P2Y12 Inhibition Suppresses Proinflammatory Platelet-Monocyte Interactions.

BACKGROUND: Monocyte-platelet aggregates (MPAs) represent the crossroads between thrombosis and inflammation, and targeting this axis may suppress thromboinflammation. While antiplatelet therapy (APT) reduces platelet-platelet aggregation and thrombosis, its effects on MPA and platelet effector properties on monocytes are uncertain. OBJECTIVES: To analyze the effect of platelets on monocyte activation and APT on MPA and platelet-induced monocyte activation. METHODS: Agonist-stimulated whole blood was incubated in the presence of P-selectin, PSGL1, PAR1, P2Y12, GP IIb/IIIa, and COX-1 inhibitors and assessed for platelet and monocyte activity via flow cytometry. RNA-Seq of monocytes incubated with platelets was used to identify platelet-induced monocyte transcripts and was validated by RT-qPCR in monocyte-PR co-incubation ± APT. RESULTS: Consistent with a proinflammatory platelet effector role, MPAs were increased in patients with COVID-19. RNA-Seq revealed a thromboinflammatory monocyte transcriptome upon incubation with platelets. Monocytes aggregated to platelets expressed higher CD40 and tissue factor than monocytes without platelets (p < 0.05 for each). Inhibition with P-selectin (85% reduction) and PSGL1 (87% reduction) led to a robust decrease in MPA. P2Y12 and PAR1 inhibition lowered MPA formation (30 and 21% reduction, p < 0.05, respectively) and decreased monocyte CD40 and TF expression, while GP IIb/IIIa and COX1 inhibition had no effect. Pretreatment of platelets with P2Y12 inhibitors reduced the expression of platelet-mediated monocyte transcription of proinflammatory SOCS3 and OSM. CONCLUSIONS: Platelets skew monocytes toward a proinflammatory phenotype. Among traditional APTs, P2Y12 inhibition attenuates platelet-induced monocyte activation.

Platelet LGALS3BP as a Mediator of Myeloid Inflammation in Systemic Lupus Erythematosus.

OBJECTIVE: Platelets are mediators of inflammation with immune effector cell properties and have been implicated in the pathogenesis of systemic lupus erythematosus (SLE). This study investigated the role of platelet-associated lectin, galactoside-binding, soluble 3 binding protein (LGALS3BP) as a mediator of inflammation in SLE and as a potential biomarker associated with clinical phenotypes. METHODS: We performed RNA sequencing on platelets from patients with SLE (n = 54) and on platelets from age-, sex-, and race/ethnicity-matched healthy controls (n = 18) and measured LGALS3BP levels in platelet releasate and in circulating serum. We investigated the association between LGALS3BP levels and the prevalence, disease severity, and clinical phenotypes of SLE and studied platelet-mediated effects on myeloid inflammation. RESULTS: Platelets from patients with SLE exhibited increased expression of LGALS3BP (fold change 4.0, adjusted P = 6.02 × 10-11 ). Platelet-released LGALS3BP levels were highly correlated with circulating LGALS3BP (R = 0.69, P < 0.0001), and circulating LGALS3BP levels were correlated with the severity of disease according to the SLE Disease Activity Index (r = 0.32, P = 0.0006). Specifically, circulating LGALS3BP levels were higher in SLE patients with lupus nephritis than in patients with inactive disease (4.0 µg/ml versus 2.3 µg/ml; P < 0.001). Interferon-α induced LGALS3BP transcription and translation in a megakaryoblastic cell line (MEG-01) in a dose-dependent manner. Recombinant LGALS3BP and platelet releasates from SLE patients enhanced proinflammatory cytokine production by macrophages. CONCLUSIONS: Our results support that platelets act as potent effector cells that contribute to the pathogenesis of SLE by secreting proinflammatory LGALS3BP, which also represents a novel biomarker of SLE clinical activity.

Diversity of Functionally Distinct Clonal Sets of Human Conventional Memory B Cells That Bind Staphylococcal Protein A.

