Objective
Anti–citrullinated protein antibodies (ACPAs) are a hallmark of rheumatoid arthritis (RA). Aside from autoantibody production, the function of autoantigen‐specific B cells remains poorly ...understood in the context of this disease. This study set out to elucidate autoantigen‐specific B cell functions through the isolation and immortalization of unique citrullinated protein/peptide (CP)–reactive B cell clones from RA patients.
Methods
B cell clones from either the blood or synovial fluid of cyclic citrullinated peptide 2 (CCP2) antibody–positive RA patients were immortalized by genetic reprogramming with Bcl‐6 and Bcl‐xL. Enzyme‐linked immunosorbent assay and flow cytometry were used to identify CCP2‐reactive clones and to further characterize surface marker and cytokine expression as well as B cell receptor signaling competence. Global gene expression profiles were interrogated by RNA sequencing.
Results
Three unique CP‐reactive memory B cell clones were generated from the blood or synovial fluid of 2 RA patients. CP‐reactive memory B cells did not appear to be broadly cross‐reactive, but rather had a fairly restricted epitope recognition profile. These clones were able to secrete both pro‐ and antiinflammatory cytokines and had a unique surface profile of costimulatory molecules and receptors, including CD40 and C5a receptor type 1, when compared to non‐CP–reactive clones from the same patient. In addition, CP‐reactive clones bound citrullinated protein, but not native protein, and could mobilize calcium in response to antigen binding.
Conclusion
CP‐reactive memory B cells comprise a rare, seemingly oligoclonal population with restricted epitope specificity and distinct phenotypic and molecular characteristics suggestive of antigen‐presenting cells. Cloning by genetic reprogramming opens new avenues to study the function of autoreactive memory B cells, especially in terms of antigen processing, presentation, and subsequent T cell polarization.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Regulatory B cells (Bregs) are immunosuppressive cells that modulate immune responses through multiple mechanisms. The signals required for the differentiation and activation of these cells remain ...still poorly understood. We have already shown that overexpression of A PRoliferation-Inducing Ligand (APRIL) reduces the incidence and severity of collagen-induced arthritis (CIA) in mice. Furthermore, we have described that APRIL, but not BAFF, promoted IL-10 production and regulatory functions in human B cells. Therefore, we hypothesized that APRIL, but not BAFF, may be involved in the induction and/or activation of IL-10 producing Bregs that suppress inflammatory responses
and
. Here, we describe that APRIL promotes the differentiation of naïve human B cells to IL-10-producing IgA
B cells. These APRIL-induced IgA
B cells display a Breg phenotype and inhibit T cell and macrophage responses through IL-10 and PD-L1. Moreover, APRIL-induced IL-10 producing Bregs suppress inflammation
in experimental autoimmune encephalitis (EAE) and contact hypersensitivity (CHS) models. Finally, we showed a strong correlation between APRIL and IL-10 in the inflamed synovial tissue of inflammatory arthritis patients. Collectively, these observations indicate the potential relevance of this novel APRIL-induced IgA
Breg population for immune homeostasis and immunopathology.
Cutaneous antigen presenting cells (APCs) are critical for the induction and regulation of skin immune responses. The human skin contains phenotypically and functionally distinct APCs subsets that ...are present at two separated locations. While CD1ahigh LCs form a dense network in the epidermis, the CD14+ and CD1a+ APCs reside in the dermal compartment. A better understanding of the biology of human skin APC subsets is necessary for the improvement of vaccine strategies that use the skin as administration route. In particular, progress in the characterization of uptake and activatory receptors will certainly improve APC-targeting strategies in vaccination. Here we performed a detailed analysis of the expression and function of glycan-binding and pattern-recognition receptors in skin APC subsets. The results demonstrate that under steady state conditions human CD1a+ dermal dendritic cells (DCs) were phenotypically most mature as measured by the expression of CD83 and CD86, whereas the CD14+ cells showed a higher expression of the CLRs DC-SIGN, mannose receptor and DCIR and had potent antigen uptake capacity. Furthermore, steady state LCs showed superior antigen cross-presentation as compared to the dermal APC subsets. Our results also demonstrate that the TLR3 ligand polyribosinic-polyribocytidylic acid (pI:C) was the most potent stimulator of cytokine production by both LCs and dDCs. These studies warrant further exploration of human CD1a+ dDCs and LCs as target cells for cancer vaccination to induce anti-tumor immune responses.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
► Leb and LeX glycans achieve an efficient antigen-targeting to DCs through DC-SIGN. ► Glycan multivalency is a necessary condition for efficient targeting. ► DC-SIGN targeting results in robust CD4+ ...and CD8+ T cell responses.
