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Background This retrospective study aims to determine the prognostic values of HLA and MICA antibodies, serum levels of sCD30 and soluble form of MHC class I related chain A (sMICA) in kidney allograft recipients. Methods Sera samples of 40 living unrelated donor kidney recipients were tested by ELISA and Flow beads techniques for the presence of anti HLA and MICA antibodies and the contents of sCD30 and sMICA. HLA and MICA antibody specification was performed by LABScreen single antigen beads to determine whether the antibodies were directed against donor mismatches. Results Within first year post operatively 9 of 40 patients (22.5%) showed acute rejection episodes (ARE) that four of them lost their grafts compared to 31 functioning transplants (P = 0.001). The presence of HLA antibodies before and after transplantation was significantly associated with ARE (P = 0.01 and P = 0.02 respectively). Sensitization to HLA class II antigens pre-transplant was strongly associated with higher incidence of ARE (P = 0.004). A significant correlation was found between ARE and appearance of non-donor specific antibodies (P = 0.02). HLA antibody positive patients either before or after transplantation showed lower graft survival rates than those without antibodies during three years follow-up (P = 0.04 and P = 0.02). Anti-MICA antibodies were observed in 8/40(20%) and 5/40(12.5%) of patients pre and post-transplant respectively. Coexistence of HLA and MICA antibodies was shown in 2 of 4 cases with graft loss. A significant increased level of sCD30 at day 14 (P = 0.001) and insignificant decreased levels of sMICA pre and post operatively were detected in rejecting transplants compared to functioning graft group. Conclusion Our findings support the view that monitoring of HLA and MICA antibodies as well as sCD30 levels early after transplant has predictive value for early and late allograft dysfunctions and the presence of these factors are detrimental to graft function and survival.

Introduction: HLA-E is a class I HLA molecule that is involved in NK and CTL functions. It has limited polymorphism but can present unconventional peptides if it is aberrantly expressed under stress conditions. Donor-recipient (D/R) mismatching may potentiate NK-mediated alloreactivity. The functional role of HLA-E in HSCT is uncertain. We used CIBMTR data and samples to investigate the effect of HLA-E polymorphism on HSCT outcome in a 10/10 HLA matched unrelated acute leukemia setting by addressing the following questions: 1) Does D/R HLA-E match status affect HSCT outcome? 2) Does a specific D/R HLA-E genotype correlate with outcome? Methods: The study included 1840 adult AML/ALL patients in complete remission who received first T-cell replete, 10/10 high resolution HLA matched transplants from unrelated donors between 2000 to 2015. Cohort characteristics are summarized in Table 1. Both D/R were HLA-E genotyped by NGS using a validated protocol on an Illumina Miseq platform. Overall survival (OS), leukemia free survival (LFS), relapse (RI), transplant related mortality (TRM), acute GvHD (aGvHD) and chronic GvHD (cGvHD) were evaluated; p< 0.01 was considered significant. Factors violating the proportional hazards assumption were adjusted via stratification, while a stepwise model building approach was used to select variables related to a given outcome with a threshold of 0.05 for both entry and retention in the model. HLA-E genotype effects in D/R were tested separately by comparing 0, 1 or 2 copies of a particular HLA-E allele (i.e. HLA-E*01:03). Transplant pairs with HLA-E genotypes including other HLA-E alleles (i.e. other than HLA-E*01:01 or *01:03) were excluded from this analysis. The joint effect of D/R HLA-E genotype was also explored through a 4-level analysis of D/R HLA-E*01:03 homozygous vs other. Results: HLA-E*01:01 and *01:03 frequencies in both D/R as well as the rate of HLA-E discrepant transplant pairs (32.7%) were in accordance with previously reported data. Distribution of HLA-E matched vs HLA-E mismatched cases was balanced for all significant clinical predictors, while no significant interactions between the HLA-E matching status variable and the adjusted covariates were detected. HLA-E mismatch had no significant association with any of the outcome endpoints in both univariate and multivariate models (data not shown). With respect to the HLA-E genotype effect on outcome, 1827/1840 transplant pairs were considered for analysis, as 13 transplant pairs were excluded due to new or rare HLA-E genotypes. When tested separately, both D/R HLA-E*01:03/01:03 genotypes were associated with significantly lower LFS (HR=1.28, p=0.0027 and HR=1.31, p=0.0017 respectively). While the impact of HLA-E genotype on other outcome endpoints was not statistically significant, relapse rates were higher in cases with D/R HLA-E*01:03/01:03 genotype. Joint D/R HLA-E genotype analysis suggest that donor HLA-E*01:03/01:03 is driving this effect. The results of LFS and RI multivariate models are summarized in Table 2. No significant interactions were observed between the HLA-E genotype variable and the adjusted covariates in the multivariate models. Conclusion: This is the largest study investigating the effect of HLA-E polymorphism on HSCT outcome. We found no apparent effect of HLA-E mismatch on HSCT outcome in AML/ALL patients in complete remission, while donor HLA-E*01:03/01:03 genotype was associated with lower LFS, contrary to some previous reports. Differential efficiency of the two HLA allelic forms in inducing the CD94/NKG2A inhibitory pathway may account for their divergent effect on leukemia control, although this hypothesis is yet to be confirmed by functional assays. Our results suggest that avoidance of HLA-E*01:03/01:03 donors may improve the outcome of HSCT from 10/10 matched unrelated grafts. This project was funded by the Else Kröner-Fresenius-Foundation and the National Marrow Donor Program® (NMDP). Disclosures Lee: Kadmon: Research Funding; Amgen: Consultancy, Research Funding; Mallinckrodt: Honoraria; Incyte: Consultancy; Pfizer: Consultancy; Onyx: Research Funding; Takeda: Research Funding. Fleischhauer:GENDX: Research Funding. Paczesny:Viracor IBT Laboratories: Patents & Royalties. Schrezenmeier:Alexion Pharmaceuticals, Inc.: Honoraria, Research Funding.

