Search

Contains fulltext : 69186.pdf (Publisher’s version ) (Closed access) We describe a novel heterozygous mutation in exon 3 of the HFE-gene that was co-inherited with Cys282Tyr in two unrelated Dutch men both presenting a classical form of hereditary hemochromatosis. Heterozygosity for this mutation was also found in one out of 100 healthy controls of Dutch descent. This c.548T>C mutation converts a leucine to a proline residue at position 183 in the alpha2-helix of the HFE-protein (Leu183Pro). Standard bioinformatics analysis shows that the mutation is likely to disturb the HFE interaction with TfR1. This disrupting role of the mutation in the iron regulatory pathway is further corroborated by the familial co-occurrence of the observed compound heterozygosity with increased serum iron parameters. Haplotype analysis strongly suggests that this novel mutation arose from a common ancestor in the distant past. These findings may have implications for HFE-testing of iron overloaded heterozygous Cys282Tyr-patients of Northern European origin and their relatives.

Contains fulltext : 52624.pdf (Publisher’s version ) (Closed access) BACKGROUND: Because of the adverse events associated with the administration of immunosuppressive drugs, reduction of immunosuppression after solid-organ transplantation is highly desirable provided that graft rejection is prevented. In our transplant center, we used an immunosuppression reduction regimen in stable renal transplant patients. The presence of human leukocyte antigen (HLA) antibodies has been negatively associated with transplant outcome. Therefore, we evaluated the impact of HLA antibodies on the occurrence of acute rejection after immunosuppression reduction. METHODS: The presence and antigen specificity of HLA immunoglobulin G antibodies in serum samples were detected using enzyme-linked immunosorbent assay and single-antigen bead assays. Donor-specific cytotoxic potential was tested by standard CDC cross-match analysis. RESULTS: The presence of donor-specific or total HLA antibodies was not predictive for the occurrence of acute rejection after the reduction of immunosuppression. In addition, the presence of HLA antibodies did not preclude successful reduction of immunosuppression. After reduction of immunosuppression, newly formed HLA antibodies were seldom detected. Interestingly, evaluation of the cytotoxic potential of the detected HLA antibodies revealed that the one patient who developed donor-specific HLA antibodies and experienced a subsequent rejection episode was the only patient who carried cytotoxic HLA antibodies. This finding fueled the notion that the functional capacity, rather than the mere presence of donor-specific HLA antibodies, is indicative for transplant outcome. CONCLUSION: The presence of HLA antibodies does not preclude the successful reduction of immunosuppression in renal transplant patients with stable graft function.

Introduction Schnitzler’s syndrome (SchS) is a disabling autoinflammatory disorder, characterized by a chronic urticarial rash, an M-protein, arthralgia, and other signs of systemic inflammation. Anti-interleukin-1 (IL-1) beta antibodies are highly effective, but the pathophysiology is still largely unknown. Here we studied the effect of in-vivo IL-1 inhibition on serum markers of inflammation and cellular immune responses. Methods Eight patients with SchS received monthly subcutaneous (s.c.) injections with 150 mg canakinumab for six months. Blood was drawn for measurement of serum markers of inflammation (12 times per patient) and for functional and phenotypic analysis of both freshly isolated and toll-like receptor (TLR)-ligand-stimulated peripheral blood mononuclear cells (PBMCs) (five times per patient). All data were compared to results of healthy controls. Results IL-6 levels in serum and in lysates of freshly isolated PBMCs and serum myeloid-related protein (MRP8)/14 and S100A12 levels correlated with disease activity. In vitro, LPS stimulation resulted in higher IL-6 and IL-1 beta production in PBMCs from symptomatic SchS patients compared to healthy controls, whereas patient cells were relatively hyporesponsive to poly:IC and Pam3Cys. The mRNA microarray of PBMCs showed distinct transcriptomes for controls, symptomatic patients and anti-IL-1-treated patients. Numbers of T- and B-cell subsets as well as M-protein concentrations were not affected by IL-1 inhibition. Free light chain levels were elevated in 4 out of 8 patients. Conclusions In conclusion, patient PBMCs are hyperresponsive to LPS, and clinical efficacy of IL-1 beta inhibition in patients with SchS is associated with in-vivo and ex-vivo suppression of inflammation. Interestingly, patient PBMCs showed divergent responses to TLR2/6, TLR3 and TLR4 ligands. Our data underscore that IL-1 beta plays a pivotal role in SchS. Electronic supplementary material The online version of this article (doi:10.1186/s13075-015-0696-0) contains supplementary material, which is available to authorized users.

Item does not contain fulltext BACKGROUND: Treatment with rituximab may be accompanied by a systemic cytokine release. We studied the effects of a single dose of rituximab on cytokine levels in transplant patients and examined the underlying mechanism. METHODS: Twenty renal transplant recipients (10 rituximab-treated, 10 placebo-treated) were recruited from a randomized clinical trial. Rituximab or placebo was infused during surgery, and blood samples were taken before, during, and after surgery and analyzed for interleukin (IL)-2, IL-4, IL-6, IL-10, IL-12, IL-17, interferon-gamma, macrophage inflammatory protein (MIP)-1beta, transforming growth factor-beta, and tumor necrosis factor-alpha. in vitro, healthy donor peripheral blood mononuclear cells, purified B cells, monocytes, natural killer (NK) cells, or combinations thereof were incubated with rituximab, rituximab-F(ab')2, or medium and MIP-1beta, IL-10, interferon-gamma, and tumor necrosis factor-alpha levels were measured in the supernatant. RESULTS: Rituximab-treated patients had higher serum levels of IL-10 (101 +/- 35 pg/mL vs 41 +/- 9 pg/mL; P < 0.01) and MIP-1beta (950 +/- 418 pg/mL vs 125 +/- 32 pg/mL; P < 0.001) compared to placebo-treated patients at 2 hours after start of infusion. There was no difference in the level of other cytokines. In vitro, the addition of rituximab, but not rituximab-F(ab')2 fragments, only led to significantly increased levels of MIP-1beta in co-cultures of B and NK cells. Levels of MIP-1beta were higher in patients with a high affinity Fc-receptor compared to those with a lower affinity FcgammaRIIIa (1356 +/- 184 pg/mL vs 679 +/- 273 pg/mL; P < 0.01). CONCLUSIONS: In addition to B-cell depletion, rituximab can modulate the immune response by inducing cytokine secretion, especially IL-10 and MIP-1beta. Rituximab-induced MIP-1beta secretion depends on the combined presence of B cells and FcR-bearing cells, especially NK cells.

Item does not contain fulltext Diminished suppressive capacity of regulatory T cells (Treg) has been demonstrated in blood and in lesional skin of psoriatic patients. Treatment with anti-TNFalpha restored the number and function of circulating Treg in psoriasis. We aimed to study Treg in the skin of psoriatic patients undergoing topical treatment with calcipotriol-betamethasone dipropionate (CBD) ointment (n = 12) or systemic treatment with anti-TNFalpha agent adalimumab (n = 10). Skin biopsies were collected from patients with chronic plaque psoriasis who responded to the above-mentioned treatments with a SUM-score improvement of at least 50% (at the end of treatment). Biopsies were processed for immunohistochemistry. As Treg function is associated with a numerical balance between Treg and effector T cells, Foxp3/CD4 ratios were calculated. It appeared that both treatments cause a significant decrease in the presence of Foxp3+ cells. However, in patients that were treated with CBD ointment, we observed lower Foxp3/CD4 ratios after 8 weeks of treatment compared to baseline (t = 0: 0.41 +/- 0.08; t = 8: 0.22 +/- 0.04, P = 0.033), whereas in patients who were treated with adalimumab we observed an increase of the Foxp3/CD4 ratios after 1.5 and 16 weeks of treatment compared to baseline (t = 0: 0.25 +/- 0.04; t = 1.5: 0.32 +/- 0.06; t = 16: 0.49 +/- 0.10, P = 0.15). Based on Foxp3/CD4 ratios, we can conclude that adalimumab treated skin differs from CBD treated skin with regard to the anti-inflammatory/inflammatory balance. We suggest that, in contrast to CBD ointment, adalimumab favours local Treg function in the skin.

