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Previous studies have shown that low-dose ultraviolet-A (UVA-1) total body irradiations were capable of improving disease activity in patients with systemic lupus erythematosus (SLE). We hypothesized that UVA-1-induced suppression of immunoglobulin production by activated B cells in the dermal capillaries could be (partly) responsible for this effect. Our experiments with donor skin demonstrated that approximately 40% of UVA-1 could penetrate through the epidermis. Irradiation of peripheral blood mononuclear cells (PBMCs) with 2 J/cm2 of UVA-1 resulted in 20% cell death. This toxic effect could be prevented totally by preincubation of the cell cultures with catalase. This indicates that the generation of hydrogen peroxide plays a role in UVA-1 cytotoxicity. T cells and B cells appeared to be less susceptible to UVA-1 cytotoxicity than monocytes. With the use of a CD40–CD40L B cell activation method we measured immunoglobulin production after various doses of UVA-1 irradiation (0–2 J/cm2). The doses of 2 J/cm2 caused a significant decrease of IgM, IgG, IgA and IgE production under the conditions of interleukin (IL)-10 or IL-4 (IgE) stimulation. Although UVA-1 can cause apoptosis of B lymphocytes, we show that relatively low doses of UVA-1 radiation also affect the function of these cells. Both effects may be responsible for the observed improvement of disease activity in SLE patients. [ABSTRACT FROM AUTHOR]

Antigen-dependent activation of B lymphocytes is mediated through surface immunoglobulins and their associated molecules Ig-α (CD79a, Mb1) and Ig-β (CD79b, B29). Here we show that an antibody directed against the extracellular part of human Ig-β can, when cross-linked by CD32-transfected L cells, induce an IL-2-dependent proliferation of tonsil B cells. With the use of L cells stably transfected with both CD32 and CD40L, anti-Ig-β activation of B cells was combined with CD40 triggering, an important component of the T cell-dependent B cell activation. This dual cellular activation resulted in two different phases, with initially synergistic proliferative effects, both without and with IL-2 or IL-10. Then, after 5-6 days of culture, cells stimulated with both anti-Ig-β and CD40L underwent massive cell death, in contrast to B cells activated with CD40L alone. Cell death was not prevented by the addition of IL-2 or IL-10, but was prevented by the addition of IL-4. These results are discussed in the context of positive and negative selection of mature B cells. [ABSTRACT FROM AUTHOR]

This paper demonstrates that CD40 is expressed on rheumatoid synovial pannus and primary fibroblast cell lines established from rheumatoid and osteoarthritic synovium as well as normal skin. Among various tested cytokines, interferon-gamma (IFN-γ) and lo a lower extent, tumour necrosis factor-alpha (TNE-α) were found to upregulate CD40 expression on fibroblasts. Synovial and skin fibroblasts cultured over CD40 Ligand transfected L cells (L-CD40L) demonstrate a CD40 specific increase of DNA synthesis as measured by tritiated thymidine incorporation. Cell-cycle analysis and enumeration of viable cells further show that CD40 induced fibroblast proliferation. Costimulation with L-CD40 L and IFN-γ, resulted in maximal proliferation. Engagement of fibroblasts CD40 increased the IL-l-induced production of granulocyte macrophage-colony stimulating factor and macrophage inflammatory protein-Iα MIP-lα, CD40L activated fibroblasts showed decreased levels of CD40, but only marginal alterations of other cell-surface antigens. Taken together, the present results indicate that fibroblasts express functional CD40 and suggest a possible role of CD40L expressing cells, such as activated T cells and mast cells, in the development of synovium hyperplasia. [ABSTRACT FROM AUTHOR]

Abnormally O-glycosylated IgA1 is likely to be involved in the pathogenesis of IgA nephropathy (IgAN). Buck et al. show that the enzyme activity and gene expression of specific glycosyltransferases, in purified B cells isolated from peripheral blood and bone marrow, is not reduced in IgAN patients. As only a small fraction of IgA in IgAN patients is abnormally glycosylated, it is probable that a more detailed molecular analysis at the single cell level is required to unravel the cause of this abnormality.Kidney International (2008) 73, 1106–1108. doi:10.1038/ki.2008.16 [ABSTRACT FROM AUTHOR]