Aim Petersdorf et al. reported in 2014 an association between patient high expressed HLA-C (C) allotypes and inferior HSCT outcome using an imputed C-expression model (Apps et al., 2013). This study aims at: a) examining the validity of Apps et al. model in Caucasians by using the same methodology in a sample of 400 healthy German blood donors. b) specifically investigating the effect of patient’s non-shared (PNS) C expression levels on outcome by applying C expression imputed data in a 9/10 HLA C-mismatched HSCT setting. Methods Buffy coats from 400 healthy German blood donors were tested by flow cytometry as previously described (Apps et al., 2013) in order to determine C expression on lymphocytes. With reference to the mean fluorescence intensity (MFI) values measured, C antigens were categorized as high- or low- expressed. In a cohort of 324 single C-mismatched transplant pairs both C expression models were used in order to investigate the impact of high- and low-expressed PNS-C on HSCT outcome. Overall survival (OS), disease free survival (DFS), relapse incidence (RI) and non-relapse mortality (NRM) were set as outcome endpoints, while statistical significance was set at p 0.05. Results With the exception of HLA-C*15, the two expression models were fully concordant as to the definition of low- and high-expressed C antigens. Analysis of PNS-C expression’s effect on HSCT outcome revealed an unexpected correlation between high expression and better OS (49% vs 33% p = 0.04; HR = 0.43, p = 0.002) due to lower NRM (37% vs 47%, p = 0.02; HR = 0.29, p 0.001). Although similar trends were observed, statistical significance was only met with the Apps et al. model. Death cause analysis revealed a tendency for lower infection-related mortality in the high expressed PNS-C (10% vs 14%), while no differences were observed for GvHD-associated death. Interestingly, overall patient’s C expression had no influence on any outcome endpoint. Conclusions a) HLA-C antigens exhibit similar expression patterns regardless of race. b) The lower OS observed in lower expressed PNS C mismatches may be attributed to inferior infection control and thus higher NRM. Albeit noteworthy, these findings must be confirmed by larger independent cohorts, before definitive conclusions can be drawn.

relapse, or transplant related mortality (TRM) (p1⁄40.2, p1⁄40.1, p1⁄40.08, respectively). To investigate why higher CPSS scores were associated with higher mortality, we performed analysis restricted to patients with relapse following HCT. Those with intermediate-2/high riskhadnearly two-fold increase in riskof death after relapse compared to thosewith low/intermediate1 CPSS scores. Corresponding rates for low/intermediate-1 risk groups,OSat1year, 3years, and5yearswere61%,48%, and44% respectivelyand for intermediate-2/high riskgroupswere 38%, 32%, and19%respectively.OSofpatientswho receivedpre-HCT treatment with hypomethylating agents, chemotherapy, or both was not different compared to those who received no therapy prior (p1⁄40.96). In summary, CPSS scores, lower KPS scores, and bone marrow as graft source were associated with poorer outcomes following HCT.

Using sequence-based typing, we have identified a novel human leukocyte antigen (HLA)-A*30 allele, HLA-A*3014L, with a low expression pattern. The sequence of HLA-A*3014L is identical to that of HLA-A*3001 except for a G to C substitution in exon 3 at nucleotide position 563, resulting in an amino acid difference at position 164 (Cys to Ser). Due to the cysteine substitution, a disulfide bridge in the alpha2 domain of the HLA class I heavy chain cannot be formed. By using the standard microlymphocytotoxicity test, the HLA-A30 antigen cannot be detected. By flow cytometric analysis of the cell-surface expression at either 37 degrees C or 30 degrees C, a temperature-sensitive expression pattern of the HLA-A*3014L antigen was observed. Only by incubating the cells at 30 degrees C, which increases the stability of HLA class I heavy chains, was a weak but clearly detectable HLA-A*3014L expression found. The mRNA expression level of the HLA-A*3014L allele was not affected by the nucleotide substitution. The intrachain disulfide bond formation in the alpha2 domain is essential for the normal expression of the HLA molecules. Reduced protein expression is probably caused by incorrect HLA class I heavy chain folding and HLA class I complex assembly.

Human leukocyte antigen (HLA)-B and HLA-DRB1 typing in two female individuals revealed reaction patterns that did not correspond to any known HLA-B specificity and appeared to identify a very rare HLA-DRB1 allele, respectively. Sequence-based analysis of these samples revealed two new HLA alleles, one similar to B*4023 and the other to DRB1*1308. The new HLA-B allele, which was assigned the name HLA-B*4051, could have been generated by a double crossing over recombination between B*4001 and B*1401 or 1402, whereas DRB1*1364, the new DRB1 allele, could have been generated either by a double crossing over recombination between DRB1*1308 and DRB1*1201, 1202, or 1203 or by two independent crossing over events between DRB1*1401, DRB1*1201, 1202, or 1203 and DRB1*1301.

Background. A computer-based approach for determining human leukocyte antigen (HLA) compatibility between kidney donors and recipients on the basis of differences of amino acid sequences as motifs for immunogenic epitopes was proposed by Duquesnoy et al. The HLAMatchmaker algorithm focuses on HLA class I polymorphisms of serologically defined antigens encoded by the HLA-A and -B loci. HLA phenotypic mismatches that represent only a few mismatches at the amino acid triplet level are held to be not or only mildly immunogenic. This approach was proposed as being especially suitable for the allocation of donor kidneys to highly sensitized patients. Methods. We reexamined this attractive concept using the data of the Collaborative Transplant Study. Intra- and interlocus comparisons for HLA-A and -B were performed according to the original HLAMatchmaker algorithm. To exclude the influence of HLA-DR, only transplants with no HLA-DR mismatch were considered. Patients who had one HLA-A and one HLA-B antigen mismatch were separated into subgroups, depending on the number of triplet mismatches as calculated by the HLAMatchmaker software. Separate analyses were performed for first transplants, retransplants, and patients with a panel-reactive antibody activity of 50% or more. A total of 16,997 white patients matched for HLA-DR who received a cadaver kidney transplant between 1991 and 2001 formed the basis of this analysis. Results. Application of the HLAMatchmaker method could not be shown to result in any statistically significant effect on graft survival. Conclusions. The HLAMatchmaker concept is theoretically attractive; however, it could not be shown to yield useful results in this analysis. Serologic HLA typing appears to provide an insufficient basis for applying epitope matching in clinical kidney transplantation.