Background HLA-G is a nonclassical MHC class I molecule and its physiological expression restricted to placental extravillous trophoblasts contributes to maternal tolerance to the semiallogeneic fetus. Aberrant expression of HLA-G in human organ grafts has been proposed to contribute to graft acceptance. Methods We studied HLA-G expression in liver tissue and serum of adult liver transplant recipients before, early, and late after transplantation in relation to liver function and operational tolerance. Results Cirrhotic explant livers showed robust HLA-G expression on hepatocytes, whereas the majority of noncirrhotic livers and graft biopsies taken before or after liver transplantation (LTX) showed no, or weak, HLA-G expression. The HLA-G expression was induced on hepatocytes in vitro by TGF-β, but not by other relevant cytokines. Serum levels of the HLA-G isoforms 1 + 5 gradually declined after LTX. Early after LTX, serum HLA-G levels were higher in patients with acute rejection episodes than nonrejectors. Late after LTX, serum HLA-G levels did not differ between operationally tolerant patients and patients on regular immunosuppressive therapy. Conclusions Our data do not support a graft-protective role for HLA-G after LTX, but show that end-stage liver diseases are associated with HLA-G expression on hepatocytes, which may determine a negative feedback to protect the liver against immunological damage.

Item does not contain fulltext BACKGROUND: Previous reports revealed the potential value of the soluble CD30 level (sCD30) as biomarker for the risk of acute rejection and graft failure after renal transplantation, here we examined its use for the prediction of safe tapering of calcineurin inhibitors as well as late acute rejection. METHODS: In a cohort of renal transplant patients receiving triple immunosuppressive therapy we examined whether sCD30 can be used as a marker for safe (rejection-free) discontinuation of tacrolimus at six months after transplantation (TDS cohort: 24 rejectors and 44 non-rejecting controls). Also, in a second cohort of patients (n=22, rejectors n=11 and non-rejectors n=11), participating in a clinical trial of rituximab as induction therapy after renal transplantation (RITS cohort), we examined whether sCD30 could predict the occurrence of late (>3months post-transplant) acute rejection episodes. sCD30 was measured by ELISA in serum taken before and at several time points after transplantation. RESULTS: Overall, in the TDS cohort sCD30 decreased after transplantation. No difference in sCD30 was observed between rejectors and non-rejecting controls at any of the time points measured. In addition, in the RITS cohort, sCD30 measured at three months after transplantation were not indicative for the occurrence of late acute rejection. CONCLUSION: In two prospectively followed cohorts of renal transplant patients we found no association between sCD30 and the occurrence of either late acute rejection or acute rejection after reduction of immunosuppression.

Regulatory T cells (Treg) are critical controllers of the immune response. Disturbed Treg function results in autoimmunity, whereas in transplantation Treg are crucial in graft survival and transplant tolerance. Hence therapeutic modalities that influence Treg numbers or function hold great clinical opportunity. Ahead of us are clinical trails studying in vivo Treg induction protocols and immunotherapy with ex vivo expanded Treg. Here we discuss the preferential use and/or induction of antigen-specific Treg subsets with high suppressive power and migratory capacity as a potential therapeutic tool to prevent solid organ transplantation rejection. Accordingly, ex vivoselection procedures to induce and isolate highly suppressive antigen-specific Treg (subsets) are needed. This subject, as well as the Treg-facilitating potential of immunosuppressive agents, is discussed.

Contains fulltext : 47575.pdf (Publisher’s version ) (Closed access) Naturally occurring CD4(+)CD25(+) regulatory T cells (Treg) are crucial in immunoregulation and have great therapeutic potential for immunotherapy in the prevention of transplant rejection, allergy, and autoimmune diseases. The efficacy of Treg-based immunotherapy critically depends on the Ag specificity of the regulatory T cells. Moreover, the use of Ag-specific Treg as opposed to polyclonal expanded Treg will reduce the total number of Treg necessary for therapy. Hence, it is crucial to develop ex vivo selection procedures that allow selection and expansion of highly potent, Ag-specific Treg. In this study we describe an ex vivo CFSE cell sorter-based isolation method for human alloantigen-specific Treg. To this end, freshly isolated CD4(+)CD25(+) Treg were labeled with CFSE and stimulated with (target) alloantigen and IL-2 plus IL-15 in short-term cultures. The alloantigen-reactive dividing Treg were characterized by low CFSE content and could be subdivided by virtue of CD27 expression. CD27/CFSE cell sorter-based selection of CD27(+) and CD27(-) cells resulted in two highly suppressive Ag-specific Treg subsets. Each subset suppressed naive and Ag-experienced memory T cells, and importantly, CD27(+) Treg also suppressed ongoing T cell responses. Summarizing, the described procedure enables induction, expansion, and especially selection of highly suppressive, Ag-specific Treg subsets, which are crucial in Ag-specific, Treg-based immunotherapy.

Contains fulltext : 57297.pdf (Publisher’s version ) (Closed access) The expression of HLA-G by invading trophoblasts suggests a role for this molecule in embryo implantation. Putative targets for HLA-G are the uterine natural killer cells (uNK) that are abundantly present at the time of implantation. Since NK cells are potent producers of a variety of cytokines, interaction with HLA-G may result in the production of cytokines involved in trophoblast differentiation or tissue remodelling. In the present study we investigated the effect of membrane-bound HLA-G (mHLA-G) on the uterine mononuclear cell population (UMC) as a whole and on uNK cells in particular by measuring proliferation and cytokine production [interferon-gamma (IFN-gamma)/vascular endothelial growth factor (VEGF)/leukaemia inhibitory factor (LIF)/interleukin-3 (IL-3)]. Uterine cells were isolated from endometrium of non-pregnant women at the time that the endometrium is thought to be receptive to implantation, and then co-cultured with HLA-class I(-)/HLA-class II(+) 721.221 B-LCL cells transfected with mHLA-G. HLA-G suppressed the alloproliferative response of unfractionated UMC to 721.221 cells. Also, IFN-gamma and IL-3 production was strongly reduced. In contrast, purified uNK cells were stimulated by mHLA-G. Proliferation as well as IFN-gamma production was increased after co-culture with mHLA-G transfected 721.221 cells. HLA-G stimulated VEGF production by UMC as well as purified uNK cells. LIF-levels were below the detection level of our enzyme-linked immunosorbent assay. In conclusion, our data show that mHLA-G stimulates proliferation and cytokine production by NK cells, while down-modulating the response of unfractionated UMC.