Moldoveanu et al. present a diagnostic test for IgA nephropathy based on the presence of undergalactosylated IgA in serum. This underglycosylated IgA appears to be overrepresented in serum of IgA nephropathy patients and is most likely related to mesangial IgA deposition. Further studies on the nature, production, regulation, and cellular and molecular interactions of this undergalactosylated IgA may facilitate disease diagnosis and provide further insight into the pathogenesis of this important disease.Kidney International (2007) 71, 1089–1091. doi:10.1038/sj.ki.5002262 [ABSTRACT FROM AUTHOR]

The complement system is an important part of the innate immune defence. It contributes not only to local inflammation, removal and killing of pathogens, but it also assists in shaping of the adaptive immune response. Besides a role in inflammation, complement is also involved in physiological processes such as waste disposal and developmental programmes. The complement system comprises several soluble and membrane-bound proteins. The bulk of the soluble proteins is produced mainly by the liver. While several complement proteins are produced by a wide variety of cell types, other complement proteins are produced by only a few related cell types. As these data suggest that local production by specific cell types may have specific functions, more detailed studies have been employed recently analysing the local and even intracellular role of these complement proteins. Here we review the current knowledge about extrahepatic production and/or secretion of complement components. More specifically, we address what is known about complement synthesis by cells of the human immune system. [ABSTRACT FROM AUTHOR]

Acute cellular rejection (ACR) occurs in 10% of renal allograft recipients and is characterized by leukocyte infiltration as observed in needle biopsies. ACR onset is subject to several risk factors, including delayed graft function (DGF). As the impact of DGF on the etiology of ACR remains unclear, this study analyzed the association between presence of leukocyte subsets and ACR onset, in DCD kidney biopsies with extensive DGF following transplantation. Immunohistochemical analysis of protocol biopsies taken 10 days after kidney transplantation revealed that patients with high levels of renal CD163+ macrophages have a decreased risk (OR = 0.021, P = 0.008) for ACR in the first 6 months after transplantation. In pre-transplant biopsies of a comparable DCD cohort, with >80% DGF, presence of donor CD163+ macrophages showed no effect on ACR risk. Therefore, leukocyte infiltrate present during the inflammatory response at the time of DGF may contain anti-inflammatory macrophages that exert a protective effect against ACR development. [ABSTRACT FROM AUTHOR]

• Complement activation in IgA nephropathy is a major pathogenic process. • Lectin and alternative pathway activation are the main complement routes involved in IgA nephropathy. • In IgA nephropathy, variation in IgA consists of isoforms, glycoforms and presence of IgA containing immune complexes. • Variations in IgA might be responsible for complement activation in both systemic and kidney compartments. • Novel complement inhibitory strategies are currently tested in phase II or III trials in IgA nephropathy. IgA Nephropathy (IgAN) is the main cause of primary glomerulonephritis, globally. This disease is associated with a wide range of clinical presentations, variable prognosis and a spectrum of histological findings. More than fifty years after its first description, this heterogeneity continues to complicate efforts to understand the pathogenesis. Nevertheless, involvement of the complement system in IgAN was identified early on. Dysfunction of the immunoglobulin A (IgA) system, the principal offender in this disease, including modification of isoforms and glycoforms of IgA1, the nature of immune complexes and autoantibodies to galactose deficient IgA1 might all be responsible for complement activation in IgAN. However, the specific mechanisms engaging complement are still under examination. Research in this domain should allow for identification of patients that may benefit from complement-targeted therapy, in the foreseeable future. [ABSTRACT FROM AUTHOR]

Anti-C1q autoantibodies are found frequently in patients with Systemic Lupus Erythematosus (SLE) and several studies indicate that these autoantibodies are associated with renal involvement. We have shown earlier that administration of anti-C1q antibodies to normal BALB/c mice results in the deposition of these antibodies and C1q in the kidney. In the present study we have investigated which factors are essential for this C1q–anti-C1q deposition. Injection of anti-C1q antibodies in C57BL/6 mice results in deposition of both C1q and anti-C1q in glomeruli, while administration of equal concentrations of anti-C1q to immunoglobulin deficient Rag2−/− mice did not result in deposition of anti-C1q antibodies. Analysis of renal sections of naive Rag2−/− mice revealed absence of mouse IgG and C1q in the glomeruli, while circulating C1q was within normal levels. Reconstitution of Rag2−/− mice with IgG, either by injection with purified mouse IgG or by splenocyte transfer, resulted in restored localization of mouse IgG together with C1q in the kidney. Subsequent injection of anti-C1q antibodies in these IgG reconstituted mice resulted in clear deposition of C1q together with anti-C1q in the kidneys comparable to that found in C57BL/6 mice receiving anti-C1q. We propose that the continuos presence of serum derived non-immune IgG in the glomerulus serves as a target for low affinity interactions with C1q, which then can serve as antigen for anti-C1q antibodies. Therefore we hypothesize that high and fluctuating levels of IgG as observed in patients with SLE may contribute to flares of renal inflammation in those patients with anti-C1q autoantibodies. [Copyright &y& Elsevier]