Staphylococcus aureus, a common cause of serious and often fatal infections, is well-armed with secreted factors that disarm host immune defenses. Highly expressed in vivo during infection, Staphylococcal protein A (SpA) is reported to also contribute to nasal colonization that can be a prelude to invasive infection. Co-evolution with the host immune system has provided SpA with an Fc-antibody binding site, and a Fab-binding site responsible for non-immune superantigen interactions via germline-encoded surfaces expressed on many human BCRs. We wondered whether the recurrent exposures to S. aureus commonly experienced by adults, result in the accumulation of memory B-cell responses to other determinants on SpA. We therefore isolated SpA-specific class-switched memory B cells, and characterized their encoding VH : VL antibody genes. In SpA-reactive memory B cells, we confirmed a striking bias in usage for VH genes, which retain the surface that mediates the SpA-superantigen interaction. We postulate these interactions reflect co-evolution of the host immune system and SpA, which during infection results in immune recruitment of an extraordinarily high prevalence of B cells in the repertoire that subverts the augmentation of protective defenses. Herein, we provide the first evidence that human memory responses are supplemented by B-cell clones, and circulating-antibodies, that bind to SpA determinants independent of the non-immune Fc- and Fab-binding sites. In parallel, we demonstrate that healthy individuals, and patients recovering from S. aureus infection, both have circulating antibodies with these conventional binding specificities. These findings rationalize the potential utility of incorporating specially engineered SpA proteins into a protective vaccine.

Hydroxychloroquine is associated with lower platelet activity and improved vascular health in systemic lupus erythematosus.

OBJECTIVE: Hydroxychloroquine (HCQ) is a mainstay of therapy in the treatment of SLE. The effect of HCQ on platelets and vascular health is uncertain. We investigated the relationship between HCQ use and dose with platelet activity, platelet transcriptomics and vascular health in patients with SLE. METHODS: Platelet aggregation, platelet mRNA expression and vascular health (sublingual capillary perfused boundary region (PBR), red blood cell filling (RBCF) and brachial artery reactivity testing) were analysed by HCQ use and dose. RESULTS: Among 132 subjects with SLE (age: 39.7±12.9 years, 97% female), 108 were on HCQ. SLE disease activity was similar between subjects on and off HCQ. Platelet aggregation in response to multiple agonists was significantly lower in patients on HCQ. There were inverse relationships between HCQ dose and gene expression pathways of platelet activity. Gene expression of P-selectin (SELP) was inversely correlated with HCQ dose (r=-0.41, p=0.003), which was validated at the protein level. Subjects on HCQ had improved vascular function correlating with HCQ dose as measured by lower PBR (r=-0.52, p=0.007), higher RBCF (r=0.55, p=0.004) and greater brachial artery reactivity (r=0.43, p=0.056). CONCLUSION: HCQ use was associated with decreased platelet activation and activation-related transcripts and improved vascular health in SLE.

Platelet-conditioned media induces an anti-inflammatory macrophage phenotype through EP4.

BACKGROUND: Platelets are increasingly recognized as immune cells. As such, they are commonly seen to induce and perpetuate inflammation; however, anti-inflammatory activities are increasingly attributed to them. Atherosclerosis is a chronic inflammatory condition. Similar to other inflammatory conditions, the resolution of atherosclerosis requires a shift in macrophages to an M2 phenotype, enhancing their efferocytosis and cholesterol efflux capabilities. OBJECTIVES: To assess the effect of platelets on macrophage phenotype. METHODS: In several in vitro models employing murine (RAW264.7 and bone marrow-derived macrophages) and human (THP-1 and monocyte-derived macrophages) cells, we exposed macrophages to media in which non-agonized human platelets were cultured for 60 minutes (platelet-conditioned media [PCM]) and assessed the impact on macrophage phenotype and function. RESULTS: Across models, we demonstrated that PCM from healthy humans induced a pro-resolving phenotype in macrophages. This was independent of signal transducer and activator of transcription 6 (STAT6), the prototypical pathway for M2 macrophage polarization. Stimulation of the EP4 receptor on macrophages by prostaglandin E2 present in PCM, is at least partially responsible for altered gene expression and associated function of the macrophages-specifically reduced peroxynitrite production, increased efferocytosis and cholesterol efflux capacity, and increased production of pro-resolving lipid mediators (ie, 15R-LXA4 ). CONCLUSIONS: Platelet-conditioned media induces an anti-inflammatory, pro-resolving phenotype in macrophages. Our findings suggest that therapies targeting hemostatic properties of platelets, while not influencing pro-resolving, immune-related activities, could be beneficial for the treatment of atherothrombotic disease.