Background. Several years ago a significant impact of matching for HLA-DPB1 alleles on the survival of cadaver kidney retransplants was shown. Here we report the results of a new approach, based on matching for HLA-DPB1 epitopes. Methods. The analysis is based on 1,478 patients who received a cadaver kidney retransplant between 1988 and 1998. DNA methodology (polymerase chain reaction, sequence-specific oligonucleotides) was used to perform HLA-DPB1 typing. Epitope matching was facilitated with the aid of sequence databases and computer calculations. Results. Matching for the HLA-DP epitopes A, B, E, and F, corresponding to the homonymous hypervariable regions of the second exon of the DPB1 gene, seems to have a greater influence on graft survival than matching for the epitopes C and D. Within a group of 529 retransplants with exactly one allelic HLA-DPB1 mismatch, a significantly better graft outcome was observed when less than two epitope mismatches were found, compared with the group with more than three epitope mismatches (at 2 years: 77.8% vs. 65.8%, P=0.0112). Importantly, patients with two DPB1 allele mismatches who had less than or equal to two epitope mismatches exhibited a significantly better graft outcome than recipients who had one HLA-DPB1 allelic mismatch but more than three epitope mismatches (at 2 years: 77.1% vs. 65.8%, P=0.0488). Conclusions. The findings indicate that the impact of HLA-DPB1 matching on the outcome of kidney retransplants is a result of the predominant immunogenicity of certain epitopes of the HLA-DP molecule. Matching for immunogenic HLA-DPB1 epitopes seems to be functionally more relevant than classical matching at the allelic level.

HLA-A typing by the PCR-SSP method in a male 21-year-old Caucasian individual revealed a very rare allele combination corresponding to a unique reaction pattern. Therefore, the result was examined using sequence-based typing. Sequencing of exons 2 and 3 of the HLA-A locus after allelic separation with specific primers revealed the sequence of a new allele, similar to A*0245. Sequencing of exons 1 and 4 resulted in no additional inconclusive positions. The sequence pattern of the new allele HLA-A*0256 might have been generated as a result of a double crossing over recombination of an A*0201 and either an A*03 or an A*11.

Background Prognostic information is essential for optimal treatment of patients with head and neck cancer. We studied the relationship of class I and class II human leukocyte antigens (HLA) on prognosis in patients with head and neck cancer. Methods HLA-A, -B, -C and -DR antigens were determined in 209 patients with squamous cell carcinoma of the oral cavity, oropharynx, hypopharynx, or larynx. The patients were subjected to follow-up investigations for a period of 5 years. Results Five-year survival rates in relation to tumor stage varied between 86% for stage I tumors and 28% for stage IV tumors (P

Aim While the role of HLA-DPB1 (DP) compatibility in uHSCT regarding graft versus host disease (GVHD)- and graft versus leukaemia (GVL)-effects has been extensively discussed, its interaction with donor CMV status (dCMV), remains elusive. The aim of this study was, to investigate the impact of dCMV status in DP matched (DPM) and mismatched (DPMM) uHSCT. Methods 1749 HSCT transplant (Tx) pairs were DP genotyped with an exon 2 amplicon based NGS approach in order to assess their DP matching status. CMV sero-status as well as clinical data were retrieved from the German Stem Cell Registry. Overall survival (OS), relapse incidence (RI) and chronic GvHD (cGvHD) were defined as endpoints, while statistical significance was set to a p Download : Download high-res image (52KB) Download : Download full-size image Results Data analysis revealed a significantly lower 5y RI in DPM dCMV(+) vs. DPM dCMV(−) Tx (34.6% vs 54.5%, p = 0.01). In DPMM cases, dCMV did not appear to positively influence this endpoint. Furthermore, dCMV(+) appeared to benefit DPM vs. DPMM Tx, as lower cGvHD incidence rates were observed in DPM compared to DPMM cases, yet not in a statistically significant manner (36.3% vs. 44.7%, p = 0.38). This beneficial association between dCMV(+) and DP matching did not appear to significantly influence OS. Our study, although not conclusive, is suggestive of a positive interaction between dCMV positivity and patient-donor DP-Match status as to relapse and cGvHD incidence rates. In a DPM setting, dCMV specific allo-reactive T-cells may account for a more pronounced GvL effect, as their T-cell Receptors are programmed to recognize “self”-DP/viral peptide complexes. Accordingly, in a DPMM setting, a CMV-induced upregulation of DP expression on patients’ non HC’s could lead to higher cGvHD rates via DP-specific CD4+ T-cell alloreactivity. Conclusions In conclusion, our study provides information regarding a possible association between donor CMV status and DP matching in patients undergoing HSCT. Larger cohort studies are needed to confirm these initial findings, before further conclusions can be drawn.

Aim The established variability of HLA-C expression on cell surface, although already investigated in African Americans by Apps et al. Science 2013(1), has not been yet accordingly researched in Caucasians. Furthermore, the possibility that HLA-C expression heterogeneity may be attributed to an overall HLA-Class I expression regulation has not been thus far experimentally addressed either. This work aims at elucidating both issues through actual protein expression analysis by flow-cytometry in 188 German healthy blood donors. Methods 188 buffy-coats provided from the Ulm Blood Donor Centre were used for leucocyte collection in order to determine by flow cytometry the protein expression levels of HLA-C and HLA-Class I on lymphocytes as previously described 1 . For HLA-C and HLA-Class I expression estimation, a DT-9 and an anti-HLA-ABC Antibody were used. Median Intensity Fluorescence (MFI) coefficients were calculated for each HLA-C allotype through implementation of a linear regression model. Results According to our findings, HLA-C03, -C07 and -C08 (MFI: 408, 450 and 481 respectively) were ranked as low-expressed HLA-C antigens contrary to HLA -C12, -C14 and -C01, which were notably higher expressed (MFI: 1261, 1280 and 1322 respectively). In addition, our data on overall HLA-Class I expression levels were suggestive of no direct correlation between HLA-C and HLA-Class I expression levels on cells as: (1) The divergence range of expression levels for HLA-Class I was markedly narrower (MFIs: 450–758 vs 408–1322). (2) Low expressed HLA-C allotypes were found linked with high HLA-Class I expression levels (e.g. C08: 758 for HLA-Class I vs 481 for HLA-C). Conclusions Our HLA-C expression results match significantly those of Apps et al. at least with respect to high-and low-expressed HLA-C antigens, despite the different race of subjects included in the two studies. Moreover, of high interest is our finding regarding the lack of association between HLA-C and HLA-Class I expression, which in turn points to an HLA-C rather than an HLA-Class I specific expression- regulating factor accounting for this marked divergence in HLA-C expression levels. Further investigation on a much greater scale is nevertheless mandatory before final conclusions can be drawn.

