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Solid-phase multiplex-bead assays are widely used in transplantation to detect anti-human leukocyte antigen (HLA) antibodies. These assays enable high resolution detection of low levels of HLA antibodies. However, multiplex-bead assays are costly and yield variable measurements that limit the comparison of results between laboratories. In the context of a Dutch national Consortium study we aimed to determine the inter-assay and inter-machine variability of multiplex-bead assays, and we assessed how to reduce the assay reagents costs. Fifteen sera containing a variety of HLA antibodies were used yielding in total 7092 median fluorescence intensities (MFI) values. The inter-assay and inter-machine mean absolute relative differences (MARD) of the screening assay were 12% and 13%, respectively. The single antigen bead (SAB) inter-assay MARD was comparable, but showed a higher lot-to-lot variability. Reduction of screening assay reagents to 50% or 40% of manufacturers' recommendations resulted in MFI values comparable to 100% of the reagents, with an MARD of 12% or 14%, respectively. The MARD of the 50% and 40% SAB assay reagent reductions were 11% and 22%, respectively. From this study, we conclude that the reagents can be reliably reduced at least to 50% of manufacturers' recommendations with virtually no differences in HLA antibody assignments.

Contains fulltext : 153318.pdf (Publisher’s version ) (Open Access) Insight into the maintenance of naive T cells is essential to understand defective immune responses in the context of aging and other immune compromised states. In humans, naive CD4+ T cells, in contrast to CD8+ T cells, are remarkably well retained with aging. Here, we show that low-affinity TCR engagement is the main driving force behind the emergence and accumulation of naive-like CD4+ T cells with enhanced sensitivity to IL-2 in aged humans. In vitro, we show that these CD45RA(+) CD25(dim) CD4(+) T cells can develop from conventional naive CD25(-) CD4+ T cells upon CD3 cross-linking alone, in the absence of costimulation, rather than via stimulation by the homeostatic cytokines IL-2, IL-7, or IL-15. In vivo, TCR engagement likely occurs in secondary lymphoid organs as these cells were detected in lymph nodes and spleen where they showed signs of recent activation. CD45RA(+) CD25(dim) CD4+ T cells expressed a broad TCRVbeta repertoire and could readily differentiate into functional T helper cells. Strikingly, no expansion of CD45RA(+) CD25(dim) CD8+ T cells was detected with aging, thereby implying that maintenance of naive CD4+ T cells is uniquely regulated. Our data provide novel insight into the homeostasis of naive T cells and may guide the development of therapies to preserve or restore immunity in the elderly.

Item does not contain fulltext Psoriasis is a common chronic inflammatory skin disease that results from interplay between the immune system and the epithelium. In the light of very successful anticytokine therapies for psoriasis, the focus has been directed towards the adaptive immune system. Expression studies, genetic studies and treatments specifically targeting players of the IL-23/IL-17 pathway, point at an important role for IL-17 in the pathogenesis of psoriasis. IL-17 stimulates the keratinocytes to produce psoriasis-associated molecules, eventually leading to chronic skin inflammation. The current opinion is that IL-17 is mainly produced by T cells, so-called T-helper 17 (Th17) cells, in psoriasis. However, evidence is accumulating that cells of the innate immune system, like neutrophils, mast cells, gammadelta T cells and innate lymphoid cells are the main source of IL-17 in psoriasis, rather than T cells. The paradigm in this field of research is shifting. With this viewpoint article, we will address this novel concept by critically summarizing the current literature on this subject. In psoriatic arthritis and atherosclerosis, important conditions related to psoriasis, it was also found that the majority of IL-17 is associated with cells of the innate immune system. This new concept changes our view of IL-17. Blocking IL-17 with targeted treatments might be more far-reaching than previously thought; not only IL-17 production by T cells but also by innate immune cells is blocked. Furthermore, therapies specifically targeting IL-17 may not only improve psoriasis, but also comorbidity that is associated with the IL-17 pathway, hereby preventing serious complications on the long term.

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.