Contains fulltext : 57804.pdf (Publisher’s version ) (Closed access) We retrospectively analysed the outcome of voluntary unrelated donor (VUD)-SCT in 56 patients after conditioning without or with ATG. All received partially lymphocyte-depleted grafts. Four of 17 patients (24%) who were not given ATG rejected their grafts, as did one of 33 (3%) conditioned with ATG (P=0.02). The incidences of acute graft-versus-host disease grade III/IV were 29 and 6%, respectively (P=0.02), and probabilities of 1-year transplant-related mortality were 64% (95% CI, 44-84%) and 27% (95% CI, 12-42%), respectively (P=0.004). Projected at 3 years, probability of survival was 18% (95% CI, 2-34%) after conditioning without ATG and 60% (95% CI, 43-70%) after conditioning with ATG (P=0.002). Probabilities of disease-free survival (DFS) were 18% (95% CI, 2-34%) and 45% (95% CI, 27-63%), respectively (P=0.005). Patients who did not receive ATG had a probability of current DFS of 18% (95% CI, 3-34%) and this was 60% (95% CI, 43-77%) for the patients conditioned with ATG (P

An important prerequisite in using regulatory T cells for immunotherapy is their ex vivo expansion without loss of suppressor function. Human anergic regulatory T cells are expandable by Ag-specific stimulation in the presence of IL-2. IL-15, like IL-2, is a T cell growth factor that, in contrast to IL-2, stimulates survival of T cells. In this study, we examined whether IL-15 could be exploited as a superior growth factor of human CD4+ anergic regulatory T cells that were generated by costimulation blockade. Next, IL-15, as compared with IL-2, was investigated with respect to expansion and function of these regulatory T cells. Optimal expansion required cognate allogeneic stimulation in the presence of exogenous IL-15. IL-15 resulted in enhanced survival that was paralleled by an increased number of Bcl-2-expressing cells. Moreover, IL-15 induced a distinct type of anergy characterized by hyperreactivity to IL-15, resulting in improved expansion. This is likely attributed to increased propensity of these cells to up-regulate both α- and γ-chains of the IL-2 and IL-15 receptor. Notably, IL-15-expanded regulatory CD4+ T cells suppressed both naive and memory T cells in a superior way. Immunosuppression required alloantigen-specific stimulation and appeared gamma-irradiation resistant and independent of IL-10, TGFβ, or CTLA-4 interactions. These regulatory T cells were stable suppressors, mediating bystander suppression upon TCR stimulation, but leaving recall responses unaffected in the absence of cognate Ag. Finally, human naturally occurring regulatory CD4+CD25+ T cells appeared important in generating regulatory T cells by costimulation blockade. In conclusion, IL-15-expanded, de novo-induced human anergic regulatory CD4+ T cells are of interest in Ag-specific immunotherapy.

Biochemical and serological methods were used to characterize sheep MHC class I polymorphism at the product level. The cells of 65 selected animals were subjected to immunoprecipitation and one-dimensional isoelectric focusing (IEF) with two cross-reacting monoclonal antibodies, B1.1G6 and HC-10. We were able to define up to 18 distinct haplotypes in the Latxa breed sample. Most of the locally defined serological specificities were confirmed or subdivided by biochemical typing. As IEF detected antigens not yet defined by serology, it provided us with a guide to producing more specific antisera by appropriate immunizations. Lastly, evidence of expression of two class I loci products was found.

We investigated whether HLA-DR2 or -DR12 alleles in 63 Javanese patients with complicated or non-complicated typhoid fever were associated with severity of disease. No association was observed between HLA type and susceptibility to disease. However, in patients we did find a negative association of DR12 (DRB1*12021) with complicated typhoid fever (P = 0.05; OR = 0.3; 95% CI: 0.1-1.0). No effect of DR2 (DRB1*1502) on outcome (P = 0.46; OR = 1.5; 95% CI: 0.5-4.5) was demonstrated. The odds ratio for DR12 remained unchanged after adjusting for DR2. Tumour necrosis factor alpha (TNF-alpha) production capacity in lipopolysaccharide (LPS)-stimulated whole blood culture, as measured by non-equilibrium radioimmunoassay, was significantly lower in complicated than in non-complicated cases (P = 0.02), confirming previous data. No significant correlation of either DR12 (P = 0.47) or DR2 (P = 0.89) was found with TNF-alpha production capacity. Apparently, protection against complications by DR12 is attributable to other mechanisms.

Item does not contain fulltext BACKGROUND: The interaction between decidual natural killer (NK) cells and alloantigens expressed on fetal trophoblast cells are thought to be essential for successful implantation and placentation. Consequently, a disturbed interaction during the first trimester of pregnancy might well lead to a subsequent pregnancy failure. METHODS: We investigated the expression of HLA-G and NK cell markers in tissue sections from recurrent miscarriage (n = 9) and ectopic tubal pregnancies (n = 5), and two hysterectomy specimens of healthy pregnancy as well as decidual biopsies (n = 9) were used as controls. RESULTS: We show in normal pregnancy not only a decrease, but also a morphological change in CD56+ NK cells upon interaction with HLA-G-expressing trophoblasts. The cells appear to be transitioning from a blast-like (activation) state into a state of apoptosis. The number of CD16+ NK cells was low. In contrast, in recurrent miscarriage tissue a sustained NK cell marker expression of both CD56 and CD16 was paralleled by a decreased expression of HLA-G. No morphological changes from the blast-like stage were apparent. Finally, in ectopic pregnancies HLA-G expression in the absence of decidual NK cells was associated with a disturbed trophoblast differentiation. CONCLUSIONS: In pathological pregnancies we show an in-situ altered phenotype of trophoblast and NK cells.

Aim Individual HLA mismatches may have differential effects on graft survival after kidney transplantation. Therefore, there is a need for a reliable tool to define permissible HLA mismatches in kidney transplantation. We previously demonstrated that donor-derived Predicted Indirectly ReCognizable HLA epitopes presented by recipient HLA class-II (PIRCHE-II) play a role in de novo donor-specific HLA antibody formation after kidney transplantation. Moreover, we also showed that PIRCHE-II is involved in HLA antibody formation after pregnancy. In the present Dutch multi-center study we evaluated the possible association between PIRCHE-II and kidney graft failure in 2918 donor-recipient couples that were transplanted between 1995 and 2005. Methods For all donors-recipients couples, PIRCHE-II numbers were determined and related to graft survival in both univariate and multivariable analyses. Results Adjusted for confounders, the natural logarithm of PIRCHE-II was associated with a higher risk for graft failure (HR:1.13, 95% CI:1.04–1.23, p = 0.003). Univariately analyzed, patients with low PIRCHE-II numbers had a better 10-years graft survival than patients with higher PIRCHE-II numbers (p = 0.006; PIRCHE-II strata: Conclusions Our data suggest that the PIRCHE-II algorithm is a valuable tool to discriminate between permissible HLA mismatches and high-risk HLA mismatches in kidney transplantation. Inclusion of PIRCHE-II in donor-selection criteria may eventually lead to an improved kidney graft survival.

BACKGROUND Cytomegalovirus(CMV) infections have a significant effect on morbidity and mortality in kidney transplants. We conducted a study to ascertain the association of natural killer cell killer immunoglobulin-like receptors and human leukocyte antigen (HLA) genotype with risk of CMV disease. METHODS The 90 CMV-negative patients receiving a first renal transplantation from a CMV-positive donor in this study received triple immunosuppressive therapy and prophylactic CMV treatment for up to 3 months after transplantation. RESULTS We observed a 43.3% incidence rate of CMV disease within the first year after transplantation. Twenty-seven recipients experienced a rejection episode, 14 of which had CMV disease, mostly after rejection, suggesting that in this group, CMV disease is not a risk factor for rejection. KIR gene or genotype distribution were similar between the CMV diseased and CMV disease-free group. Twenty-seven recipients (30%) carried KIR-AA genotype, of which nine (33%) had CMV disease. Of the remaining 63 (70%) recipients with KIR-BX genotype, 30 (48%) had CMV disease. There was no significant difference between the two genotype groups with regard to occurrence of CMV disease, although there was a trend toward a lower incidence of CMV disease in recipients carrying the KIR-AA genotype. For CMV disease, we found no significant risk associated with the number of activating or inhibitory KIRs. Neither was missing KIR ligands for the inhibitory KIRs (HLA-C1/C2/Bw4) in recipients associated with lower rates of CMV disease. CONCLUSION In CMV-negative recipients, genotypic analysis of KIR repertoire and HLA ligands does not provide risk factors for primary CMV disease after renal transplantation.