Genotoxic stress can promote antitumor NK cell responses by upregulating the surface expression of activating ligands on cancer cells. Moreover, a number of studies suggested a role for soluble NK group 2D ligands in the impairment of NK cell tumor recognition and killing. We investigated whether genotoxic stress could promote the release of NK group 2D ligands (MHC class I–related chain [MIC]A and MICB), as well as the molecular mechanisms underlying this event in human multiple myeloma (MM) cells. Our results show that genotoxic agents used in the therapy of MM (i.e., doxorubicin and melphalan) selectively affect the shedding of MIC molecules that are sensitive to proteolytic cleavage, whereas the release of the short MICA*008 allele, which is frequent in the white population, is not perturbed. In addition, we found that a disintegrin and metalloproteinase 10 expression is upregulated upon chemotherapeutic treatment both in patient-derived CD138+/CD38+ plasma cells and in several MM cell lines, and we demonstrate a crucial role for this sheddase in the proteolytic cleavage of MIC by means of silencing and pharmacological inhibition. Interestingly, the drug-induced upregulation of a disintegrin and metalloproteinase 10 on MM cells is associated with a senescent phenotype and requires generation of reactive oxygen species. Moreover, the combined use of chemotherapeutic drugs and metalloproteinase inhibitors enhances NK cell–mediated recognition of MM cells, preserving MIC molecules on the cell surface and suggesting that targeting of metalloproteinases in conjunction with chemotherapy could be exploited for NK cell–based immunotherapeutic approaches, thus contributing to avoid the escape of malignant cells from stress-elicited immune responses.

Non-lipidated spirochetal antigens (OspA,OspC and P39) including those selectively expressed in the mammalian host (pG and BapA)were used for antigenic stimulation and autologous dendritic cells served as antigen-presenting cells. The majority of patients with well-defined early and late manifestations ofLyme disease exhibited specific T cell proliferative responses to one or more of the indicatedantigens, however at distinct levels. Most notably, among the five antigens tested, pG wasspecifically recognized by the majority of T cell populations ( >70%) – mainly Th1 cells – frompatients but not control individuals. These data indicate a causal relationship between

It has long been suspected that pentachlorophenol (PCP) exerts a damaging influence on the immune system. In this study, the possible relationship between blood levels of PCP and immune function was studied in 190 patients who had been exposed for more than 6 mo to PCP-containing pesticides. The patients suffered from frequent respiratory infections and general fatigue. Lymphocyte subpopulations, in-vitro responses to mitogens, allogeneic stimulator cells, plasma neopterin, cytokines, soluble cytokine receptors, soluble adhesion molecules, and immunoglobulin autoantibodies were determined. A dose-response relationship between blood levels of PCP and cellular and humoral immune parameters was established. Blood levels of PCP were associated negatively with (a) total lymphocyte counts (p = .0002), CD4/CD8 ratios (p = .0015), and absolute counts of CD3+ (p < .0001), CD4+ (p < .0001), CD16+ (p < .0001), CD25+ (p = .0003), DR+ (p < .0001), CD8+/56+ (p = .020), and CD19+ cells (p = .092); (b) plasma levels of interleukin-2 (IL-2) (p < .0001), soluble IL-2R (p < .0001), IL-6 (p < .0001), IL-10 (p = .0039), interferon-gamma (IFN-gamma) (p < .0001), tumor necrosis factor-alpha (TNF-alpha) (p < .0001), transforming-growth factor-beta2 (p = .023), soluble IL-1 receptor antagonist (sIL-1 RA) (p < .0001), soluble intercellular adhesion molecule-1 (p = .0003); and (c) immunoglobulin (Ig) M-anti-Fab type autoantibodies (p = .0353). PCP levels were associated positively with (a) number of impaired stimulation assays per patient (p = .041); (b) number of circulating CD11b+ monocytes (p = .0015); and (c) plasma levels of neopterin (p < .0001), IL-4 (p = .020), and sIL-6R (p = .020). Compared with patients who had PCP plasma levels that were less than or equal to 10 microg/l, patients with blood levels of PCP that exceeded 10 microg/l experienced the following more often: low numbers of total blood lymphocytes (p = .054), CD3+ (p = .0014), CD4+ (p = .0001), DR+ (p = .0003), CD16+ (p = .0033), and CD25+ cells (p = .0033). In addition, the same aforementioned patients experienced the following more frequently: undetectable plasma levels of IL-2 (p = .0057), IL-6 (p = .042), IL-8 (p = .038), IL-10 (p = .0001), TNF-alpha (p = .0062), and IFN-gamma (p = .016); and impaired in-vitro responses of lymphocytes (p = .071). The authors concluded that increased blood levels of PCP were associated significantly with cellular and humoral immunodeficiencies. Recurrent respiratory infections and general fatigue could originate from PCP-associated immunosuppression.

Lymphoid and dendritic cells of donor origin can be detected in the recipient several years after a solid organ transplantation. This phenomenon is termed microchimerism and could play a role in the induction of tolerance. The fate of other hematopoietic cells transferred by liver transplantation, in particular of stem and progenitor cells, is unknown. For this reason, we studied peripheral blood and bone marrow samples of 12 patients who had received a liver transplant from an HLA-DR mismatched donor. Eight patients were long-term survivors between 2.8 and 10.1 years after allografting. CD34+ cells from bone marrow were highly enriched with the use of a 2-step method, and a nested polymerase chain reaction was applied to detect donor cells on the basis of allelic differences of the HLA-DRB1 gene. Rigorous controls with DRB1 specificities equal to the donor and host were included. In 5 of 8 long-term liver recipients, donor-specific CD34+ cells could be detected in bone marrow. Microchimerism in the CD34+ cell fraction did not correlate to the chimeric status in peripheral blood. In conclusion, our results demonstrate a frequent microchimerism among bone marrow–derived CD34+ cells after liver transplantation. The functional role of this phenomenon still needs to be defined.