Cellular immunotherapy has proven to be effective in the treatment of hematological cancers by donor lymphocyte infusion after allogeneic hematopoietic stem cell transplantation and more recently by targeted therapy with chimeric antigen or T-cell receptor-engineered T cells. However, dependent on the tissue distribution of the antigens that are targeted, anti-tumor responses can be accompanied by undesired side effects. Therefore, detailed tissue distribution analysis is essential to estimate potential efficacy and toxicity of candidate targets for immunotherapy of hematological malignancies. We performed microarray gene expression analysis of hematological malignancies of different origins, healthy hematopoietic cells and various non-hematopoietic cell types from organs that are often targeted in detrimental immune responses after allogeneic stem cell transplantation leading to graft-versus-host disease. Non-hematopoietic cells were also cultured in the presence of IFN-γ to analyze gene expression under inflammatory circumstances. Gene expression was investigated by Illumina HT12.0 microarrays and quality control analysis was performed to confirm the cell-type origin and exclude contamination of non-hematopoietic cell samples with peripheral blood cells. Microarray data were validated by quantitative RT-PCR showing strong correlations between both platforms. Detailed gene expression profiles were generated for various minor histocompatibility antigens and B-cell surface antigens to illustrate the value of the microarray dataset to estimate efficacy and toxicity of candidate targets for immunotherapy. In conclusion, our microarray database provides a relevant platform to analyze and select candidate antigens with hematopoietic (lineage)-restricted expression as potential targets for immunotherapy of hematological cancers. [ABSTRACT FROM AUTHOR]

There is a growing interest in the monitoring of complement activation, not only in clinical settings but also in experimental models. However, for rodents only a limited number of tools are available to assess complement activity and activation. Here we describe three ELISAs for the measurement of rat classical (CP), MB-lectin (LP) and alternative (AP) pathway activities in serum and plasma. Moreover, we optimised a soluble C5b-9 (sC5b-9) ELISA for the detection of low level complement activation in rat. We determined the conditions for correct sample handling and showed that the assays had low inter- and intra-assay variation. We applied these assays to monitor complement activation in an experimental rat model of renal ischemia/reperfusion injury. We did not observe major complement consumption following reperfusion in CP or LP, and only minor AP consumption at 24 h post reperfusion. However, MBL depletion prior to ischemia/reperfusion using a monoclonal antibody, transiently and specifically inhibited 75% of LP activity and ameliorated the AP consumption at 24 h. To further assess complement activation during renal IRI, we monitored serum sC5b-9 and found that it was only significantly increased 72 h post-reperfusion, but not when rats were pre-treated with anti-MBL or after sham surgery. In conclusion the described assays enable sensitive, reproducible and comprehensive assessment of complement activation in experimental rat models. [ABSTRACT FROM AUTHOR]

In general, humoral immune responses depend critically upon T cell help. In transplantation, prevention or treatment of humoral rejection therefore require drugs that ideally inhibit both B cell and T helper cell activity. Here, we studied the effects of commonly used immunosuppressive drugs [tacrolimus, cyclosporin, mycophenolic acid (MPA) and rapamycin] on T cell helper activity and on T cell-dependent B cell responses. T cells were activated polyclonally in the presence of immunosuppressive drugs in order to analyse the effect of these drugs on T cell proliferation, co-stimulatory ligand expression and cytokines. The impact of immunosuppressive drugs on T cell-dependent immunoglobulin production by B cells was addressed in T–B cell co-cultures. All drugs affected T cell proliferation and attenuated T cell co-stimulatory ligand (CD154 and CD278) expression when T cells were activated polyclonally. Tacrolimus, cyclosporin and rapamycin also attenuated B cell stimulatory cytokine mRNA levels in T cells. As a consequence, a decrease in immunoglobulin levels was observed in autologous T–B cell co-cultures, where T cell help is essential for immunoglobulin production. In contrast, when pre-activated T cells were used to stimulate autologous B cells, calcineurin inhibitors failed to inhibit B cell immunoglobulin production, whereas MPA and rapamycin did show inhibition. From these studies, it is evident that calcineurin inhibitors affect the humoral immune response by interfering with T helper signals, but not by targeting B cells directly. Furthermore, our studies support the necessity of intervening in T cell helper function to attenuate humoral responses. [ABSTRACT FROM AUTHOR]