In the consortium "ALLOVIR" we aim to characterize the effect of CMV and BKV infections on the innate immune responses to viral and alloantigens. Furthermore, we want to characterize the interplay between adaptive immune responses to viral and alloantigens with emphasis on the role of heterologous immunity. Thirdly, we will characterize the manifestations of these immune responses in the allograft, as reflected in tissue and urine, and their correlation with graft function. Finally, we will assess how immunosuppressive drugs interfere with these cross-reactive immune responses.

Clinical translation of tolerance-inducing cell therapies requires a novel approach focused on innovative networks, patient involvement, and, foremost, a fundamental paradigm shift in thinking from both Academia, and Industry and Regulatory Agencies. Tolerance-inducing cell products differ essentially from conventional drugs. They are personalized and target interactive immunological networks to shift the balance toward tolerance. The human cell products are often absent or fundamentally different in animals. This creates important limitations of pre-clinical animal testing for safety and efficacy of these products and calls for novel translational approaches, which require the combined efforts of the different parties involved. Dedicated international and multidisciplinary consortia that focus on clinical translation are of utmost importance. They can help in informing and educating regulatory policy makers on the unique requirements for these cell products, ranging from pre-clinical studies in animals to in vitro human studies. In addition, they can promote reliable immunomonitoring tools. The development of tolerance-inducing cell products requires not only bench-to-bedside but also reverse translation, from bedside back to the bench.

Item does not contain fulltext Traditionally, antirejection therapy in organ transplantation has mainly been directed at T cells. During recent years, the role of B cells in acute rejection has attracted more attention. In the Radboud University Medical Center (Nijmegen, The Netherlands) we performed a randomized, placebo controlled study to assess the efficacy and safety of rituximab as induction therapy after renal transplantation. In parallel we investigated the effects of rituximab on the numbers and function of B and T cells. An overview of the results, which have largely been published in peer reviewed papers, is presented below.

Item does not contain fulltext In allogeneic stem cell transplantation (SCT), natural killer (NK) cells lacking their cognate inhibitory ligand can induce graft-versus-leukemia responses, without the induction of severe graft-versus-host disease (GVHD). This feature can be exploited for cellular immunotherapy. In this study, we examined selective expansion of NK cell subsets expressing distinct killer immunoglobulin-like receptors (KIRs) within the whole human peripheral blood NK cell population, in the presence of HLA-Cw3 (C1) or Cw4 (C2) transfected K562 stimulator cells. Coculture of KIR(+) NK cells with C1 or C2 positive K562 cells, in the presence of IL-2+IL-15, triggered the outgrowth of NK cells that missed their cognate ligand. This resulted in an increased frequency of alloreactive KIR(+) NK cells within the whole NK cell population. Also, after preculture with K562 cells lacking their cognate ligand, we observed that this alloreactive NK population revealed higher numbers of CD107(+) cells when cocultured with the relevant K562 HLA-C transfected target cells, as compared to coculture with untransfected K562 cells. This enhanced reactivity was confirmed using primary leukemic cells as target. This study demonstrates that HLA class I expression can mediate the skewing of the NK cell repertoire and enrich the population for cells with enhanced alloreactivity towards leukemic target cells. This feature may support future clinical applications of NK cell-based immunotherapy.

The generation of immunoregulatory T cells that block the B7(CD86/CD80)-CD28 and/or CD40-CD154 costimulatory pathways has great potential for the induction of long-term transplantation tolerance. In a human polyclonal in vitro model, combined monoclonal antibody (mAb) blocking of the costimulatory ligands CD40 and CD86 lead to allospecific T-cell anergy that cannot be reversed by antigenic rechallenge in the presence of IL-2. Although antigenic restimulation with IL-2 restored the proliferative response, subsequent antigenic restimulation of the restored anergic cells in a tertiary mixed lymphocyte culture still resulted in nonresponsiveness. Importantly, these anergic T cells suppress the response of naive alloreactive T cells in an antigen-specific way via linked recognition. Suppression may partially depend on local IL-10 production, while transforming growth factor–β (TGF-β) did not play a role. Irrespective of the monoclonal antibody combination used, blast formation occurred in a subset of CD4+ cells. These cells were characterized by a sustained CD45RA expression, an increased T-cell receptor density, and a lower level of CD4 expression. A reduced number of CD45RO+/CD8+ T cells was observed whenever anti-CD86 was combined with anti-CD40, which was reflected by an even more attenuated cytotoxic T-cell function. This indicates the importance of CD40-CD154 in the generation of cytotoxic T cells in this transplantation model. We hypothesize that in our model, anergy is induced in the CD4+ T-cell subset, whereby CD8+ cytotoxic effector function is impaired by the lack of both CD40-CD154 signaling and cytokine-mediated help. This costimulatory ligand–directed mAb approach might well be used for the ex vivo generation of antigen-specific immunoregulatory T cells applicable in adoptive immunotherapy.

Selection procedures in bone marrow transplantation (BMT) would benefit from the development of easy-to-perform cellular assays with high discriminative power. We tested a cytokine-based mixed lymphocyte culture (MLC) and compared its outcome to the routinely used MLC, helper T-lymphocyte precursor (HTLp)-f and cytotoxic T-lymphocyte precursor (CTLp)-f assays. Interferon (IFN)gamma was selected as a marker cytokine for (deleterious) T-helper 1 like responses and 36 (potential) BMT donor-recipient pairs were analysed. The IFNgamma-MLC appeared sensitive to HLA class II (subtype) differences, but not to isolated class I differences, or to mismatches other than HLA (identical siblings). The test enabled a distinction between combinations with positive MLC (proliferation) and HTLp-f, exemplified by the fact that although high IFNgamma levels were observed in the class II mismatched group, certain DRB3, DQB1-subtype and DRB1-subtype mismatches did not give rise to IFNgamma production. This might be of relevance for the detection of so-called permissible mismatches. With regard to prediction of acute graft-versus-host disease (aGVHD) in unrelated BMT, the data indicated that high levels of IFNgamma coincided with severe aGVHD, whereas low levels were largely associated with grades 0-I. However, in the case of isolated class I mismatches the test had no predictive value. The cell-saving IFNgamma-MLC provides an alternative for the assays currently in use, but should be employed along with an assay that is sensitive to class I differences to correct for false negatives. Consequently, a combination of IFNgamma-MLC and CTLp-f assays seems most promising for donor selection, other than identical siblings.

The Lewis rat, an inbred rat strain susceptible to several well-characterized experimental autoimmune diseases, provides a good model to study peptide-mediated immunotherapy. Peptide immunotherapy focussing on the modulation of T cell responses by interfering with TCR-peptide-MHC complex formation requires the elucidation of the molecular basis of TCR-peptide-MHC interactions for an efficient design of modulatory peptides. In the Lewis rat most autoimmune-associated CD4+ T cell responses are MHC class II RT1.BL restricted. In this study, the characteristics of RT1.BL-peptide interactions were explored. A series of substitution analogs of two Lewis rat T cell epitopes was examined in a direct peptide-MHC binding assay on isolated RT1.BL molecules. Furthermore, other autoimmune-related as well as non-disease-related T cell epitopes were tested in the binding assay. This has led to the definition of an extended RT1.BL-peptide binding motif. The RT1.BL-peptide binding motif established in this study is the first described rat MHC-peptide binding motif based on direct MHC-peptide binding experiments. To predict good or intermediate RT1.BL binding peptides, T cell epitope search profiles were deduced from this motif. The motif and search profiles will greatly facilitate the prediction of modulatory peptides based on autoimmune-associated T cell epitopes and the identification of target structures in experimental autoimmune diseases in Lewis rats.