Dendritic cells are the most efficient professional antigen-presenting cells in pathogen recognition and play a pivotal role in the control of the immune response. Pathogen recognition is ensured by the expression of a vast variety of pattern-recognition receptors. Amongst them are C-type lectins, a large family of receptors characterized by a domain that – in many cases – mediates calcium-dependent glycan binding. C-type lectins facilitate antigen uptake for efficient processing and presentation and, in some cases, also trigger signaling to modulate T cell responses. These properties make C-type lectin receptors ideal candidates for the targeting of antigens to dendritic cells for vaccination. DC-SIGN is a paradigmatic example of C-type lectin receptors on dendritic cells that facilitate vaccination strategies. DC-SIGN is highly expressed on immature conventional dendritic cells, particularly at the mucosa and the dermis, where DCs first encounter pathogens, but also can easily be accessed for vaccination. Upon ligand binding, DC-SIGN rapidly internalizes and directs its cargo into the endo-lysosomal pathway, which results in MHC-II presentation. But antigens targeted to DC-SIGN are also presented efficiently to CD8+ T cells, suggesting there is an additional endocytic route that leads to cross-presentation. Simultaneous triggering of DC-SIGN and TLRs results in the modulation of cytokine responses and facilitates cross-presentation to enhance CD4+ and CD8+ T cell responses. Because the glycan specificity of DC-SIGN has been characterized in detail, glycans can be used for the targeting of antigens to DCs in a DC-SIGN-dependent manner. Glycans represent a great advantage over monoclonal antibodies, they diminish the risk of side effects, are very small, and their production can rely entirely in organic chemistry approaches. Here, we discuss the capacity of glycan-based vaccines to enhance antigen-specific CD4+ and CD8+ T cell responses in human skin and mouse model systems.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Antigen cross-presentation, the process in which exogenous antigens are presented on MHC class I molecules, is crucial for the generation of effector CD8(+) T cell responses. Although multiple cell ...types are being described to be able to cross-present antigens, in vivo this task is mainly carried out by certain subsets of dendritic cells (DCs). Aspects such as the internalization route, the pathway of endocytic trafficking, and the simultaneous activation through pattern-recognition receptors have a determining influence in how antigens are handled for cross-presentation by DCs. In this review, we will summarize new insights in factors that affect antigen cross-presentation of human DC subsets, and we will discuss the possibilities to exploit antigen cross-presentation for immunotherapy against cancer.
Dendritic cells (DCs) and Langerhans cells (LC) are professional antigen presenting cells (APCs) that initiate humoral and cellular immune responses. Targeted delivery of antigen towards DC- or ...LC-specific receptors enhances vaccine efficacy. In this study, we compared the efficiency of glycan-based antigen targeting to both the human DC-specific C-type lectin receptor (CLR) DC-SIGN and the LC-specific CLR langerin. Since DC-SIGN and langerin are able to recognize the difucosylated oligosaccharide Lewis Y (Le(Y)), we prepared neoglycoconjugates bearing this glycan epitope to allow targeting of both lectins. Le(Y)-modified liposomes, with an approximate diameter of 200nm, were significantly endocytosed by DC-SIGN(+) DCs and mediated efficient antigen presentation to CD4(+) and CD8(+) T cells. Surprisingly, although langerin bound to Le(Y)-modified liposomes, LCs exposed to Le(Y)-modified liposomes could not endocytose liposomes nor mediate antigen presentation to T cells. However, LCs mediated an enhanced cross-presentation when antigen was delivered through langerin using Le(Y)-modified synthetic long peptides. In contrast, Le(Y)-modified synthetic long peptides were recognized by DC-SIGN, but did not trigger antigen internalization nor antigen cross-presentation. These data demonstrate that langerin and DC-SIGN have different size requirements for antigen uptake. Although using glycans remains an interesting option in the design of anti-cancer vaccines targeting multiple CLRs, aspects such as molecule size and conformation need to be taken in consideration.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
To investigate the presence of different isotypes of anti-carbamylated protein (CarP) antibodies in systemic sclerosis (SSc) patients and its association with skin involvement.
Sera of 194 SSc ...patients from the Leiden CCISS cohort, fulfilling ACR/EULAR 2013 criteria and a clinical diagnosis of SSc, 83 patients with other connective tissue diseases/Raynaud's Phenomenon, 24 rheumatoid arthritis patients and 98 age and sex-matched healthy controls were tested for the presence of anti-CarP IgG, IgA and IgM, determined by ELISA. Clinical characteristics, that were evaluated in SSc patients, included age, anti-topoisomerase antibodies (ATA), anti-centromere antibodies (ACA) and modified Rodnan Skin Score (mRSS).