Sequence-specific primed polymerase chain reaction (PCR-SSP) is widely used in HLA laboratories. The TaqMan method, which is described here for high-resolution typing of HLA-DRB1*15 and -DRB1*16, does not require elaborate and time-consuming post-PCR detection steps. In this one-tube assay, conventional PCR-SSP and fluorescence detection of the amplicon with a doubly labeled fluorescent probe are combined: a fluorogenic hybridization probe (FHP) labeled with a spectral resolvable fluorescent reporter dye (FAM or TET) at its 5' terminus and a common quencher dye (TAMRA) at its 3' terminus is cleaved by the 5' nuclease activity of Taq DNA polymerase only if the target sequence is amplified. An increase of fluorescence intensity indicates a successful amplification. For high-resolution typing of HLA-DRB1*15 and -DRB1*16 alleles we designed two FHPs and 14 specific primer mixes (7 for DR15 and 7 for DR16). Amplification of the specific sequence was detected by a FAM-labeled FHP, whereas amplification of the internal control was detected by a TET-labeled FHP. We were able to type all heterozygous DRB1*15/DRB1*16 subtype combinations. For evaluation, 60 HLA-DRB1*15-positive and 40 HLA-DRB1*16-positive individuals were typed and the results were compared with conventional PCR-SSP DR15/16 subtyping. There were no discrepancies between the two methods. The TaqMan method is an alternative to conventional PCR-SSP typing which is suitable for routine use in HLA laboratories.

We report the distribution of the human platelet antigens HPA-1, -2, -3, -4 and -5 as determined using the polymerase chain reaction with sequence-specific primers in 100 random, healthy Chinese in Hong Kong. The HPA-1a, -2a, -4a and -5a genes were present in every sample tested, HPA-1b, -2b and -5b were rare, and the sample was monomorphic for HPA-4a. HPA-3a and -3b genes showed frequencies of 0.525 and 0.475 respectively. There was no departure from Hardy-Weinberg equilibrium in any of the five HPA systems studied.

Human leukocyte antigen (HLA)-Cw*0740 differs from HLA-Cw*070101 by one nucleotide substitution at codon 73 (GCT>ACT).

In a recent study, we observed a discrepancy rate of 8.5% between the results of molecular and serological HLA class I typing in Caucasian kidney donors and recipients. In the present study we addressed the question how often black individuals are mistyped using the serological technique. 421 Blacks whose HLA typing results were reported to the Collaborative Transplant Study (CTS) were typed retrospectively for HLA-A and -B using a PCR-SSP method. 78 of the 421 individuals (18.5%) showed a discrepancy for HLA-A and 107 individuals (25.4%) for HLA-B. 36.3% of all individuals tested showed either an HLA-A or an HLA-B discrepancy. 13.1% of the discrepancies at the HLA-A locus were due to antigen misassignments and 4.8% were due to missed antigens. HLA-B discrepancies were caused in 15.7% by antigen misassignments and in 10.5% by missed antigens. These results demonstrate an impressive advantage of the PCR-SSP method for HLA-A and HLA-B locus typing over serological typing in black individuals. The high typing discrepancy rate observed in Blacks provides a strong argument for replacing serological typing by the DNA method. It is likely that this will improve the HLA matching correlation in clinical transplantation in Blacks.

The association of HLA class II alleles with multiple sclerosis (MS) has been amply documented. In the present study, the role of HLA class II (DRB1, DQA1 and DQB1) alleles and haplotypes was investigated in 43 unrelated Iranian chronic progressive multiple sclerosis (CP-MS) patients compared with 100 healthy individuals. HLA typing for DRB1, DQA1 and DQB1 was performed by restriction fragment length polymorphism (RFLP). Subtypes of DR4, DR15 and DR16 were defined using polymerase chain reaction (PCR) amplification with sequence-specific primers (PCR-SSP). The results show that, among DR2-positive MS patients and the control group, a positive association with the DRB1*1503, DQA1*0102, DQB1*0602 haplotype (21% vs. 2.7%, P = 0.057, RR = 9.8) and a negative association with the most frequent DR15 haplotype in the control group, DRB1*15021, DQA1*0103, DQB1*0601 (7% vs. 24.3%, P = 0.001), were observed. No significant association was found with the analysed HLA-DRB1, DQA1 and DQB1 alleles.

DNA typing for HLA class II improves the typing quality and this was shown previously to be relevant for kidney graft survival. In this project we addressed the question whether molecular typing for HLA class I also increases the efficacy of HLA matching in kidney transplantation. 215 HLA-A,-B,-DR zero-mismatched donor/recipient pairs as defined by serological typing were selected. Retrospective HLA-A and HLA-B typing was performed both by the PCR-SSP and the PCR-SSOP method. DNA typing for HLA-A revealed discrepant results to serology in 5.7% of the donors and 2.8% of the recipients. HLA-B typing discrepancies were found in 6.6% of the donors and 5.6% of the recipients. 10.4% of the donors and 6.5% of the recipients showed either an HLA-A or an HLA-B discrepancy Nearly one-third of the HLA-A discrepancies affected A19 splits. The most common reason for HLA-A discrepancies was the erroneous assignment of serological blanks, whereas HLA-B errors were caused mainly by the assignment of incorrect specificities. DNA typing allowed the definition of HLA-A and -B split specificities in all 118 "splitable" cases for which only broad specificities were reported based on serological typing. A total of 183 DNA class I compatible transplants had a 15% higher one-year graft survival rate than 32 transplants for which DNA typing revealed a class I incompatibility