Secretory immunoglobulin A (SIgA), although generated at mucosal surfaces, is also found in low concentrations in the circulation. Recently, SIgA was demonstrated in mesangial deposits of patients with immunoglobulin A nephropathy (IgAN), suggesting a role in the pathogenesis. This finding is in line with the belief that high molecular weight (HMW) immunoglobulin A (IgA) is deposited in the kidney. However, there is little information on the size distribution of antigen-specific IgA in circulation upon mucosal challenge. In this study we measured antigen-specific IgA, including SIgA, in serum following challenge of IgAN patients and controls via intranasal vaccination with a neoantigen, cholera toxin subunit B (CTB). We size-fractionated serum and nasal washes to study the size distribution of total IgA, SIgA and CTB-specific IgA. Finally, we compared the size distribution of antigen-specific IgA after mucosal immunization with the distribution upon systemic immunization. A significant induction of antigen-specific SIgA was detectable in serum of both patients with IgAN and controls after mucosal immunization with CTB. Independent of the route of immunization, in both groups the antigen-specific IgA response was predominantly in the polymeric IgA fractions. This is in contrast to total IgA levels in serum that are predominantly monomeric. We conclude that mucosal challenge results in antigen-specific SIgA in the circulation, and that the antigen-specific IgA response in both IgAN patients and in controls is of predominantly HMW in nature. No differences between IgAN patients and controls were detected, suggesting that the size distribution of antigen-specific IgA in the circulation is not disturbed specifically in IgAN patients. [ABSTRACT FROM AUTHOR]

Dendritic cells (DCs) play critical roles in immune responses and can be distinguished in two major subsets, myeloid and plasmacytoid DCs. Although the presence of DC in all peripheral organs, including the kidney, has been well documented, no accurate estimates of DC subsets in human kidneys have been reported. This study shows a detailed analysis of DC subsets in cryosections of human renal tissue. The cortex of normal kidneys contains at least two different HLA-DR+ myeloid DC subtypes characterized by BDCA-1+DC-SIGN+ and BDCA-1+DC-SIGN−. The staining for DC-SIGN completely overlapped with CD68 in the renal interstitium. Unexpectedly, BDCA-2+DC-SIGN− plasmacytoid DCs are also abundantly present. Both subsets are located in the tubulo–interstitium often with a high frequency around, but rarely observed within glomeruli. Quantification of BDCA-1+, DC-SIGN+, and BDCA-2+ cells in normal human renal tissue (pretransplant biopsy living donors; n=21) revealed that BDCA-1 is about four times as frequently present as BDCA-2. A preliminary cross-sectional analysis of DC in diseased kidneys, including rejection and immunoglobulin A nephropathy, revealed that the number of DC as well as their anatomical distribution might change under pathophysiological conditions. In conclusion, we show that human kidneys contain a dense network of myeloid and plasmacytoid DCs and provide the tools for phenotyping and enumeration of these cells to better understand interindividual differences in immune responses.Kidney International (2007) 71, 1001–1008. doi:10.1038/sj.ki.5002187; published online 14 March 2007 [ABSTRACT FROM AUTHOR]

