower with abatacept versus adalimumab), and polymorphonuclear leukocytes (higher with abatacept versus adalimumab). CONCLUSIONS: In AMPLE, despite similar clinical outcomes, data showed that pharmacodynamic/genetic changes after 2 years of abatacept or adalimumab were consistent with drug MoAs. Further assessment of the relationship between such changes and clinical outcomes, including prediction of response, is warranted. TRIAL REGISTRATION: ClinicalTrials.gov identifier, NCT00929864.

Efficacy of Abatacept and Adalimumab in Patients with Early Rheumatoid Arthritis With Multiple Poor Prognostic Factors: Post Hoc Analysis of a Randomized Controlled Clinical Trial (AMPLE).

INTRODUCTION: Patients with rheumatoid arthritis (RA) with poor prognostic factors, such as seropositivity for anti-citrullinated protein antibodies and early erosions, may benefit from early intensive treatment. However, information to guide physicians on the best choice of therapy in these patients is limited. The objective of this study was to describe the efficacy of subcutaneous abatacept versus adalimumab over 2 years in patients with seropositive, erosive early RA in the AMPLE study. METHODS: This exploratory post hoc analysis compared clinical, functional and radiographic outcomes in two subsets of patients: patients with early RA (≤ 6 months' disease duration) who were seropositive for rheumatoid factor and/or anti-citrullinated protein antibodies and had > 1 radiographic erosion (Cohort 1); and patients with RA and absence of ≥ 1 of these inclusion criteria (Cohort    2). RESULTS: Of the 646 randomized patients, Cohort 1 included 38 patients receiving abatacept and 45 receiving adalimumab, and Cohort 2 included 280 patients receiving abatacept and 283 receiving adalimumab. Baseline demographics and disease characteristics were generally similar between treatment groups in both cohorts. Over 2 years, in Cohort 1, the adjusted mean change from baseline in the Disease Activity Score in 28 joints (using C-reactive protein) was numerically greater for abatacept than for adalimumab (mean difference at day 365 was 0.9, 95% confidence interval - 1.47 to - 0.33). Similar patterns of improvement were observed for other disease activity measures and physical function, but not for radiographic outcomes. No treatment-related differences were observed in Cohort 2. CONCLUSION: This analysis indicates a trend towards improved disease activity and physical function with abatacept versus adalimumab in patients with seropositive, erosive early RA. TRIAL REGISTRATION: ClinicalTrials.gov NCT00929864. FUNDING: Bristol-Myers Squibb.

Re-treatment with abatacept plus methotrexate for disease flare after complete treatment withdrawal in patients with early rheumatoid arthritis: 2-year results from the AVERT study.

Objectives: To complete reporting of outcomes after total withdrawal of all rheumatoid arthritis (RA) therapy and re-treatment after flare in Assessing Very Early Rheumatoid arthritis Treatment study (NCT01142726). Methods: Patients with early RA were initially randomised to double-blind, weekly subcutaneous abatacept plus methotrexate, or abatacept or methotrexate monotherapy. At month 12, patients with Disease Activity Score (DAS)28 C reactive protein (CRP) <3.2 had all RA treatments rapidly withdrawn and were observed for ≤12 months or until flare. After ≥3 months' withdrawal, patients with protocol-defined RA flare received open-label abatacept plus methotrexate for 6 months (re-treatment). Results: Proportion of patients in DAS28-CRP-defined remission remained numerically higher in original abatacept plus methotrexate and abatacept arms versus methotrexate arm up to day 253 of withdrawal. At the end of the withdrawal period, few patients remained in remission across all arms: 9/73 (12.3%), 7/50 (14.0%) and 6/53 (11.3%), respectively. For patients entering re-treatment, after 6 months' re-treatment, 95/124 (76.6%) and 78/124 (62.9%) patients achieved DAS28-CRP <3.2 and <2.6, respectively; mean changes in DAS28-CRP and Health Assessment Questionnaire-Disability Index scores from re-treatment baseline were -2.87 and 0.76, respectively. Re-treatment was well tolerated; exposure-adjusted infection rates per 100 patient-years were lower with abatacept plus methotrexate during withdrawal (7.2) and re-treatment (17.2) versus initial treatment periods of months 0-6 (116.6) and 6-12 (64.6). Conclusions: Most patients flared within 6 months of therapy withdrawal and few sustained major responses for 1 year. Re-treatment with abatacept plus methotrexate was effective and well tolerated in this controlled setting.