Associations of human leukocyte antigens (HLA) with the idiopathic nephrotic syndrome (NS) have mainly been described for alleles of the HLA-DR locus. In the present study the polymorphism of HLA-DQ and -DP at the molecular level was investigated in 167 children with NS (129 steroid-sensitive) using the polymerase chain reaction and sequence-specific oligonucleotides in a French and a German cohort. HLA-DR typing was also performed by classical serology. In steroid-sensitive patients we observed an increased frequency of the alleles HLA-DQA1*0201 and -DQB1*0201 in both populations with relative risks ranging from 3.8 to 8.5 (Pb

To validate current donor selection strategies based on previous international studies, we retrospectively analyzed 2646 transplantations performed for hematologic malignancies in 28 German transplant centers. Donors and recipients were high resolution typed for HLA-A, -B, -C, -DRB1, and -DQB1. The highest mortality in overall survival analysis was seen for HLA-A, -B, and DRB1 mismatches. HLA-DQB1 mismatched cases showed a trend toward higher mortality, mostly due to HLA-DQB1 antigen disparities. HLA incompatibilities at >1 locus showed additive detrimental effects. HLA mismatching had no significant effect on relapse incidence and primary graft failure. Graft source had no impact on survival end points, neither in univariate nor in multivariate analysis. Higher patient age, advanced disease, transplantations before 2004, patient C2C2 killer cell immunoglobulin-like receptor (KIR)-ligand phenotype, and unavailability of a national donor adversely influenced outcomes in multivariate analysis. Our study confirms the association of HLA-A, -B, -C, and -DRB1 incompatibilities with adverse outcome in hematopoietic stem cell transplantation (HSCT). The relevance of HLA-DQB1 disparities in single mismatched transplantations remains unclear. Similar hazard ratios for allele and antigen mismatches (possibly with an exception for HLA-DQB1) highlight the importance of allele level typing and matching in HSCT. The number of incompatibilities and their type significantly impact survival.

Continuing a project presented at the 15th International HLA and Immunogenetics Workshop (IHIWS) on the rarity of HLA alleles, we sought to expand the number of data sources and bioinformatics tools available in the Allele Frequencies Net Database website (AFND, www.allelefrequencies.net). In this 16th IHIWS Rare Alleles project, HLA alleles described in the latest IMGT/HLA Database (release 3.8.0) were queried against different sources including data from registries (stem cell) and from 74 different laboratories around the world. We demonstrated that approximately 40% of the alleles officially named in the IMGT/HLA Database have been reported only once across all different sources. To facilitate the large-scale analysis of rare alleles, we have produced an online tool called the Rare Allele Detector that simplifies the detection of alleles that are considered to be 'very rare', 'rare' or 'frequent'. Tools and associated data can be accessed via the www.allelefrequencies.net website.

Summary In this multicentre study, sera from 803 retransplant candidates, including 775 kidney transplant recipients, were analysed with regard to the presence and specificity of anti-HLA alloantibodies of the IgA isotype using a modified microsphere-based platform. Of the kidney recipients, nearly one-third (n = 237, 31%) had IgA alloantibodies. Mostly, these antibodies were found in sera that also harboured IgG alloantibodies that could be found in a total of 572 (74%) of patients. Interestingly, IgA anti-HLA antibodies were preferentially targeting HLA class I antigens in contrast to those of the IgG isotype, which targeted mostly both HLA class I and II antigens. Donor specificity of the IgA alloantibodies could be established for over half of the 237 patients with IgA alloantibodies (n = 124, 52%). A further 58 patients had specificities against HLA-C or HLA-DP, for which no information regarding donor typing was available. In summary, these data showed in a large cohort of retransplant candidates that IgA alloantibodies occur in about one-third of patients, about half of these antibodies being donor specific.

Contrary to other HLA loci, allele vs antigen mismatches in HLA-C appear to differentially impact HSCT outcome. Aim of this study was to investigate the independent role of other factors characterizing a patient’s HLA-C non-shared allele, in HSCT outcome as well as their distribution in allele vs antigen mismatched cases. 288 9/10 HLA-C mismatched unrelated transplant pairs, were additionally genotyped by sequence based typing for rs9264942(C/T) and rs67384697(ins/del), which are considered surrogate markers of HLA-C expression levels. A proxy MFI model as previously described (Petersdorf et al., Blood 2014), was also implemented in the analysis. All patient non-shared alleles were characterized in regard to mismatch level, expression-relevant polymorphisms’ genotype and proxy MFI levels as well as differences in residues 77, 80 (C1/C2 KIR epitopes) and 116 (immunogenic spot). In order to assess the independent impact of each factor on HSCT outcome, overall survival, disease free survival, non-relapse mortality and relapse incidence were set as endpoints. The balance in distribution of the aforementioned parameters in allele vs antigen mismatched cases was estimated by Chi-square and Wilcoxon tests. Data analysis revealed no statistically significant impact of any of the parameters tested individually on HSCT outcome. Their distribution with respect to mismatch level, however, was markedly skewed. Antigen mismatches were associated with more highly-expressed HLA-C alleles according to the MFI proxy model as well as the rs9264942C and rs67384697del genotypes (MFI: p