Contains fulltext : 137431.pdf (Publisher’s version ) (Closed access) STUDY QUESTION: Is menstrual blood a suitable source of endometrial derived lymphocytes? SUMMARY ANSWER: Mononuclear cells isolated from menstrual samples (menstrual blood mononuclear cells (MMC)) are clearly distinct from peripheral blood mononuclear cells (PBMC) and show a strong resemblance with biopsy-derived endometrial mononuclear cells. WHAT IS KNOWN ALREADY: A critical event in the onset of pregnancy is the implantation of the embryo in the uterine wall. The immune cell composition in the endometrium at the time of implantation is considered pivotal for success. Despite advancing knowledge on the composition of the immune cell population in the uterus, the role of endometrial immune cells in reproductive disorders is still not fully resolved, mainly due to the fact that this type of research requires invasive techniques. Here, we collected menstrual fluid and validated this unique non-invasive technique to obtain and study the endometrium-derived immune cells which would be present around the time of implantation. STUDY DESIGN, SIZE, DURATION: Five healthy non-pregnant females with regular menstruation cycles and not using oral contraceptives collected their menstrual blood using a menstrual cup in five consecutive cycles. Sampling took place over the first 3 days of menses, with 12 h intervals. Peripheral blood samples, taken before and after each menstruation, were obtained for comparative analysis. PARTICIPANTS/MATERIALS, SETTING, METHODS: MMC and PBMC samples were characterized for the different lymphocyte subsets by flow cytometry, with emphasis on NK cells and T cells. Next, the functional capacity of the MMC-derived NK cells was determined by measuring intracellular production of IFN-gamma, granzyme B and perforin after culture in the presence of IL-2 and IL-15. MAIN RESULTS AND THE ROLE OF CHANCE: In support of their endometrial origin, MMC samples contained the typical composition of mononuclear cells expected of endometrial tissue, were phenotypically similar to the reported phenotype for biopsy-derived endometrial cells, and were distinct from PBMC. Increased percentages of NK cells and decreased percentages of T cells were found in MMC when compared with PBMC from the same female. The MMC-derived NK cells were pre-dominantly CD56(bright)/CD16(-), in contrast to the primarily CD56(dim)/CD16(+) peripheral blood NK cells. MMC-derived NK cells expressed CD103, indicating their mucosal origin. In addition, the pattern of natural cytotoxicity receptor (NCR) expression in MMC-derived NK cells was comparable with that in endometrial biopsy-derived NK cells. Compared with PBMC, the NKp30 expression was decreased, while the percentage of NKp44 positive cells was increased in MMC samples. CXCR3 and CXCR4 were hardly expressed by MMC-derived NK cells, indicating that these cells are not of PBMC origin. NK cells from MMC samples were functional as shown by their capacity to produce IFN-gamma, granzyme B and perforin, upon stimulation with IL-2 and IL-15. MMC-derived T cells revealed an increased expression of CD103, CD69 and CXCR4 compared with PBMC-derived T cells. Importantly, MMC collection using a menstrual cup proved highly reliable and reproducible between women and between cycles. LIMITATIONS, REASONS FOR CAUTION: Based on the parameters we studied, MMC appear similar to biopsy-derived endometrial mononuclear cells. However, sampling is not done at the exact same time in the menstrual cycle, and thus we cannot exclude some, as yet undetected, differences. Also, it should be considered that for some women, the use of the menstrual cup may be unpleasant. WIDER IMPLICATIONS OF THE FINDINGS: Menstrual blood may be a source of endometrial cells and may create new opportunities to study uterine immunological cells in fertility issues. STUDY FUNDING/COMPETING INTEREST(S): No external funding was obtained for the present study. None of the authors have any conflict of interest to declare. TRIAL REGISTRATION NUMBER: NA.

Item does not contain fulltext Clinical trials successfully using antibodies targeting IL-17 in psoriasis support the importance of IL-17 in the pathophysiology of this disease. However, there is a debate concerning the source and dynamics of IL-17 production in inflamed skin. Here we characterized IL-17-producing immune cells over time, using two established in vivo models of human skin inflammation that share many histological features with psoriasis, i.e., leukotriene B4 application and tape-stripping. Both treatments revealed a clear influx of neutrophils and T cells. Staining for IL-17 revealed that the majority of IL-17 was expressed by neutrophils and mast cells, in both models. Neutrophils, but not mast cells, coexpressed the IL-17-associated transcription factor RORgammat and were able to form extracellular traps. While the presence of mast cells remained steady during the skin inflammatory process, the presence of neutrophils was clearly dynamic in time. Therefore, it is attractive to hypothesize that IL-17+/RORgammat+ neutrophils contribute to human skin inflammation in vivo and possibly to the pathogenesis of skin diseases such as psoriasis. Surprisingly, T cells represented a minority of the IL-17-expressing cell population. These observations challenge the classical opinion that IL-17 is predominantly associated with T cells in skin inflammation.

Contains fulltext : 118163.pdf (Publisher’s version ) (Open Access) Topical application of coal tar is one of the oldest therapies for atopic dermatitis (AD), a T helper 2 (Th2) lymphocyte-mediated skin disease associated with loss-of-function mutations in the skin barrier gene, filaggrin (FLG). Despite its longstanding clinical use and efficacy, the molecular mechanism of coal tar therapy is unknown. Using organotypic skin models with primary keratinocytes from AD patients and controls, we found that coal tar activated the aryl hydrocarbon receptor (AHR), resulting in induction of epidermal differentiation. AHR knockdown by siRNA completely abrogated this effect. Coal tar restored filaggrin expression in FLG-haploinsufficient keratinocytes to wild-type levels, and counteracted Th2 cytokine-mediated downregulation of skin barrier proteins. In AD patients, coal tar completely restored expression of major skin barrier proteins, including filaggrin. Using organotypic skin models stimulated with Th2 cytokines IL-4 and IL-13, we found coal tar to diminish spongiosis, apoptosis, and CCL26 expression, all AD hallmarks. Coal tar interfered with Th2 cytokine signaling via dephosphorylation of STAT6, most likely due to AHR-regulated activation of the NRF2 antioxidative stress pathway. The therapeutic effect of AHR activation herein described opens a new avenue to reconsider AHR as a pharmacological target and could lead to the development of mechanism-based drugs for AD.

Item does not contain fulltext Microparticles (MPs) are small plasma membrane-derived vesicles that can expose molecules originating from their parental cells. As vectors of biological information they are likely to play an active role in both homeostasis and pathogenesis, making them promising biomarkers and nanomedicine tools. Therefore, there is an urgent need for standardization of MP isolation and analysis protocols to propel our understanding of MP biology to the next level. Based on current methodology and recent insights, this review proposes an optimized protocol for the isolation and biochemical characterization of MPs.