Identification of biomarkers of response to abatacept in patients with SLE using deconvolution of whole blood transcriptomic data from a phase IIb clinical trial.

OBJECTIVE: To characterise patients with active SLE based on pretreatment gene expression-defined peripheral immune cell patterns and identify clusters enriched for potential responders to abatacept treatment. METHODS: This post hoc analysis used baseline peripheral whole blood transcriptomic data from patients in a phase IIb trial of intravenous abatacept (~10 mg/kg/month). Cell-specific genes were used with a published deconvolution algorithm to identify immune cell proportions in patient samples, and unsupervised consensus clustering was generated. Efficacy data were re-analysed. RESULTS: Patient data (n=144: abatacept: n=98; placebo: n=46) were grouped into four main clusters (C) by predominant characteristic cells: C1-neutrophils; C2-cytotoxic T cells, B-cell receptor-ligated B cells, monocytes, IgG memory B cells, activated T helper cells; C3-plasma cells, activated dendritic cells, activated natural killer cells, neutrophils; C4-activated dendritic cells, cytotoxic T cells. C3 had the highest baseline total British Isles Lupus Assessment Group (BILAG) scores, highest antidouble-stranded DNA autoantibody levels and shortest time to flare (TTF), plus trends in favour of response to abatacept over placebo: adjusted mean difference in BILAG score over 1 year, -4.78 (95% CI -12.49 to 2.92); median TTF, 56 vs 6 days; greater normalisation of complement component 3 and 4 levels. Differential improvements with abatacept were not seen in other clusters, except for median TTF in C1 (201 vs 109 days). CONCLUSIONS: Immune cell clustering segmented disease severity and responsiveness to abatacept. Definition of immune response cell types may inform design and interpretation of SLE trials and treatment decisions. TRIAL REGISTRATION NUMBER: NCT00119678; results.

Brief Report: Estimating Disease Activity Using Multi-Biomarker Disease Activity Scores in Rheumatoid Arthritis Patients Treated With Abatacept or Adalimumab.

OBJECTIVE: To assess the ability of a multi-biomarker disease activity (MBDA) test (Vectra DA) to reflect clinical measures of disease activity in patients enrolled in the AMPLE (Abatacept Versus Adalimumab Comparison in Biologic-Naive RA Subjects with Background Methotrexate) trial. METHODS: In the AMPLE trial, patients with active rheumatoid arthritis (RA) who were naive to biologic agents and had an inadequate response to methotrexate were randomized (1:1) to receive subcutaneous abatacept (125 mg every week) or subcutaneous adalimumab (40 mg every 2 weeks), with background methotrexate, for 2 years. The MBDA score was determined using serum samples collected at baseline, month 3, and years 1 and 2. The adjusted mean change from baseline in the MBDA score was compared between the abatacept and adalimumab treatment groups. Cross-tabulation was used to compare the MBDA score with the following clinical measures of disease activity: Clinical Disease Activity Index (CDAI), Simplified Disease Activity Index (SDAI), Disease Activity Score in 28 joints using the C-reactive protein level (DAS28-CRP), and Routine Assessment of Patient Index Data 3 (RAPID-3). RESULTS: In total, 318 patients were randomized to receive abatacept, and 328 were randomized to receive adalimumab; MBDA data were available for 259 and 265 patients, respectively. No association between the MBDA score and disease activity defined by the CDAI, SDAI, DAS28-CRP, or RAPID-3 in the abatacept and adalimumab treatment groups was observed. CONCLUSION: The MBDA score did not reflect clinical disease activity in patients enrolled in AMPLE and should not be used to guide decision-making in the management of RA, particularly for patients who receive abatacept or adalimumab as the first biologic agent.

Patient-Reported Outcomes From a Two-Year Head-to-Head Comparison of Subcutaneous Abatacept and Adalimumab for Rheumatoid Arthritis.