IgA nephropathy (IgAN) is characterized by deposits of IgA in the renal mesangium. It is thought that deposits of IgA mainly involve high molecular weight (HMW) IgA1. However, there is limited information on the exact composition of HMW IgA in these deposits. In this study, we investigated the presence of secretory IgA (SIgA) in human serum and in the glomerular deposits of a patient with IgAN. Furthermore, we analyzed the interaction of SIgA with mesangial cells. With enzyme-linked immunosorbent assay, SIgA concentrations in the serum of IgAN patients and healthy controls were measured. Both patients and controls had circulating SIgA that was restricted to the HMW fractions. Patients tended to have higher levels of SIgA, but this difference was not significant. However, in patients with IgAN, high serum SIgA concentrations were associated with hematuria. Binding of size-fractionated purified serum IgA and SIgA to mesangial cells was investigated with flow cytometry. These studies showed stronger binding of SIgA to primary mesangial cells compared to binding of serum IgA. Importantly, after isolation and elution of glomeruli from a nephrectomized transplanted kidney from a patient with recurrent IgAN, we demonstrated a 120-fold accumulation of SIgA compared to IgA1 in the eluate. In conclusion, we have demonstrated that SIgA strongly binds to human mesangial cells, and is present in significant amounts in serum. Furthermore, we showed that SIgA is accumulated in the glomeruli of an IgAN patient. These data suggest an important role for SIgA in the pathogenesis of IgAN.Kidney International (2006) 69, 1131–1138. doi:10.1038/sj.ki.5000074; published online 4 January 2006 [ABSTRACT FROM AUTHOR]

Chronic renal allograft rejection is characterized by alterations in the extracellular matrix compartment and in the proliferation of various cell types. These features are controlled, in part by the metzincin superfamily of metallo-endopeptidases, including matrix metalloproteinases (MMPs), a disintegrin and metalloproteinase (ADAM) and meprin. Therefore, we investigated the regulation of metzincins in the established Fisher to Lewis rat kidney transplant model. Studies were performed using frozen homogenates and paraffin sections of rat kidneys at day 0 (healthy controls) and during periods of chronic rejection at day +60 and day +100 following transplantation. The messenger RNA (mRNA) expression was examined by Affymetrix Rat Expression Array 230A GeneChip and by real-time Taqman polymerase chain reaction analyses. Protein expression was studied by zymography, Western blot analyses, and immunohistology. mRNA levels of MMPs (MMP-2/-11/-12/-14), of their inhibitors (tissue inhibitors of metalloproteinase (TIMP)-1/-2), ADAM-17 and transforming growth factor (TGF)-β1 significantly increased during chronic renal allograft rejection. MMP-2 activity and immunohistological staining were augmented accordingly. The most important mRNA elevation was observed in the case of MMP-12. As expected, Western blot analyses also demonstrated increased production of MMP-12, MMP-14, and TIMP-2 (in the latter two cases as individual proteins and as complexes). In contrast, mRNA levels of MMP-9/-24 and meprin α/β had decreased. Accordingly, MMP-9 protein levels and meprin α/β synthesis and activity were downregulated significantly. Members of metzincin families (MMP, ADAM, and meprin) and of TIMPs are differentially regulated in chronic renal allograft rejection. Thus, an altered pattern of metzincins may represent novel diagnostic markers and possibly may provide novel targets for future therapeutic interventions.Kidney International (2006) 69, 358–368. doi:10.1038/sj.ki.5000049 [ABSTRACT FROM AUTHOR]

Autoantibodies against C1q can be found in the circulation of patients with several autoimmune diseases including systemic lupus erythematosus (SLE). In SLE there is an association between the occurrence of these antibodies and renal involvement. How anti-C1q autoantibodies contribute to renal disease is currently unknown. Cohorts of MRL-lpr mice, which are known to develop age-dependent SLE-like disease, were used to study the relationship between levels of anti-C1q autoantibodies and renal disease. We collected serum, urine and renal tissue and analysed autoantibodies, complement levels and renal deposition as well as renal function. At 2 months of age all mice already had elevated levels of anti-C1q autoantibodies, and elution of kidneys revealed the presence of these antibodies in renal immune deposits in MRL-lpr mice and not in control MRL+/+ mice. In conclusion, anti-C1q antibodies are already present in serum and immune deposits of the kidney early in life and therefore can play a role in nephritis during experimental SLE-like disease in mice. [ABSTRACT FROM AUTHOR]