Introduction/Background: Head and neck squamous cell carcinoma (HNSCC) is the fifth most common cancer type and is associated with a poor prognosis. Cancer-testis antigens (CTA) are specifically expressed in cancer tissue, but not in healthy normal tissue with the exception of testis. Their tumor-restricted expression and immunogenicity makes them an interesting target for specific cancer immunotherapy. We have previously shown that CTA of the MAGE-A family and NY-ESO-1 serve as independent prognosticators of poor survival. The addition of immunotherapeutic strategies targeting CTA may specifically improve the survival of this group of patients with an increased risk of death. The IRECT trial focuses on tumor host interactions to CTA and human papilloma virus (HPV) during conventional therapy of HNSCC to determine the most promising strategies for integration of immunotherapy targeting CTA or HPV in combination with immune checkpoint modulating drugs. Material/Methods: The IRECT trial is a single institution non-interventional, prospective clinical trial. HNSCC patients treated with curative intent by surgery followed by (chemo-)radiation as indicated or by primary chemoradiation are monitored over the course of standard-of-care treatment for HNSCC. Snap-frozen and formalin-fixed paraffin embedded tumor tissue was collected at the time of diagnosis/ surgery. Serum, plasma and peripheral blood mononuclear cells (PBMC) are collected at defined time points before and during treatment and at every follow-up after treatment for the first 12 months. Patients were typed for HLA class I and II. CTA mRNA expression for MAGE-A1, -A3, -A4, -C1, -C2, NY-ESO-1 and PRAME was determined by PCR from baseline tumor tissue. Results: Patient recruitment started 09/2013 at the University Medical Center Ulm. Patient accrual was scheduled for 18 months. A total of 22 patients have been recruited. Mean follow-up was 11.2 months (range: 1-22 months). The primary sites were oropharynx (12/22), larynx (2/22), hypopharynx (3/22) and oral cavity (5/22). Eighteen patients were treated surgically and four patients received primary chemoradiation. To date, two patients had a recurrence (1 locoregional, 1 distant) and two patients developed a second primary tumor (1 HNSCC, 1 pulmonary adenocarcinoma). Six of 21 evaluable tumors were positive for HPV DNA and 14/21 for at least one of the tested CTA. Ten of the 14 CTA positive patients expressed >1 antigen. Of the 21 evaluable tumors, expression frequencies were as follows: MAGE-A1: 4, MAGE-A3: 11, MAGE-A4: 8, MAGE-C1: 1, MAGE-C2: 1, NY-ESO-1: 2 and PRAME: 10. Conclusion: Two thirds of the recruited patients expressed at least one of the seven antigens tested. The analysis of immune responses to CTA and HPV during conventional therapy is crucial for a rational integration of cancer immunotherapy into curative HNSCC treatment. This trial will lay the groundwork for a rational integration of cancer-testis antigen or HPV specific vaccines and immune-modulating drugs into the conventional treatment of HNSCC. As a next step, CTA expression will be confirmed by immunohistochemistry (IHC). A T cell related cytokine panel including Perforin, GM-CSF, sCD137, IFNγ, sFas, sFasL, Granzyme A, Granzyme B, IL-2, IL-4, IL-5, IL-6, IL-10, IL-13, MIP-1α, MIP-1β, TNF-α will be quantified by Luminex from 10 defined time points over the course of treatment and for the first 12 months. DAMP will also be quantified over the course of treatment. Immune infiltrates in the primary tumor will be quantified by IHC. After screening for antibody responses to a panel of tumor associated antigens (primarily CTA) and HPV by Luminex, quantities of selected antigen-specific T cells will be monitored over the course of treatment and during the first 12 months of follow-up by Elispot and tetramer analysis. Citation Format: Simon Laban, Marie-Nicole Theodoraki, Daniel Fürst, Joannis Mytilineos, Ramin Lotfi, Djordje Atanackovic, Tim Luetkens, Thomas K. Hoffmann. Immunoprofiling head and neck cancer: Preliminary data of the IRECT trial (Immune response evaluation to curative conventional therapy). [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B142.

The interaction of the MHC class I-related chain molecules A and B (MICA and MICB) with the corresponding natural killer group 2, member D (NKG2D) receptor triggers cytotoxic effector activity of natural killer cells and certain T-cell subsets and provides a costimulatory signal for cytokine production. Thus, the presence of MICA/B on transformed cells contributes to tumor immunosurveillance. Consequently, the proteolytic cleavage of MICA/B is regarded as an important immune escape mechanism of various cancer cells. To investigate the molecular machinery responsible for the shedding of endogenous MICA/B, we analyzed different human tumor entities including mammary, pancreatic and prostate carcinomas. Flow cytometry and enzyme-linked immunosorbent assay (ELISA) revealed that all tested tumor cells constitutively expressed MICA and MICB on the cell surface and also released NKG2D ligands into the supernatant. We demonstrate that the "a disintegrin and metalloproteases" (ADAMs) 10 and 17 are largely responsible for the generation of soluble MICA/B. Pharmacological inhibition of metalloproteases reduced the level of released MICA/B and increased cell surface expression. Studies using RNA interference not only revealed a prominent role of ADAM10 and ADAM17 in NKG2D ligand shedding but also a tumor cell-specific role of ADAM10 and/or ADAM17 in shedding of MICA or MICB. Moreover, we report that in the prostate carcinoma cell line PC-3, MICA was not shed at all but rather was secreted in exosomes. These data indicate that the release of NKG2D ligands from individual tumor entities is by far more complex than suggested in previously reported MICA/B transfection systems.

Mutations in the nucleophosmin gene (NPM1mut) are one of the most frequent molecular alterations in acute myeloid leukemia (AML), and immune responses may contribute to the favorable prognosis of AML patients with NPM1mut. In the present study, we were able to demonstrate both CD4+ and CD8+ T-cell responses against NPM1mut. Ten peptides derived from wild-type NPM1 and NPM1mut were subjected to ELISPOT analysis in 33 healthy volunteers and 27 AML patients. Tetramer assays against the most interesting epitopes were performed and Cr51-release assays were used to show the cytotoxicity of peptide-specific T cells. Moreover, HLA-DR–binding epitopes were used to test the role of CD4+ T cells in NPM1 immunogenicity. Two epitopes (epitopes #1 and #3) derived from NPM1mut induced CD8+ T-cell responses. A total of 33% of the NPM1mut AML patients showed immune responses against epitope #1 and 44% against epitope #3. Specific lysis of leukemic blasts was detected. To obtain robust immune responses against tumor cells, the activation of CD4+ T cells is crucial. Therefore, overlapping (OL) peptides were analyzed in ELISPOT assays and OL8 was able to activate both CD8+ and CD4+ T cells. The results of the present study show that NPM1mut induces specific T-cell responses of CD4+ and CD8+ T cells and therefore is a promising target for specific immunotherapies in AML.