Item does not contain fulltext BACKGROUND: Both keratinocytes and T-cells are crucial players in cutaneous immune responses. We hypothesized that direct interactions between keratinocytes and T-cell subsets could shape the nature or strength of the local immune response. OBJECTIVE: We investigated direct interactions between keratinocytes and T-cell subsets, focused on keratinocyte chemokine production and T-cell phenotype and cytokine production. METHODS: A newly developed in vitro serum free co-culture model using primary keratinocytes and T-cells subsets from healthy human donors was used. Keratinocyte chemokine production was analyzed with luminex, T-cell phenotype and cytokine production were analyzed with flow cytometry. RESULTS: Our data show that upon co-culture with CD4(pos) or CD8(pos) T-cells primary human keratinocytes increased production of functionally active chemokines CCL2, CCL20 and CXCL10 and that regulatory T-cells did not regulate keratinocyte chemokine production. Next to that, we found that keratinocytes skewed CD4(pos) and CD8(pos) T-cell populations toward an IL-17(pos) CCR6(pos) RORgammat(pos) phenotype in a cell-cell contact independent manner, and that Treg were able to decrease the absolute number of IL-17 producing T-cells in keratinocyte/T-cell co-cultures. Correspondingly, freshly isolated skin-derived T-cell populations contained relatively high percentages of IL-17(pos) cells. CONCLUSION: We provide evidence that keratinocyte/T-cell communication may regulate leukocyte influx in the skin, and that keratinocytes enrich T-cell populations for Th17/Tc17 cells. Accumulation of Th17/Tc17 cells, but not chemokine production, appears under the control of regulatory T-cells. Dysregulation of these processes may well contribute to the pathophysiology of inflammatory skin diseases.

Contains fulltext : 118695.pdf (Publisher’s version ) (Open Access) Hepcidin regulation by competing stimuli such as infection and iron deficiency has not been studied in infants and it's yet unknown whether hepcidin regulatory pathways are fully functional in infants. In this cross-sectional study including 339 Kenyan infants aged 6.0+/-1.1 months (mean+/-SD), we assessed serum hepcidin-25, biomarkers of iron status and inflammation, and fecal calprotectin. Prevalence of inflammation, anemia, and iron deficiency was 31%, 71%, 26%, respectively. Geometric mean (+/-SD) serum hepcidin was 6.0 (+/-3.4) ng/mL, and was significantly lower in males than females. Inflammation (C-reactive protein and interleukin-6) and iron status (serum ferritin, zinc protoporphyrin and soluble transferrin receptor) were significant predictors of serum hepcidin, explaining nearly 60% of its variance. There were small, but significant differences in serum hepcidin comparing iron deficient anemic (IDA) infants without inflammation to iron-deficient anemic infants with inflammation (1.2 (+/-4.9) vs. 3.4 (+/-4.9) ng/mL; P

Item does not contain fulltext Vitamin D(3) is known to have an effect on the immune function. We investigated the immunomodulatory capability of vitamin D(3) in HIV-infected patients and studied the expression of chemokine receptors on regulatory T cells (Treg). Vitamin D(3)-deficient HIV-1-seropositive subjects were treated with cholecalciferol (vitamin D(3)) at a dose of 800 IU daily for 3 months (n=9) or 25,000 IU weekly for 2 months (n=7). Peripheral blood mononuclear cells (PBMCs) were isolated and analyzed for skin-homing (CCR4 and CCR10) and gut-homing (CCR9 and integrin alpha(4)beta(7)) marker expression on Treg, by flow cytometry, before and after supplementation. Serum 25(OH)D(3) and parathyroid hormone (PTH) levels were determined at baseline and after the treatment period. Weekly doses of 25,000 IU cholecalciferol effectively achieved the optimal target serum 25(OH)D(3) concentration of >75 nmol/liter (30 ng/ml) in HIV-infected patients. High-dose cholecalciferol supplementation differentially influenced skin-homing markers on Treg with an increased level of CCR10 expression and while a reduction in CCR4 expression level was observed together with a lower percentage of Treg expressing CCR4. For both dosing regimens, there were no significant differences in the expression of gut-homing markers, CCR9, and integrin alpha(4)beta(7). High-dose vitamin D(3) supplementation is needed to reverse vitamin D(3) deficiency in HIV-infected individuals and this results in modulation of skin-homing markers but not gut-homing markers expression on Treg. At a standard dose of 800 IU/day, vitamin D(3) is not effective in achieving an optimal 25(OH)D(3) concentration in patients with an underlying T cell dysfunction and is unable to exert any immunomodulatory effects.

Contains fulltext : 109407.pdf (Publisher’s version ) (Closed access) The use of tissue-engineered human skin equivalents (HSE) for fundamental research and industrial application requires the expansion of keratinocytes from a limited number of skin biopsies donated by adult healthy volunteers or patients. A pharmacological inhibitor of Rho-associated protein kinases, Y-27632, was recently reported to immortalize neonatal human foreskin keratinocytes. Here, we investigated the potential use of Y-27632 to expand human adult keratinocytes and evaluated its effects on HSE development and in vitro gene delivery assays. Y-27632 was found to significantly increase the life span of human adult keratinocytes (up to five to eight passages). The epidermal morphology of HSEs generated from high-passage, Y-27632-treated keratinocytes resembled the native epidermis and was improved by supplementing Y-27632 during the submerged phase of HSE development. In addition, Y-27632-treated keratinocytes responded normally to inflammatory stimuli, and could be used to generate HSEs with a psoriatic phenotype, upon stimulation with relevant cytokines. Furthermore, Y-27632 significantly enhanced both lentiviral transduction efficiency of primary adult keratinocytes and epidermal morphology of HSEs generated thereof. Our study indicates that Y-27632 is a potentially powerful tool that is used for a variety of applications of adult human keratinocytes. 01 september 2012

Contains fulltext : 107961.pdf (Publisher’s version ) (Open Access) Inflammation enhances the secretion of sphingomyelinases (SMases). SMases catalyze the hydrolysis of sphingomyelin into phosphocholine and ceramide. In erythrocytes, ceramide formation leads to exposure of the removal signal phosphatidylserine (PS), creating a potential link between SMase activity and anemia of inflammation. Therefore, we studied the effects of SMase on various pathophysiologically relevant parameters of erythrocyte homeostasis. Time-lapse confocal microscopy revealed a SMase-induced transition from the discoid to a spherical shape, followed by PS exposure, and finally loss of cytoplasmic content. Also, SMase treatment resulted in ceramide-associated alterations in membrane-cytoskeleton interactions and membrane organization, including microdomain formation. Furthermore, we observed increases in membrane fragility, vesiculation and invagination, and large protein clusters. These changes were associated with enhanced erythrocyte retention in a spleen-mimicking model. Erythrocyte storage under blood bank conditions and during physiological aging increased the sensitivity to SMase. A low SMase activity already induced morphological and structural changes, demonstrating the potential of SMase to disturb erythrocyte homeostasis. Our analyses provide a comprehensive picture in which ceramide-induced changes in membrane microdomain organization disrupt the membrane-cytoskeleton interaction and membrane integrity, leading to vesiculation, reduced deformability, and finally loss of erythrocyte content. Understanding these processes is highly relevant for understanding anemia during chronic inflammation, especially in critically ill patients receiving blood transfusions.

Purpose Keyhole limpet hemocyanin (KLH) attracts biomedical interest because of its remarkable immunostimulatory properties. Currently, KLH is used as vaccine adjuvant, carrier protein for haptens and as local treatment for bladder cancer. Since a quantitative human anti-KLH assay is lacking, it has not been possible to monitor the dynamics of KLH-specific antibody (Ab) responses after in vivo KLH exposure. We designed a quantitative assay to measure KLH-specific Abs in humans and retrospectively studied the relation between vaccination parameters and the vaccine-induced anti-KLH Ab responses. Experimental design Anti-KLH Abs were purified from pooled serum of melanoma patients who have responded to KLH as a vaccine adjuvant. Standard isotype-specific calibration curves were generated to measure KLH-specific Ab responses in individual serum samples using ELISA. Results KLH-specific IgM, IgA, IgG and all IgG-subclasses were accurately measured at concentrations as low as 20 μg/ml. The intra- and inter-assay coefficients of variation of this ELISA were below 6.7 and 9.9 %, respectively. Analyses of 128 patients demonstrated that mature DC induced higher levels of KLH-specific IgG compared to immature DC, prior infusion with anti-CD25 abolished IgG and IgM production and patients with locoregional disease developed more robust IgG responses than advanced metastatic melanoma patients. Conclusions We present the first quantitative assay to measure KLH-specific Abs in human serum, which now enables monitoring both the dynamics and absolute concentrations of humoral immune responses in individuals exposed to KLH. This assay may provide a valuable biomarker for the immunogenicity and clinical effectiveness of KLH-containing vaccines and therapies. Electronic supplementary material The online version of this article (doi:10.1007/s00262-012-1263-z) contains supplementary material, which is available to authorized users.