OBJECTIVE: To report 2-year patient-reported outcomes (PROs) from the head-to-head Abatacept versus Adalimumab Comparison in Biologic-Naive RA Subjects with Background Methotrexate (MTX) (AMPLE) trial. METHODS: AMPLE was a phase IIIb, randomized, investigator-blinded trial. Biologic-naive patients with rheumatoid arthritis (RA) and an inadequate response to MTX were randomized to subcutaneous (SC) abatacept (125 mg/week) or adalimumab (40 mg every 2 weeks) with background MTX. PROs (pain, fatigue, ability to perform work, and ability to perform daily activities) were compared up to year 2 for patients in each treatment group, as well as those who achieved low disease activity at both years 1 and 2 (responders) and those who did not (nonresponders). RESULTS: A total of 646 patients were randomized and treated with SC abatacept (n = 318) or adalimumab (n = 328). Baseline characteristics were balanced between the 2 treatment arms. Comparable improvements in PROs were observed in the abatacept and adalimumab groups over 2 years, with both groups achieving clinically meaningful improvements in PROs from baseline. At year 2, fatigue improved by 23.4 mm and 21.5 mm on a 100-mm visual analog scale with abatacept and adalimumab, respectively. Clinical responders achieved greater improvements in PROs than nonresponders. CONCLUSION: In biologic-naive patients with active RA, despite prior MTX, treatment with SC abatacept or adalimumab with background MTX resulted in comparable improvements in PROs, which were highly correlated with physician-reported clinical response end points.

Impact of baseline anti-cyclic citrullinated peptide-2 antibody concentration on efficacy outcomes following treatment with subcutaneous abatacept or adalimumab: 2-year results from the AMPLE trial.

OBJECTIVES: To examine whether baseline anti-cyclic citrullinated peptide-2 (CCP2) antibody status and concentration correlated with clinical outcomes in patients treated with abatacept or adalimumab on background methotrexate (MTX) in the 2-year AMPLE (Abatacept versus adaliMumab comParison in bioLogic-naïvE rheumatoid arthritis subjects with background MTX) study. METHODS: In this exploratory analysis, anti-CCP2 antibody concentration was measured at baseline, and antibody-positive patients were divided into equal quartiles, Q1-Q4, representing increasing antibody concentrations. Clinical outcomes analysed by baseline anti-CCP2 status and quartile included change from baseline in disease activity and disability and remission rates. RESULTS: Baseline characteristics were generally comparable across quartiles and treatment groups. In both treatment groups, anti-CCP2 antibody-negative patients responded less well than antibody-positive patients. At year 2, improvements in disease activity and disability and remission rates were similar across Q1-Q3, but were numerically higher in Q4 in the abatacept group; in contrast, treatment effects were similar across all quartiles in the adalimumab group. CONCLUSIONS: In AMPLE, baseline anti-CCP2 positivity was associated with a better response for abatacept and adalimumab. Patients with the highest baseline anti-CCP2 antibody concentrations had better clinical response with abatacept than patients with lower concentrations, an association that was not observed with adalimumab. TRIAL REGISTRATION NUMBER: NCT00929864.

Absence of CD59 exacerbates systemic autoimmunity in MRL/lpr mice.

CD59 is a GPI-anchored membrane regulator of complement expressed on blood cells as well as peripheral tissues. It protects host cells from complement injury by inhibiting formation of the membrane attack complex. Recent studies in mice have suggested also a role of CD59 in T cell immune response that was mechanistically independent of complement. In the present study, we investigated the function of CD59 in the MRL/lpr model of murine lupus. We backcrossed the Cd59a knockout (Cd59a(-/-)) mouse onto the MRL/lpr background and compared Cd59a(+/+)-MRL/lpr and Cd59a(-/-)-MRL/lpr littermates for the development of systemic autoimmunity. We found that CD59a deficiency significantly exacerbated the skin disease and lymphoproliferation characteristic of MRL/lpr mice. It also increased autoantibody titers and caused a higher level of proteinuria in male MRL/lpr mice. Bone marrow transfer experiments indicated that CD59a expression on both bone marrow-derived cells and peripheral tissues played a role in lymphoproliferation, whereas the skin disease phenotype is determined mainly by local CD59a expression. Importantly, C3 gene deletion or C5 neutralization with a blocking mAb in Cd59a(-/-)-MRL/lpr mice did not rescue the proautoimmune phenotype associated with CD59a deficiency. These results together suggest that CD59a inhibits systemic autoimmunity in MRL/lpr mice through a complement-independent mechanism.