The SSc patients were 55 (SD:13) years and 155 (80%) were female. Forty-four (23%) patients tested positive for ATA, and 80 (42%) ACA. The median mRSS was 2 (range: 0; 47). Prevalence of anti-CarP IgG was higher in SSc patients than in healthy controls (8% vs 3%, p = 0.007. Prevalence of anti-CarP IgA and IgM and levels of anti-CarP isotypes were comparable between SSc patients and healthy controls. Fifteen (8%) SSc patients tested positive for anti-CarP IgG, 16 (8%) for anti-CarP IgA, and 36 (19%) for anti-CarP IgM. There were no significant correlations between age and levels of anti-CarP isotypes. No correlation between anti-CarP IgG levels and mRSS was found (r = 0.141, p = 0.049), nor for anti-CarP IgM and IgA levels. Anti-CarP IgA levels were higher in ATA compared to ACA positive SSc patients (ATA: 616 aU/ml 359; 1103; ACA: 424 aU/ml 300; 673, p = 0.015).
SSc patients can test positive for Anti-CarP IgG, IgA and IgM. We do not observe a relevant clinical association between anti-CarP antibody response and skin involvement in SSc.
The potential of the skin immune system to generate immune responses is well established, and the skin is actively exploited as a vaccination site. Human skin contains several antigen-presenting cell ...subsets with specialized functions. In particular, the capacity to cross-present exogenous antigens to CD8^＋ T cells is of interest for the design of effective immunotherapies against viruses or cancer. Here, we show that primary human Langerhans cells （LCs） were able to cross-present a synthetic long peptide （SLP） to CD8^＋ T cells. In addition, modification of this SLP using antibodies against the receptor langerin, but not dectin-1, further enhanced the cross-presenting capacity of LCs through routing of internalized antigens to less proteolytic early endosome antigen 1 ^＋ early endosomes. The potency of LCs to enhance CD8^＋ T-cell responses could be further increased through activation of LCs with the toll-like receptor 3 ligand polyinosinic：polycytidylic acid （phC）. Altogether, the data provide evidence that human LCs are able to cross-present antigens after langerin-mediated internalization. Furthermore, the potential for antigen modification to target LCs specifically provides a rationale for generating effective anti-tumor or anti-viral cytotoxic T lymphocyte responses.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
The prostaglandin transporter (PGT, SLCO2A1) mediates transport of prostanoids (a.o. prostaglandin E2 (PGE
)) into cells and thereby promotes their degradation. Overexpression of PGT leads to low ...extracellular PGE
levels and has been linked to impaired wound healing of diabetic foot ulcers. Inhibition of PGT could thus be beneficial, however, no PGT inhibitors are currently on the market and drug discovery efforts are hampered by lack of high-through screening assays for this transporter. Here we report on a label-free impedance-based assay for PGT that measures transport activity through receptor activation (TRACT) utilizing prostaglandin E2 receptor subtype EP3 and EP4 that are activated by PGE
. We found that induction of PGT expression on HEK293-JumpIn-SLCO2A1 cells that also express EP3 and EP4 leads to an over 10-fold reduction in agonistic potency of PGE
. PGE
potency could be recovered upon inhibition of PGT-mediated PGE
uptake with PGT inhibitors olmesartan and T26A, the potency of which could be established as well. Moreover, the TRACT assay enabled the assessment of transport function of PGT natural variants. Lastly, HUVEC cells endogenously expressing prostanoid receptors and PGT were exploited to study wound healing properties of PGE
and T26A in real-time using a novel impedance-based scratch-induced wound healing assay. These novel impedance-based assays will advance PGT drug discovery efforts and pave the way for the development of PGT-based therapies.
Rheumatoid arthritis (RA) is a chronic inflammatory disease of synovial joints, characterized by the presence of the highly disease-specific anti-citrullinated protein antibodies (ACPA) in ...approximately 70% of patients. Epstein-Barr virus (EBV) has previously been suggested to be involved in the pathophysiology of RA. However, given the high incidence of EBV in the general population and the difficulty of detecting initial infection, providing a direct link between EBV infection and RA development has remained elusive. We hypothesized that primary EBV infection may be a trigger for the development of the ACPA response in vivo. Using a unique cohort of 26 kidney transplant patients with a primary EBV infection, the presence of ACPA before and following infection was determined. No increase in IgG anti-CCP2 titers was detected following EBV infection. IgG anti-CCP2 antibodies were present in two patients and borderline positive in another. These three patients were HLA-DR4 negative. To test whether EBV infection may trigger a non-class switched anti-CCP2 response, IgM anti-CCP2 antibodies were analyzed. No general trend in the IgM anti-CCP2 response was observed following EBV infection. Since two out of the three IgG anti-CCP2 (borderline) positive patients were diagnosed with IgA nephropathy, 23 additional IgA nephropathy patients were tested for IgG anti-CCP2, regardless of their EBV status. All of these patients were IgG anti-CCP2 negative, indicating that IgG anti-CCP2 is not commonly present in IgA nephropathy patients. Collectively, these data do not support the hypothesis that EBV does trigger the highly RA specific ACPA response.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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