Human low-affinity IgG receptor FcγRIIA polymorphism H131R associates with subclinical atherosclerosis and increased platelet activity in systemic lupus erythematosus.

Essentials Systemic lupus erythematosus (SLE) patients are at increased risk for premature CVD. Platelet activity, vascular dysfunction and carotid artery plaque are associated with FcγRIIA genotype in SLE. FcγRIIA genotype was not associated with platelet activity or carotid plaque in healthy controls. FcγRIIA represents a link that connects platelet activity, vascular health and CVD in SLE. SUMMARY: Background Systemic lupus erythematosus (SLE) is a complex autoimmune disease associated with an elevated risk of premature cardiovascular disease. Platelets express receptors contributing to inflammation and immunity, including FcγRIIA, the low affinity receptor of the Fc portion of IgG antibodies. The variation at a single amino acid substitution, H131R, in the extracellular binding domain alters the affinity for IgG, which may account for individual variation in platelet activity and platelet-mediated disease. Objectives This study was performed to investigate the association between FcγRIIA genotype, preclinical atherosclerosis, platelet reactivity and vascular health. Methods FcγRIIA was genotyped in 80 SLE patients and 30 healthy controls. Carotid ultrasound plaque, soluble E-selectin and platelet aggregability were evaluated in SLE and matched controls. Results Carotid plaque was significantly more prevalent in SLE patients carrying a variant allele compared to those with a homozygous ancestral allele (58% vs. 25%, P = 0.04). In contrast, prevalent carotid plaque was not associated with genotype in controls. Consistently, SLE variant FcγRIIA carriers vs. ancestral allele carriers had a significant increase in the levels of soluble E-selectin, which was not observed in controls. Monocyte and leukocyte-platelet aggregation and platelet aggregation in response to submaximal agonist stimulation were significantly elevated in SLE patients with the variant vs. ancestral genotype. Conclusions Carotid ultrasound plaque, soluble E-selectin levels and platelet activity were more frequently prevalent in SLE patients carrying variant FcγRIIA. The interplay between FcγRIIA-mediated platelet activation and endothelial cells might represent a mechanism underlying the pathogenesis of cardiovascular disease in SLE patients.

Human Memory B Cells Targeting Staphylococcus aureus Exotoxins Are Prevalent with Skin and Soft Tissue Infection.

Staphylococcus aureus is 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. aureus possesses 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. aureus isolates, 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. aureus antigens, 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. aureus antigens 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. aureus exotoxins, suggesting a relationship between the cellular and humoral compartments. Overall, although there was no global defect in the representation of anti-S. aureus memory B cells, there was evidence of restrictions in the range of epitopes recognized, which may suggest potential therapeutic approaches for augmenting host defenses.IMPORTANCE The contribution of B-cell memory and long-term antibody responses to host defenses against S. aureus exotoxins 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. aureus secreted 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. aureus secreted 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.

Unbiased RACE-Based Massive Parallel Surveys of Human IgA Antibody Repertoires.