Decay-accelerating factor ameliorates systemic autoimmune disease in MRL/lpr mice via both complement-dependent and -independent mechanisms.

Decay-accelerating factor (DAF) is a glycosylphosphatidylinositol-anchored membrane protein that restricts complement activation on autologous cells. Previous studies have established a significant protective activity of DAF in the MRL/lpr murine model of human systemic lupus erythematosus. To dissect the mechanism of protection by DAF in this disease model, we evaluated the effect of C3 gene ablation on disease development in MRL/lpr-Daf-1(-/-) mice. We found no significant difference in lymphadenopathy, splenomegaly, or anti-chromatin autoantibody titer between complement-sufficient and complement-deficient MRL/lpr-Daf-1(-/-) mice. On the other hand, complement deficiency strikingly reduced the incidence and severity of dermatitis in MRL/lpr-Daf-1(-/-) mice. To assess the contribution of DAF expression on lymphocytes versus local tissues in suppressing dermatitis, we generated BM chimeric mice between MRL/lpr-Daf-1(-/-) and MRL/lpr-Daf-1(+/+) mice. Compared with MRL/lpr-Daf-1(-/-) --> MRL/lpr-Daf-1(-/-) controls, MRL/lpr-Daf-1(-/-) --> MRL/lpr-Daf-1(+/+) chimeras developed significantly attenuated dermatitis, suggesting that the protective effect of DAF in suppressing dermatitis is primarily attributable to its local expression. We conclude that DAF works as a complement regulator in the skin to protect MRL/lpr mice from skin inflammation, whereas its inhibitory role in the induction phase of MRL/lpr autoimmunity is complement-independent. Together, these results reveal multiple mechanisms of action for DAF in ameliorating systemic autoimmunity.

The ubiquitin-proteasome system and its role in inflammatory and autoimmune diseases.

Protein degradation through the ubiquitin-proteasome system is the major pathway of non-lysosomal proteolysis of intracellular proteins. It plays important roles in a variety of fundamental cellular processes such as regulation of cell cycle progression, division, development and differentiation, apoptosis, cell trafficking, and modulation of the immune and inflammatory responses. The central element of this system is the covalent linkage of ubiquitin to targeted proteins, which are then recognized by the 26S proteasome, an adenosine triphosphate-dependent, multi-catalytic protease. Damaged, oxidized, or misfolded proteins as well as regulatory proteins that control many critical cellular functions are among the targets of this degradation process. Aberration of this system leads to the dysregulation of cellular homeostasis and the development of multiple diseases. In this review, we described the basic biochemistry and molecular biology of the ubiquitin-proteasome system, and its complex role in the development of inflammatory and autoimmune diseases. In addition, therapies and potential therapeutic targets related to the ubiquitin-proteasome system are discussed as well.

The role of host CD4 T cells in the pathogenesis of the chronic graft-versus-host model of systemic lupus erythematosus.

Systemic lupus erythematosus is characterized by production of autoantibodies and glomerulonephritis. The murine chronic graft-vs-host (cGVH) model of systemic lupus erythematosus is induced by allorecognition of foreign MHC class II determinants. Previous studies have shown that cGVH could not be induced in CD4 knockout (CD4KO) mice. We have further explored the role of host CD4 T cells in this model. Our studies now show that B cells in CD4KO mice have intrinsic defects that prevent them from responding to allohelp. In addition, B cells in CD4KO mice showed phenotypic differences compared with congeneic C57BL/6 B cells, indicating some degree of in vivo activation and increased numbers of cells bearing a marginal zone B cell phenotype. The transfer of syngeneic CD4 T cells at the time of initiation of cGVH did not correct these B cell abnormalities; however, if CD4 T cells were transferred during the development and maturation of B cells, then the B cells from CD4KO mice acquire the ability to respond in cGVH. These studies clearly indicate that B cells need to coexist with CD4 T cells early in their development to develop full susceptibility to alloactivation signals.

Animal models for SLE.