SUMMARY Anti-C1q autoantibodies are present in the serum of patients with different autoimmune diseases such as systemic lupus erythematosus (SLE). The occurrence of these autoantibodies correlates with renal involvement. In the present study we examined whether injection of rabbit antimouse C1q antibodies in mice leads to deposition in kidneys. Injection of healthy mice with a single dose of rabbit IgG antimouse C1q antibodies resulted in deposition of both C1q and IgG anti-C1q in glomeruli. The pattern of deposition observed in the glomeruli of mice injected with antimouse C1q antibodies both at 24 h and 2 weeks was both glomerular basement membrane (GBM)-associated and mesangial. Injection of control IgG did not have a detectable effect on circulating C1q levels, and no deposition of either C1q or rabbit IgG was seen at 24 h. The deposition of rabbit antimouse C1q and C1q in glomeruli resulted in complement activation, as assessed by C3 deposition, and influx of leucocytes associated with albuminuria in some, but not all mice. In none of the control mice was albuminuria observed. This report is the first to show that anti-C1q antibodies deposit in the healthy glomerulus together with autologous C1q. This deposition is stable for at least 2 weeks, causes complement activation, leucocyte influx and can lead to mild albuminuria. [ABSTRACT FROM AUTHOR]

SUMMARY The persistent presence of rheumatoid factors (RFs) in the circulation is a characteristic phenomenon in patients with rheumatoid arthritis (RA). Recent data indicate that RFs associated with seropositive RA are derived from terminally differentiated CD20– , CD38+ plasma cells (PCs) present in synovial fluids of the inflamed joints. These cells were shown to secrete RFs actively and are thought to originate from germinal centre (GC)-like structures present in the inflamed synovium. To obtain a representative image of the structural properties of IgM and IgG RFs associated with RA, phage antibody display libraries were constructed from CD38+ PCs isolated from the inflamed joints of RF-seropositive patients with RA. Subsequently, human IgG Fc-binding monoclonal phage antibodies were selected and analysed. The data suggest that RA-associated RFs are encoded by a diverse set of VL and a more restricted set of VH regions. VH gene family usage of PC-derived IgM- and IgG-RFs was found to be restricted to the VH1 and 3 gene families, with a preference for VH3, and many different VL genes were shown to contribute to RF specificity. Clonally related VH as well as VL sequences were identified, based on the presence of identical CDR3 regions and shared somatic mutations. In this B cell selection process base-pair substitutions as well as deletions of triplets in CDR regions, leaving the transcripts in frame, were involved. Together, these data provide further evidence for an Ag-driven immune response in the terminal differentiation into RF-producing PCs in patients with RA, including expansion of clonally related B cells, selection and isotype switching, all hallmarks of a GC reaction. [ABSTRACT FROM AUTHOR]

Recent data indicate that rheumatoid factors (RFs) that occur in patients with rheumatoid arthritis (RA) are derived from Ig-producing terminally differentiated CD20−, CD38+ plasma cells present in synovial fluids (SFs). Phage antibody display libraries were constructed using CD38+ plasma cells isolated from SFs of two RF-seropositive RA patients. The libraries were enriched for phage antibodies (Phabs) binding to human IgG (HuIgG) Fc fragments and the sequences of their V genes were analysed. These data provided further evidence for an Ag-driven immune response in patients with RA, including expansion of clonally related B cells, selection and isotype switching, all hallmarks of a germinal center reaction. In the present study, the functional characteristics of these HuIgG Fc-binding monoclonal (mo) Phabs were further analysed in order to provide more insight into the specificity of HuIgG Fc-binding Phabs. Remarkably, all HuIgG Fc-binding moPhabs tested (n=48; derived from four different libraries) displayed polyreactivity. Structural analysis of the CDR3 regions revealed characteristic features of polyreactive Igs. Most H chain CDR3 regions harboured tryptophan/tyrosine-rich parts and approximately 60% of the L chain CDR3 regions of both RA patients displayed an identical stretch of amino acids (W/Y-D-S-S). Supportive for a dominant role of VH in specificity, exchange of VL regions with a single VH region yielded moPhabs with similar specificities. All together, the data suggest the presence of an Ag-driven process in the joints of patients with RA, including somatic mutation and clonal selection entailing isotype switching, resulting in the differentiation of B cells into polyreactive RF-secreting plasma cells. [Copyright &y& Elsevier]

Presents a study that compares the requirements by physiological rheumatoid factors (RF) production by peripheral blood B cells to those of total Ig production. Methods and materials; Discussion; Conclusion.