For investigations of human B-cell receptor (BCR) repertoires, we have developed a protocol for large-scale surveys of human antibody heavy chain (VH) rearrangements. Here we study IgA repertoires, as more IgA antibodies are synthesized in the human body on a daily level than all other isotypes combined. In fact, IgA is secreted at all mucosal surfaces, and it is also secreted in the perspiration that coats our cutaneous surfaces. In these studies we can characterize the IgA clonal diversity of B-cell populations obtained from any donor. To recover representative repertoire libraries, we make our libraries from antibody gene transcript templates (i.e., cDNA), as these are closer reflections of the immune repertoire expressed at the antibody protein level. To avoid biases potentially introduced by upstream oligonucleotide primers that hybridize to variable region framework regions, our approach also uses rapid amplification of cDNA ends (RACE) of antibody transcripts. For exploration of human IgA responses, we have designed a duplexing antisense constant region primer that efficiently amplifies, side-by-side, heavy chain transcripts of both the IgA1 and IgA2 subclasses. By these methods we have begun to define the molecular differences in the IgA1 and IgA2 responses occurring simultaneously in different donors. These methods will be used to investigate the effects of microbial virulence factors on host defenses, during autoimmune responses, and in B-cell malignancies.

Association analysis of copy numbers of FC-gamma receptor genes for rheumatoid arthritis and other immune-mediated phenotypes.

Segmental duplications (SDs) comprise about 5% of the human genome and are enriched for immune genes. SD loci often show copy numbers variations (CNV), which are difficult to tag with genotyping methods. CNV in the Fcγ receptor region (FCGR) has been suggested to be associated with rheumatic diseases. The objective of this study was to delineate association of FCGR-CNV with rheumatoid arthritis (RA), coeliac disease and Inflammatory bowel disease incidence. We developed a method to accurately quantify CNV in SD loci based on the intensity values from the Immunochip platform and applied it to the FCGR locus. We determined the method's validity using three independent assays: segregation analysis in families, arrayCGH, and whole genome sequencing. Our data showed the presence of two separate CNVs in the FCGR locus. The first region encodes FCGR2A, FCGR3A and part of FCGR2C gene, the second encodes another part of FCGR2C, FCGR3B and FCGR2B. Analysis of CNV status in 4578 individuals with RA and 5457 controls indicated association of duplications in the FCGR3B gene in antibody-negative RA (P=0.002, OR=1.43). Deletion in FCGR3B was associated with increased risk of antibody-positive RA, consistently with previous reports (P=0.023, OR=1.23). A clear genotype-phenotype relationship was observed: CNV polymorphisms of the FCGR3A gene correlated to CD16A expression (encoded by FCGR3A) on CD8 T-cells. In conclusion, our method allows determining the CNV status of the FCGR locus, we identified association of CNV in FCGR3B to RA and showed a functional relationship between CNV in the FCGR3A gene and CD16A expression.

Abatacept decreases disease activity in a absence of CD4(+) T cells in a collagen-induced arthritis model.

INTRODUCTION: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Bridging autoantibodies and arthritis: the role of Fc receptors.

Autoantibodies represent a hallmark of Rheumatoid arthritis (RA), which is a chronic inflammatory autoimmune disease characterized by inflammation and damage in the joints. Anti-Citrullinated Protein Antibodies (ACPA) are the most prominent autoantibodies present in RA patients. These autoantibodies have been intensively investigated during the last 20 years due to their diagnostic and predictive value. Furthermore, they are believed to be involved in mediating the damage associated with RA. Antibodies of the IgG isotype interact with the immune system via Fcγ receptors expressed on immune cells as well as nonimmune cells. These receptors, therefore, form the bridge between Fcγ receptor-positive cells and antibodies complexed to antigen allowing the modulation and activation of cellular immune responses that are involved in immune defense against invading microorganisms. However, in case triggered by antibodies against self-antigens, they can also play a pivotal role in the induction and perpetuation of autoimmune diseases such as RA. Mouse models have been indispensably important for understanding the role of Fcγ receptors in the development of arthritis. Here we discuss the contribution of autoantibodies to the pathogenesis of arthritis in preclinical animal models, as well as RA, in relation to their interaction with the different (immune inhibitory and activating) Fcγ receptors.

DX5+ CD4+ T cells modulate CD4+ T-cell response via inhibition of IL-12 production by DCs.

