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Abatacept is a fusion protein of human cytotoxic T-lymphocyte–associated protein (CTLA)-4 and the Fc portion of human immunoglobulin G1 (IgG1). It is believed to be effective in the treatment of rheumatoid arthritis by inhibiting costimulation of T cells via blocking CD28–B7 interactions as CTLA-4 binds to both B7.1 (CD80) and B7.2 (CD86). However, the interaction of CD28 with B7 molecules is crucial for activation of naive cells, whereas it is unclear whether the action of already activated CD4+T cells, which are readily present in established disease, also depends on this interaction. The aim of this study was to determine whether the mode of action of abatacept depends solely on its ability to halt T cell activation in established disease. Arthritis was induced in thymectomized male DBA/1 mice by immunisation with bovine collagen type II. The mice were subsequently depleted for CD4+T cells. Abatacept or control treatment was started when 80 % of the mice showed signs of arthritis. Arthritis severity was monitored by clinical scoring of the paws, and anti-collagen antibody levels over time were determined by enzyme-linked immunosorbent assay. Treatment with abatacept in the absence of CD4+T cells resulted in lower disease activity. This was associated with decreasing levels of collagen-specific IgG1 and IgG2a antibodies, whereas the antibody levels in control or CD4+T cell–depleted mice increased over time. These results show that abatacept decreased disease activity in the absence of CD4+T cells, indicating that the mode of action of abatacept in established arthritis does not depend entirely on its effects on CD4+T cell activation.

T-SPOT.TB is a specific assay for the diagnosis of tuberculosis. The assay needs to be performed with freshly isolated cells, and interpretation requires training. T-SPOT.TB has been used in various clinical-epidemiological settings, but so far no studies have evaluated the effect of interobserver variation in test reading. Our aim was to evaluate variation between different observers in reading T-SPOT.TB results. The study was nested within an ongoing cohort study, in which part of the T-SPOT.TB had been performed with frozen material. Culture plates were read visually by four different observers from two laboratories and by two automated readers. Of 313 T-SPOT.TB assays, 235 were performed with fresh cells and 78 were performed with frozen cells. No significant difference was found between results obtained with fresh cells and those obtained with frozen cells. The percentage of positive results varied between readers by maximally 15%; five/six raters were within a 6% difference in positive results. Analysis of the observed interrater differences showed that some individuals systematically counted more spots than others did. Because test interpretation includes subtraction of background values, this systematic variance had little influence on interindividual differences. The test result as positive or negative varied between independent raters, mainly due to samples with values around the cutoff. This warrants further study regarding determinants affecting the reading of T-SPOT.TB.

ABSTRACTT-SPOT.TBis a specific assay for the diagnosis of tuberculosis. The assay needs to be performed with freshly isolated cells, and interpretation requires training. T-SPOT.TBhas been used in various clinical-epidemiological settings, but so far no studies have evaluated the effect of interobserver variation in test reading. Our aim was to evaluate variation between different observers in reading T-SPOT.TBresults. The study was nested within an ongoing cohort study, in which part of the T-SPOT.TBhad been performed with frozen material. Culture plates were read visually by four different observers from two laboratories and by two automated readers. Of 313 T-SPOT.TBassays, 235 were performed with fresh cells and 78 were performed with frozen cells. No significant difference was found between results obtained with fresh cells and those obtained with frozen cells. The percentage of positive results varied between readers by maximally 15%; five/six raters were within a 6% difference in positive results. Analysis of the observed interrater differences showed that some individuals systematically counted more spots than others did. Because test interpretation includes subtraction of background values, this systematic variance had little influence on interindividual differences. The test result as positive or negative varied between independent raters, mainly due to samples with values around the cutoff. This warrants further study regarding determinants affecting the reading of T-SPOT.TB.

ABSTRACTStaphylococcus aureusis a Gram-positive opportunistic pathogen that causes superficial and invasive infections in the hospital and community. High mortality from infection emphasizes the need for improved methods for prevention and treatment. Although S. aureuspossesses an arsenal of virulence factors that contribute to evasion of host defenses, few studies have examined long-term humoral and B-cell responses. Adults with acute-phase skin and soft tissue infections were recruited; blood samples were obtained; and S. aureusisolates, including methicillin-resistant strains, were subjected to genomic sequence analysis. In comparisons of acute-phase sera with convalescent-phase sera, a minority (37.5%) of patients displayed 2-fold or greater increases in antibody titers against three or more S. aureusantigens, whereas nearly half exhibited no changes, despite the presence of toxin genes in most infecting strains. Moreover, enhanced antibody responses waned over time, which could reflect a defect in B-cell memory or long-lived plasma cells. However, memory B cells reactive with a range of S. aureusantigens were prevalent at both acute-phase and convalescent-phase time points. While some memory B cells exhibited toxin-specific binding, those cross-reactive with structurally related leucocidin subunits were dominant across patients, suggesting the targeting of conserved epitopes. Memory B-cell reactivity correlated with serum antibody levels for selected S. aureusexotoxins, suggesting a relationship between the cellular and humoral compartments. Overall, although there was no global defect in the representation of anti-S. aureusmemory B cells, there was evidence of restrictions in the range of epitopes recognized, which may suggest potential therapeutic approaches for augmenting host defenses.IMPORTANCEThe contribution of B-cell memory and long-term antibody responses to host defenses against S. aureusexotoxins remains poorly understood. Our studies confirmed that infection did not commonly lead to enhanced long-term humoral responses. Whereas circulating memory B cells against S. aureussecreted exotoxins were prevalent, they were dominated by cross-reactivity with structurally related leucocidin subunits, consistent with recognition of conserved epitopes. These findings also provide the first evidence of a relationship between the reactivity of antistaphylococcal circulating memory B cells and serum antibody levels. In general, infection was not associated with a global defect in B-cell memory for S. aureussecreted factors, and responses were highly dominated by cross-reactivity to structurally related exotoxins, which arguably may alone be suboptimal in providing host defenses. Our studies illuminate aspects of the S. aureus-host relationship that may better inform strategies for the development of an effective protective vaccine.