Item does not contain fulltext Vitamin D(3) affects both the innate as well as adaptive immune responses. Epidemiological studies have established that vitamin D(3) deficiency plays an important role in tuberculosis (TB) and viral influenza prevalence as well as susceptibility to active disease in TB. Vitamin D(3) status has been associated with the clinical course of HIV infection and drug interaction with anti-retroviral therapy. This article reviews the immunomodulatory capacity of vitamin D(3) and examines the impact of vitamin D(3) supplementation as a preventive or therapeutic intervention with the intent to uncover its potential therapeutic application in infectious diseases and to identify novel areas for future research. We present a review of randomized, controlled clinical studies conducted in humans which included assessment of the immune function or clinical outcome as study end points. Current data support vitamin D(3) supplementation as risk-modifying intervention in tuberculosis and viral respiratory tract infection, but the optimal dosage regimen remains to be determined. However, to date the knowledge on its role in fungal infection and sepsis is limited although a potential benefit could be harnessed from its ability to curtail the unrestrained pro-inflammatory response and therefore prevent excessive collateral tissue damage. 01 mei 2012

Contains fulltext : 109459.pdf (Publisher’s version ) (Open Access) It is well-recognized that vitamin D(3) has immune-modulatory properties and that the variation in ultraviolet (UV) exposure affects vitamin D(3) status. Here, we investigated if and to what extent seasonality of vitamin D(3) levels are associated with changes in T cell numbers and phenotypes. Every three months during the course of the entire year, human PBMC and whole blood from 15 healthy subjects were sampled and analyzed using flow cytometry. We observed that elevated serum 25(OH)D(3) and 1,25(OH)(2)D(3) levels in summer were associated with a higher number of peripheral CD4+ and CD8+ T cells. In addition, an increase in naive CD4+CD45RA+ T cells with a reciprocal drop in memory CD4+CD45RO+ T cells was observed. The increase in CD4+CD45RA+ T cell count was a result of heightened proliferative capacity rather than recent thymic emigration of T cells. The percentage of Treg dropped in summer, but not the absolute Treg numbers. Notably, in the Treg population, the levels of forkhead box protein 3 (Foxp3) expression were increased in summer. Skin, gut and lymphoid tissue homing potential was increased during summer as well, exemplified by increased CCR4, CCR6, CLA, CCR9 and CCR7 levels. Also, in summer, CD4+ and CD8+ T cells revealed a reduced capacity to produce pro-inflammatory cytokines. In conclusion, seasonal variation in vitamin D(3) status in vivo throughout the year is associated with changes in the human peripheral T cell compartment and may as such explain some of the seasonal variation in immune status which has been observed previously. Given that the current observations are limited to healthy adult males, larger population-based studies would be useful to validate these findings.

Previous studies on expressed bovine MHC class II polymorphism using one-dimensional isoelectric focusing (1D-IEF) allowed the identification of at least 12 allelic variants of the DRB3 gene. So far, only limited data have been available on the expression of other class II genes. The present study involved biochemical analysis of bovine MHC class II molecules using a set of monoclonal antibodies presupposed to be bovine DR and DQ reactive. After essential modification of the standard electrophoresis conditions used for 1D-IEF typing of bovine DR products, biochemical polymorphism was observed for non-DR molecules, revealing polymorphic sets of basic and acidic focusing bands. Because of the extensive DNA polymorphism described for bovine DQA and DQB genes, and the apparent similarity with the focusing pattern of human DQ products, these molecules were considered to be the bovine DQ homologues. The definition of the DQ-associated banding patterns was made possible by using two half-sib sire families. Four different DQA-like patterns and nine DQB-like patterns were detected. Segregation of the DQ types was supported by serological class I and class II typing. These results show that it is now possible to discriminate between expressed bovine DR and DQ polymorphism.

SUMMARY Polymorphism of the bovine DRB, DQA, DQB, DYA, DOB and DIB genes was investigated using restriction fragment length polymorphism (RFLP) analysis, isoelectric focusing (IEF), class II serology and polymerase chain reaction (PCR) based typing techniques. The simultaneous application of multiple typing techniques and the characterization of multiple genes resulted in a greatly enhanced picture of the bovine class II regions. Thirty-eight class IIa (DR-DQ) and 5 class lib (DYA-DOB-DIB) haplotypes were defined. It was found that IEF types were associated with DRB3 polymorphism defined by DRB3 PCR-RFLP and DRB3 microsatellite PCR. Serologically defined polymorphism was associated with distinct molecular/IEF motifs and, therefore, DR and DQ specificities could be tentatively distinguished. Although the DR and DQ genes are tightly linked, neither DR nor DQ typing defined all of the class IIa region polymorphism. Furthermore, even the most powerful DRB3 typing technique, DRB3 PCR-RFLP, failed to detect all expressed DRB3 polymorphism. All detected DRB3 polymorphism could, however, be distinguished with a combination of two molecular techniques: DRB3 PCR-RFLP and DRB3 microsatellite PCR. RFLP typing with transmembrane probes detected significantly less polymorphism than typing with cDNA or exon probes. However, the transmembrane probes were useful because they were locus specific. The presence of only 5 of 12 possible class lib haplotypes was unexpected and indicates that the DYA, DOB and DIB genes are tightly linked.

Previous studies have shown that major histocompatibility complex (MHC) blockade by competitor peptides with high MHC class II binding affinity can prevent peptide-induced experimental autoimmune encephalomyelitis (EAE). However, none of these studies addressed the question whether this approach could also be used to prevent EAE induced with a multivalent antigen. In this report we show the effect of competitor peptides co-immunized during EAE induction with entire guinea pig myelin basic protein (MBP) in Lewis rats. As MHC class II binding competitor peptides we used one nonimmunogenic disease-nonrelated peptide, and two immunogenic peptides, one EAE-related and one non-EAE-related. The respective efficacy of these three competitor peptides to inhibit MBP-induced proliferation of an encephalitogenic T cell line in vitro correlated with their respective MHC binding affinity. Co-immunization of the competitor peptides during disease induction with entire MBP resulted in a competitor concentration-dependent inhibition of clinical signs of EAE. These results demonstrate that, although polyclonal T cell responses to MBP were not completely inhibited, co-administration of immunogenic or nonimmunogenic either EAE-related or non-EAE-related MHC class II binding competitor peptides can inhibit the development of EAE induced with entire MBP.

Vitamin D3 is known to induce regulatory T (Treg) cells by rendering antigen-presenting cells tolerogenic, its direct effect on human naturally occurring Treg cells is unclear. Here, we investigated if and how 1,25-dihydroxyvitamin D(3) [1,25(OH)2D3] can directly affect the proliferation and function of human naturally occurring Treg cells in vitro. First, we demonstrated that these Treg cells express vitamin D receptors that were up-regulated following anti-CD3/CD28-bead stimulation. 1,25(OH)2D3 inhibited proliferation of Treg cells even when exogenous interleukin-2 was provided. Treg cells were more susceptible to the inhibitory effect of 1,25(OH)2D3 than conventional T cells(.) 1,25(OH)2D3 neither affected the anergic state nor the suppressive function of Treg cells but induced a subtle increase in interleukin-10-secreting cells. The cell-division-inhibiting effect of 1,25(OH)2D3 on Treg cells was also demonstrated in vivo by supplementing vitamin D-deficient HIV-1-infected patients with 2000 IU cholecalciferol (vitamin D3). Increased serum 1,25(OH)2D3 levels were associated with a drop in the number and percentage of Treg cells, which may be attributed to a decrease in the proliferating Foxp3+ Treg cell population. In conclusion, 1,25(OH)2D3 directly affects Treg cell growth and promotes interleukin-10 production without apparent effects on activation status and suppressive phenotype whereas in vivo, high serum 1,25(OH)2D3 levels are associated with reduced Treg cell proliferation and a reduced number of Treg cells.