DX5(+) CD4(+) T cells have been shown to dampen collagen-induced arthritis and delayed-type hypersensitivity reactions in mice. These cells are also potent modulators of T-helper cell responses through direct effects on CD4(+) T cells in an IL-4 dependent manner. To further characterize this T-cell population, we studied their effect on DCs and the potential consequences on T-cell activation. Here, we show that mouse DX5(+) CD4(+) T cells modulate DCs by robustly inhibiting IL-12 production. This modulation is IL-10 dependent and does not require cell contact. Furthermore, DX5(+) CD4(+) T cells modulate the surface phenotype of LPS-matured DCs. DCs modulated by DX5(+) CD4(+) T-cell supernatant express high levels of the co-inhibitor molecules PDL-1 and PDL-2. OVA-specific CD4(+) T cells primed with DCs exposed to DX5(+) CD4(+) T-cell supernatant produce less IFN-γ than CD4(+) T cells primed by DCs exposed to either medium or DX5(-) CD4(+) T-cell supernatant. The addition of IL-12 to the co-culture with DX5(+) DCs restores IFN-γ production. When IL-10 present in the DX5(+) CD4(+) T-cell supernatant is blocked, DCs re-establish their ability to produce IL-12 and to efficiently prime CD4(+) T cells. These data show that DX5(+) CD4(+) T cells can indirectly affect the outcome of the T-cell response by inducing DCs that have poor Th1 stimulatory function.

Anti-cyclic citrullinated peptide antibodies are a collection of anti-citrullinated protein antibodies and contain overlapping and non-overlapping reactivities.

OBJECTIVE: Anti-citrullinated protein antibodies (ACPA) and anti-cyclic citrullinated peptide (anti-CCP) antibodies are a hallmark of rheumatoid arthritis and are believed to play a role in disease pathogenesis. These antibodies are typically detected in ELISA with citrullinated peptides (eg, CCP2) or proteins as antigens. The absolute concentration of anti-CCP antibodies in serum is unknown. Although antibodies to several citrullinated proteins can mainly be detected within anti-CCP-positive sera, it is currently unknown whether anti-CCP antibodies are in fact ACPA. Likewise, it is unknown to what extent antibody responses to different citrullinated antigens are crossreactive. METHODS: An affinity purification method was established in which citrullinated antigen-specific antibodies were eluted from ELISA plates and then used for detection of other citrullinated antigens in ELISA or western blot. For additional crossreactivity studies, ELISA-based inhibition assays were performed with citrullinated or control peptides as inhibitors. RESULTS: The concentration of anti-CCP IgG antibodies was estimated to be at least 30 µg/ml in patients with high anti-CCP levels (>1600 µg/ml). Affinity-purified anti-CCP antibodies were able to recognise citrullinated fibrinogen (cit-fib) and citrullinated myelin basic protein (cit-MBP) on western blot. Furthermore, antibodies specific for cit-fib and cit-MBP were crossreactive. However, additional crossreactivity studies indicated that non-overlapping antibody responses to citrullinated peptides can also exist in patients. CONCLUSIONS: This report shows for the first time that anti-CCP antibodies recognise multiple citrullinated proteins and are thus a collection of ACPA. More importantly, the data indicate that different ACPA responses are crossreactive, but that crossreactivity is not complete, as distinct non-crossreactive responses can also be detected in patients with RA.

DX5(+)CD4(+) T cells modulate cytokine production by CD4(+) T cells towards IL-10 via the production of IL-4.

CD4(+) Th cells play a critical role in orchestrating the adaptive immune response. Uncontrolled Th1 responses are implicated in the pathogenesis of autoimmune diseases. T cells with immune-modulatory properties are beneficial for inhibiting such inflammatory responses. Previously we demonstrated that repetitive injections of immature DC induce expansion of DX5(+)CD4(+) T cells, which upon adoptive transfer show potent regulatory properties in murine collagen-induced arthritis as well as in delayed-hypersensitivity models. However, their regulatory mechanism remains to be defined. Here, we analyzed the effect of DX5(+)CD4(+) T cells on other CD4(+) T cells in vitro. Although proliferation of naïve CD4(+) T cells upon antigenic triggering was not altered in the presence of DX5(+)CD4(+) T cells, there was a striking difference in cytokine production. In the presence of DX5(+)CD4(+) T cells, an IL-10-producing CD4(+) T-cell response was induced instead of a predominant IFN-γ-producing Th1 response. This modulation did not require cell-cell contact. Instead, IL-4 produced by DX5(+)CD4(+) T cells was primarily involved in the inhibition of IFN-γ and promotion of IL-10 production by CD4(+) T cells. Together, our data indicate that DX5(+)CD4(+) T cells modulate the outcome of Th-responses by diverting Th1-induction into Th responses characterized by the production of IL-10.