Systemic lupus erythematosus (SLE) in humans is characterized by inflammatory lesions in skin, joints, kidneys, the central nervous system (CNS), and elsewhere. The clinical manifestations are accompanied by autoantibodies to diverse self antigens, mainly but not exclusively derived from the cell nucleus. Autoantibodies are generally believed to cause most of the tissue damage, although direct injury via cell-mediated immunity is also important. This unit describes protocols for the quantitation of SLE-associated autoantibody levels in mouse serum: detection of anti-chromatin antibodies, detection of anti-single and anti-double-stranded DNA antibodies, and detection of rheumatoid factor. Support protocols for preparing chicken chromatin and dsDNA are included. Also included is an immunoglobulin allotype-specific adaptation of the basic autoantibody ELISA which is useful for measuring antibody production in chimeric animals. The unit includes an ELISPOT protocol for quantitating cells producing anti-chromatin, as well as a method for genotyping mice for the faslpr and fasLgld mutations.

Complement-mediated clearance of erythrocytes: mechanism and delineation of the regulatory roles of Crry and DAF. Decay-accelerating factor.

The role of complement in the pathogenesis of autoimmune hemolytic anemia (AIHA) has been controversial and may depend on a number of factors, including the affinity and isotype of the pathogenic antibodies involved. We have recently shown that mouse erythrocytes deficient in the membrane C3 regulatory protein, complement receptor 1-related gene/protein y (Crry), but not decay-accelerating factor (DAF), were spontaneously eliminated in vivo by complement. Here, by generating a mouse deficient in both DAF and Crry, we further delineated the roles of Crry and DAF in regulating alternative and classical pathway C3 activation. By using immunoglobulin-, Fcgamma receptor (FcgammaR)-, C3-, C4-, and C5-deficient mice, we also determined the mechanism by which membrane C3 regulator-deficient erythrocytes are cleared from the circulation. Finally, we evaluated the relative importance of the Fc receptor versus the complement pathway in disposing antibody-opsonized DAF/Crry-deficient erythrocytes. We conclude that (1) Crry plays a more dominant role than DAF in regulating the alternative pathway of complement, whereas DAF and Crry are equally effective in preventing antibody-induced runaway complement activation on mouse erythrocytes; (2) DAF/Crry-deficient erythrocytes are eliminated by the alternative pathway of complement via complement receptor-mediated erythrophagocytosis in the spleen; and (3) when opsonized with an immunoglobulin G2a (IgG2a) autoantibody, Crry/DAF-deficient erythrocytes are eliminated more rapidly by complement than by the Fc receptor pathway. These results shed new light on the relative activities of Crry and DAF and underscore the critical roles of membrane C3 regulators in preventing spontaneous and antibody-induced erythrocyte damage in vivo.

Deletion of decay-accelerating factor (CD55) exacerbates autoimmune disease development in MRL/lpr mice.

Decay-accelerating factor (DAF, CD55) is a glycosylphosphatidylinositol-anchored membrane protein that restricts complement activation on autologous cells. It is also a ligand for CD97, an activation-associated lymphocyte antigen with seven transmembrane domains. It is widely expressed on cells of both the hematopoietic and nonhematopoietic lineages. Although deficiency of DAF on human erythrocytes is associated with the hemolytic anemia syndrome paroxysmal nocturnal hemoglobinuria, the in vivo biology of DAF is still poorly understood. We addressed the in vivo function of DAF in a knockout mouse model and describe here that deletion of DAF exacerbates autoimmune disease development in MRL/lpr mice, a model for human systemic lupus erythematosus. Compared to DAF-sufficient littermate controls, DAF-deficient female MRL/lpr mice developed exacerbated lymphadenopathy and splenomegaly, higher serum anti-chromatin autoantibody levels, and aggravated dermatitis. Consistent with the phenotype of aggravated dermatitis in DAF-deficient mice, Northern and Western blots and immunofluorescence studies showed DAF to be expressed abundantly in the mouse skin, suggesting that it may play a particularly important role in this tissue. Histology and immunostaining demonstrated inflammatory infiltrate and focal C3 deposition in early skin lesions, mostly along the dermal-epidermal junction. These results reveal a protective function of DAF in the development of a systemic autoimmune syndrome and suggest that dysfunction or down-regulation of DAF may contribute to autoimmune disease pathogenesis and manifestation.