• SP can skew the differentiation of monocyte-derived DC cultured in human serum (HS) towards alternatively activated DC. • This immune regulatory phenotype is less pronounced compared to SP-treated DC in fetal bovine serum containing medium. • These findings highlight the importance of the serum source used in SP treated cell cultures in vitro. Dendritic cells (DCs) are key in shaping immune responses and are recruited to the human cervix after coitus by seminal plasma (SP). SP has been shown to skew the differentiation of monocyte-derived DCs towards an anti-inflammatory profile when cultured in medium containing fetal calf serum (FCS). Here, we confirmed that SP skewed DCs cultured in fetal bovine serum (FBS) towards a tolerogenic profile. To create a setting more similar to the in vivo situations in humans, we tested the immune regulatory effect of SP on DCs in cell cultures containing human serum (HS). SP-DCs cultured in HS did show increased CD14 and decreased CD1a, indicating an inhibited maturation phenotype. Gene expression of TGF-β and IL-10 and IL-10 protein expression were elevated in LPS-activated SP-DCs, whereas IL-12p70 protein levels were decreased compared to LPS-activated control DCs. In contrast to FBS culture conditions, in the presence of HS co-cultures of SP-DCs with allogeneic peripheral blood mononuclear cells (PBMCs) did not result in decreased T cell proliferation and inflammatory cytokine production. Thus, under HS culture conditions SP can skew the differentiation of monocyte-derived DCs phenotypically towards alternatively activated DCs, but this immune regulatory phenotype is functionally less pronounced compared to SP-treated DCs cultured in FBS containing medium. These findings highlight the importance of the source of the serum that is used in SP treated cell cultures in vitro. [ABSTRACT FROM AUTHOR]

Introduction: IL-35 is a novel cytokine of the IL-12 family, existing as a heterodimer of IL-12p35 and EBV-induced gene 3 (Ebi3). IL-35 is produced by natural and inducible regulatory T cells in humans and mice and seems required for optimal suppression. Dexamethasone (Dex) and 1, 25-dihydroxyvitamin D3 (D3) have previously been shown to inhibit IL-12 production in activated dendritic cells (DC) and suppress allogenic T cell responses. Tolerogenic DCs are potentially useful to specifically alleviate unwanted immune responses and induce immune tolerance in transplantation and autoimmunity. In this study, we investigated the regulation of production of various IL-12 family members in human tolerogenic DC (tDC), with a main focus on IL-35. Methods: Monocytes were isolated from human PBMC and cultured for 6days in the presence of IL-4 and GM-CSF to obtain immature DC. Tolerogenic dendritic cells were generated using the same culture conditions but with the addition of Dexamethasone for DexDC, or Dexamethasone with 1, 25-dihydroxyvitamin D3 for D3Dex. RT-PCR quantified IL-12A, IL-12B, IL-27A, IL-23A, EBI3 and IL-10. ELISA was used to measure IL-12p40, IL-12p70, IL-10 and IFN-γ in cellular supernatants. Intracellular expression of IL-12p35, Ebi3 and IL-12p40 was measured by flow cytometry. Expression of IL-12p35 and Ebi3 was evaluated using confocal microscopy and western blot. For T cell suppression assays naive CD4+ T cells were isolated form cord blood or PBMCs and stimulated with anti-CD3/28 in the presence or absence of DC derived supernatants. Proliferation was measured by 3H- thymidine incorporation. Results: We demonstrate by Q-PCR that, although tDC completely lack the expression of IL-12p40, they maintain mRNA expression of Ebi3 and IL-12p35. In line with this, tDC do not produce bioactive IL-12p70 or the homodimer IL-12p40. Using intracellular FACS and western blot we show that tDC maintain the protein expression of both Ebi3 and IL-12-p35. Expression of these proteins can be further enhanced upon stimulation with pro-inflammatory cytokines, TLR agonists and CD40 ligation. Functional inhibition studies show that tDC derived supernatants have the ability to suppress T cell proliferation. At present we are performing blocking experiments in tDC supernatants to fully evaluate the extent at which IL-35 contributes to the tolerogenic characteristic these cells possess in regulating T cell responses. Conclusion: Taken together, our results suggest that aside from increased expression of previously described tolerance inducing markers (B7-H1, ILT3, etc.) and increased IL-10 production, tDC produce IL-35, providing an additional novel mechanism by which these cells elicit their tolerogenic potential. [Copyright &y& Elsevier]