Glycan profiling of anti-citrullinated protein antibodies isolated from human serum and synovial fluid.

OBJECTIVE: Anti-citrullinated protein antibodies (ACPA) exhibit unique specificity for rheumatoid arthritis. However, it is incompletely understood whether and how ACPA contribute to disease pathogenesis. The Fc part of human IgG carries 2 N-linked glycan moieties that are crucial for the structural stability of the antibody and that modulate both its binding affinity to Fcgamma receptors and its ability to activate complement. We undertook this study to analyze Fc glycosylation of IgG1 ACPA in serum and synovial fluid (SF) in order to further characterize the immune response to citrullinated antigens. METHODS: ACPA were isolated by affinity purification using cyclic citrullinated peptides as antigen. IgG1 Fc glycosylation was analyzed by mass spectrometry. ACPA IgG1 glycan profiles were compared with glycan profiles of total serum IgG1 obtained from 85 well-characterized patients. Glycan profiles of paired SF and serum samples were available from 11 additional patients. RESULTS: Compared with the pool of serum IgG1, ACPA IgG1 lacked terminal sialic acid residues. In SF, ACPA were highly agalactosylated and lacked sialic acid residues, a feature that was not detected for total SF IgG1. Moreover, differential ACPA glycan profiles were detected in rheumatoid factor (RF)-positive and RF-negative patients. CONCLUSION: ACPA IgG1 exhibit a specific Fc-linked glycan profile that is distinct from that of total serum IgG1. Moreover, Fc glycosylation of ACPA differs markedly between SF and serum. Since Fc glycosylation directly affects the recruitment of Fc-mediated effector mechanisms, these data could further our understanding of the contribution of ACPA to disease pathogenesis.

Identification of citrullinated vimentin peptides as T cell epitopes in HLA-DR4-positive patients with rheumatoid arthritis.

OBJECTIVE: Antibodies directed against citrullinated proteins (ACPAs) are highly specific for rheumatoid arthritis (RA). The production of ACPAs is most likely dependent on the presence of T cells, since ACPAs undergo isotype switching and are associated with the shared epitope (SE)-containing HLA-DRB1 alleles. Vimentin is a likely candidate protein for T cell recognition, since >90% of patients positive for ACPAs that are reactive with (peptides derived from) citrullinated vimentin carry SE-containing HLA-DRB1 alleles. The aim of this study was to identify citrullinated vimentin peptides that are presented to HLA-DRB1*0401-restricted T cells. METHODS: HLA-DR4-transgenic mice were immunized with all possible citrulline-containing peptides derived from vimentin, and T cell reactivity was analyzed. Peptides recognized in a citrulline-specific manner by T cells were selected and analyzed for their ability to be processed from the entire vimentin protein. A first inventory of the selected epitopes recognized by T cells was performed using peripheral blood mononuclear cells (PBMCs) from ACPA+, HLA-DR4+ patients with RA. RESULTS: A citrulline-specific response was observed for 2 of the peptides analyzed in DR4-transgenic mice. These peptides were found to be naturally processed from the vimentin protein, since citrullinated vimentin was recognized by peptide-specific T cells. T cell reactivity against these peptides was also observed in cultures of PBMCs from RA patients. CONCLUSION: This study identifies, for the first time, 2 naturally processed peptides from vimentin that are recognized by HLA-DRB1*0401-restricted T cells in a citrulline-specific manner. These peptides can be recognized by T cells in ACPA+, HLA-DR4+ patients with RA, as shown in a first inventory.

Variation in T-SPOT.TB spot interpretation between independent observers from different laboratories.

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.