Summary. The possibility was examined that in cattle compatibility of major histocompatibility complex (MHC) products between dam and calf might negatively influence the placental maturation and expulsion, and therefore increase the risk of retained placenta in healthy, normally calving cattle. Fifteen combinations of a single dam and two offspring were selected; the placenta of the first offspring was normally expelled (control) and the placenta of the second one was retained (case). The MHC class I and class II antigens of the animals were typed by immunoprecipitation and by one-dimensional isoelectric focusing (1D-IEF). Compatibility or incompatibility of class I or class II antigens was established by comparison of the IEF banding patterns of dam and calves. Analysis revealed that MHC class I compatibility between dam and calf increased the risk of retained placenta. In this study, the effect of class II compatibility was not significant, nor was the effect of the interaction of class I and class II. In a subsequent, additional sample the experimental design was extended: induction of tolerance against non-inherited maternal antigens (NIMA) might be implicated in the occurrence of the disorder within the group of class I incompatible cases. In three out of the five class I incompatible retained placenta cases, the banding pattern of the incompatible haplotype of the calf was identical to that of the haplotype of the granddam that was not inherited by the dam (NIMA). Notably, within the nine class I incompatible controls, there were none in which the offspring shared a paternal class I type with the granddam. This might suggest a toleranceinducing effect of NIMA in cattle in relation to retained placenta. The results for the nine cases and 11 controls analysed in this second set confirmed the results of the first set: again there appeared to be an increased risk of retained placenta due to class I compatibility (direct or through NIMA). We suggest that retention of the placenta is, at least in part, the result of the lack of alloreactive mechanisms.

Summary We have used biochemical methods to extend and improve serological class I typing using a panel of 77 Swiss goats of the Saanen breed, comprising dam-offspring combinations from six half-sib sire families and several unrelated animals. Of these animals class I molecules were precipitated from cell lysates with the mAb B1.1G6 and HC10. Immunoprecipitates were analysed by SDS-PAGE and 1D-IEF. There was a good agreement between class I serological types and IEF banding patterns. We have identified three new class I specificities and subdivided the Bel 7 specificity. IEF has enabled us to make planned immunizations to produce antisera to the new specificities. New evidence for the expression of a second class I locus product in the Be7 haplotype has been found.

Summary. For a long time, the bovine major histocompatibility complex (MHC) (BOLA) class I region was characterized, rather uniquely among mammalian species, as having one expressed locus. Recent reports have suggested otherwise. Selective immunoprecipitation and molecular characteriztion of products enable a decisive answer to the question of whether there is indeed more than one locus expressed. Therefore, we characterized serologically defined w10 encoding haplotypes in European and African cattle by immunoprecipitation of [35S]-methioninelabelled peripheral blood mononuclear cells (PBMC), followed by one- and two-dimensional isoelectric focusing (1D/2D-IEF) of cell lysates. Monoclonal antibodies (mAb) used were directed against either human class I monomorphic determinants (W6/32 and B1.1G6) or bovine polymorphic determinants expressed on products encoded by serologically defined w10 encoding haplotypes of Boran and Friesian cattle. Sequential immunoprecipitations with W6/32 and B1.1G6 using lysates of PBMC of British Friesian cattle, revealed that from this haplotype W6/32 precipitated one product, whereas B1.1G6 precipitated two products. The product precipitated in addition appeared to be the one that was selectively precipitated by the mAb directed against polymorphic determinants on a product of w10 encoding haplotypes. Additionally, peptide maps of protease V8-digested precipitates showed that this particular ‘w10’ associated product was distinctly different from the product recognized by W6/32. Thus, we suggest that the two products are distinct gene products and that the product with higher PI is associated with the serologically defined A-locus product, whereas the product with lower PI is the putative second locus product. In the African Boran breed, variants of the serologically defined w10 specificity were found on the basis of IEF typing. These variants appeared to be associated with different second locus products. Therefore, we conclude that serologically defined w10 encoding haplotypes encode at least two independent class I locus products, expressed on normal bovine PBMC. In IEF analysis the additional use of mAb recognizing polymorphic determinants on serologically defined A-locus products highly facilitated the detection and typing of second locus products.

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.

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.

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.

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.

Summary Although HLA-G is thought to play a modulatory role in the immune system, its function and expression require to be elucidated. We analysed soluble HLA-G levels in mid-trimester amniotic fluid (n = 64) from uncomplicated pregnancies. We found a decrease in soluble HLA-G levels for female offspring as compared to male offspring (P