It is well-recognized that vitamin D₃ has immune-modulatory properties and that the variation in ultraviolet (UV) exposure affects vitamin D₃ status. Here, we investigated if and to what extent seasonality of vitamin D₃ levels are associated with changes in T cell numbers and phenotypes. Every three months during the course of the entire year, human PBMC and whole blood from 15 healthy subjects were sampled and analyzed using flow cytometry. We observed that elevated serum 25(OH)D₃ and 1,25(OH)(2)D₃ levels in summer were associated with a higher number of peripheral CD4+ and CD8+ T cells. In addition, an increase in naïve CD4+CD45RA+ T cells with a reciprocal drop in memory CD4+CD45RO+ T cells was observed. The increase in CD4+CD45RA+ T cell count was a result of heightened proliferative capacity rather than recent thymic emigration of T cells. The percentage of Treg dropped in summer, but not the absolute Treg numbers. Notably, in the Treg population, the levels of forkhead box protein 3 (Foxp3) expression were increased in summer. Skin, gut and lymphoid tissue homing potential was increased during summer as well, exemplified by increased CCR4, CCR6, CLA, CCR9 and CCR7 levels. Also, in summer, CD4+ and CD8+ T cells revealed a reduced capacity to produce pro-inflammatory cytokines. In conclusion, seasonal variation in vitamin D₃ status in vivo throughout the year is associated with changes in the human peripheral T cell compartment and may as such explain some of the seasonal variation in immune status which has been observed previously. Given that the current observations are limited to healthy adult males, larger population-based studies would be useful to validate these findings.

Whether or not Natural Killer (NK) cells affect the immune response to solid organ allografts is still controversial. Main determinants of NK-cell activation are specific HLA/killer-cell immunoglobulin-like receptors (KIR) interactions that, in transplantation, may induce NK-cell alloreactivity. So far, in liver transplantation (LTX) donor-versus-recipient alloreactivity has not been investigated; in addition, studies of predicted recipient-versus-donor NK-cell alloreactivity have led to contradicting results. We typed a cohort of LTX donors and recipients for HLA-C/Bw4 and KIRs. We estimated the effect of NK-cell alloreactivity, as predicted by classically used models, in the donor-versus-recipient direction. The results indicate that HLA/KIR mismatches in the donor-versus-recipient direction do not predict graft rejection nor graft or patient survival, suggesting that donor-derived NK cells do not play a major role in LTX outcome. In addition, when considering predicted NK-cell alloreactivity in the reverse direction (recipient-versus-donor), we first confirmed that donor HLA-C genotype was not associated with acute rejection, graft or patient survival and secondly we found that none of the models describing NK-cell alloreactivity could predict LTX outcome. Overall our observations suggest that, in contrast to what is shown in haematopoietic stem cell transplantation, donor-derived NK cells may not contribute in preventing liver graft rejection, and that recipient-versus-donor NK-cell alloreactivity does not predict LTX outcome.

Item does not contain fulltext Mycophenolic acid is the active ingredient of the immunosuppressant mycophenolate mofetil that is widely used in transplantation medicine and autoimmunity. Mycophenolic acid inhibits inosine monophosphate dehydrogenase, an enzyme involved in biosynthesis of guanine nucleotides required for lymphocyte clonal expansion. Here, we present novel insights into the mechanisms underlying mycophenolic acid-mediated suppression of human CD4+ T cells. Upon CD3/CD28 stimulation, mycophenolic acid inhibited T cell IL-17, IFN-gamma and TNF-alpha production but not IL-2 production. Phenotypic analysis showed that drug treatment enhanced the expression of negative co-stimulators PD-1, CTLA-4 and the transcription factor FoxP3 and decreased the expression of positive co-stimulators CD27 and CD28, whereas CD25 was unaffected. Mycophenolic acid-treated cells were anergic, but not suppressive, and at the same time proved hyperblastoid with high metabolic activity. Moreover, a reduced Akt/mTOR and STAT5 signaling was observed. Interestingly, the co-stimulatory molecule CD70 was uniquely and dose-dependently upregulated on mycophenolic acid-treated T cells and found to be directly linked to target enzyme inhibition. CD70 on mycophenolic acid-treated cells proved functional: an anti-CD70 agonist was found to restore both STAT5 and Akt/mTOR signaling and may thereby prevent apoptosis and promote survival. These novel insights may contribute to optimization of protocols for MPA-based immunosuppressive regimens.

Contains fulltext : 95557.pdf (Publisher’s version ) (Closed access) BACKGROUND: Our interest in immunological effects produced by vitamin D(3) (1,25(OH)(2)D(3)) and its therapeutic potential prompted us to examine the role of 1,25(OH)(2)D(3) on cytokine production by Candida albicans. METHODS: Peripheral blood mononuclear cells (PBMC) with stimulated C. albicans and 1,25(OH)(2)D(3), cytokine concentrations were measured in supernatant. Quantitative polymerase chain reaction (qPCR) was performed for T cell transcription factors, SOCS1 and 3. TLR2/4, Dectin-1, and mannose receptor expression was studied using flow cytometry and qPCR. An ex-vivo stimulation study was carried out in healthy volunteers to investigate the seasonality of immune response to C. albicans. RESULTS: Upon in vitro C. albicans stimulation, 1,25(OH)(2)D(3) induced a dose-dependent, down-regulation of IL-6, TNFalpha, IL-17, and IFNgamma. It also increased IL-10 production. The shift in cytokine profile was not due to 1,25(OH)(2)D(3) augmenting expression of either Thelper differentiation factors or SOCS1 and SOCS3 mRNA. 1,25(OH)(2)D(3) inhibited TLR2, TLR4, Dectin-1, and MR mRNA and protein expression. In our seasonality study, both IL-17 and IFNgamma levels were suppressed in summer when 25(OH)D(3) levels were elevated. CONCLUSION: Vitamin D(3) skews cytokine responses toward an antiinflammatory profile, mediated by suppression of TLR2, TLR4, Dectin-1, and MR transcription, leading to reduced surface expression. The biological relevance of these effects has been confirmed by the seasonality of cytokine responses.

SUMMARY The production of B-cell specific alloantisera by lymphocyte transfusion between class I matched, MLR positive goats is described. Furthermore, the usefulness of preceding IEF typing is demonstrated in the selection of immunization pairs. After suitable absorptions and cluster analysis the detected B-cell specific antigens were designated BeDl-BeD6. The specificity BeD3 could also be detected by two class II-specific murine anti-H2 monoclonal antibodies. IEF class II typing of 34 animals gave concordant results between the two techniques. One additional allelic variant could be detected by IEF typing. The detected products segregated in close linkage to the earlier described caprine class I antigens. One recombinant has been found in 78 offspring. The reactivity of cells in mixed lymphocyte cultures (MLC) was correlated with the compatibility of the test cells for their B-cell specific antigens. Three of the B-cell specific sera were characterized by immunoprecipitation. The precipitated antigens corresponded in molecular weight to MHC class II products as described in other species indicating that the here described caprine products are of class II nature.