The interaction of the MHC class I-related chain molecules A and B (MICA and MICB) and UL-16 binding protein (ULBP) family members expressed on tumor cells with the corresponding NKG2D receptor triggers cytotoxic effector functions in NK cells and γδ T cells. However, as a mechanism of tumor immune escape, NKG2D ligands (NKG2DLs) can be released from the cell surface. In this study, we investigated the NKG2DL system in different human glioblastoma (GBM) cell lines, the most lethal brain tumor in adults. Flow cytometric analysis and ELISA revealed that despite the expression of various NKG2DLs only ULBP2 is released as a soluble protein via the proteolytic activity of “a disintegrin and metalloproteases” (ADAM) 10 and 17. Moreover, we report that temozolomide (TMZ), a chemotherapeutic agent in clinical use for the treatment of GBM, increases the cell surface expression of NKG2DLs and sensitizes GBM cells to γδ T cell-mediated lysis. Both NKG2D and the T-cell receptor (TCR) are involved. The cytotoxic activity of γδ T cells toward GBM cells is strongly enhanced in a TCR-dependent manner by stimulation with pyrophosphate antigens. These data clearly demonstrate the complexity of mechanisms regulating NKG2DL expression in GBM cells and further show that treatment with TMZ can increase the immunogenicity of GBM. Thus, TMZ might enhance the potential of the adoptive transfer of ex vivo expanded γδ T cells for the treatment of malignant glioblastoma.

The occasional familial occurrence of idiopathic nephrotic syndrome (NS) points to a genetic predisposition. Reports on associations with certain HLA class II antigens support this hypothesis. In order to define the immunogenetic background of NS more precisely, HLA class II allele frequencies in 161 children with NS were studied by restriction fragment length polymorphism (RFLP) typing. The patient cohorts consisted of 87 children from Southwest-France and 74 from Southwest-Germany. The control group consisted of 118 French and 101 German unrelated individuals from the same geographical areas. HLA alleles were defined in patients with steroid-sensitive (SS) and steroid-resistant (SR) NS and in controls. RFLP typing revealed that the previously reported association between SSNS and HLA-DR7 is confined to the RFLP split 7.1 (DRB1*07) with a combined relative risk (RRcomb) of 6.2. HLA-DQB typing showed an increased frequency of the allele DQB2b (DQB1*0201) (RRcomb = 7.8). HLA-DQA typing showed an association of SSNS with DQA3 (DQA1*0201,0301,0302) (RRcomb = 4.1). The highest RR (16.5) for SSNS was found in German patients who carried the two DRB1 specificities 17.1 (DRB1*0301) and 7.1 (DRB1*07). All associations were stronger in SS patients with frequent relapses or steroid dependency than in non- or infrequent relapsers. SR patients exhibited no significant associations with HLA class II alleles.

Thalidomide (Thal) is a drug with antiangiogenic, anti-inflammatory, and immunomodulatory properties that was found to inhibit the production of tumor necrosis factor-α (TNF-α) in vitro. We studied single nucleotide polymorphisms at positions −308 and −238 of the TNF-α gene promoter and measured the corresponding TNF-α cytokine levels in 81 patients (pts) with refractory and relapsed multiple myeloma (MM) who were treated with Thal. In myeloma pts carrying the TNF-238A allele (n = 8), we found a correlation with higher pretreatment TNF-α levels in peripheral blood (P = .047). After Thal administration, this TNF-238A group had a prolonged 12-month progression-free and overall survival of 86% and 100% versus 44% and 84% (P = .003 andP = .07) in pts with the TNF-238G allele, respectively. These findings suggest that regulatory polymorphisms of the TNF-α gene can affect TNF-α production and predict the outcome after Thal therapy, particularly in those MM pts who are genetically defined as “high producers” of TNF-α.

The polymorphic MICA (major histocompatibility complex class I chain-related gene A) (Gene ID: 100507436) gene products are a ligand of the activating natural killer cell receptor, NKG2D. Their clinical importance spans from solid organ transplantation to bone marrow transplantation and disease susceptibility. Typing of MICA genes by sequencing is hampered by an exon 5 short tandem repeat, the definition of which is critical for the final allelic and functional assignment. We present a novel sequencing approach, which uses group-specific (7T/8T) exon 5 polymerase chain reactions (PCRs) and facilitates hemizygous exon 5 MICA-PCR in approximately 70% of the tested individuals. With this method we typed the International Histocompatibility Workshop Group MICA reference panel (40 cell lines) as well as 110 healthy South German blood donors. All ambiguities, with the exception of MICA*008:01/008:04 (synonymous substitution in exon 1) and MICA*009:01/049 (nonsynonymous substitution in exon 6), could be resolved with our method. Analysis of Hardy-Weinberg equilibrium for our cohort showed no significant difference between expected and observed frequencies of MICA alleles (P = 0.6142). The three most frequent alleles in our blood donor cohort were MICA*008:01/008:04 (40.5%), MICA*002:01 (13.2%), and MICA*009:01/049 (8.6%). The 7T polymorphism was observed in 67.7% and the 8T polymorphism in 32.3% of our blood donor cohort. Individuals (24.5%) tested were homozygous. The approach described in this paper is suitable for accurate sequencing of large sample numbers, including direct readout of exon 5 sequences. It is compatible with laboratory automation and commercial human leukocyte antigen analysis software tools. It may therefore be applied in large clinical trials.

Recipients (4076) and donors (3325) of kidney transplants performed at 110 transplant centers were typed for HLA-DRB by the DNA RFLP method. The discrepancy rate of replicate samples distributed among 8 participating laboratories was a low 2.6%. The discrepancy rate between RFLP-DRB and serological HLA-DR typings was 25.0% for organ donors and 27.6% for kidney recipients. Discrepancy rates at the different transplant centers ranged from 9.7% to 86.7%. The discrepancies consisted of antigens being incorrectly interpreted by serology (16.8%), and of serological "blanks" turning out to be definable alleles by the DNA method (10.8%). The alleles that were mainly affected by discrepancies were DR1, DR8, DR10, DR12, DR13, DR14, DR16, DR17.2, and DR18.

Human leukocyte antigen (HLA)-DRB1*1454 differs from HLA-DRB1*1401 in only one position in exon 3. Before description of this polymorphism both alleles were routinely typed as HLA-DRB1*1401. This prompted our study on relative frequency of these alleles. We determined relative frequencies of DRB1*1454 and DRB1*1401 by sequence-based tying (SBT) in 106 samples and found DRB1*1454 in 87.9% and DRB1*1401 in 12.1% of previously DRB1*1401/54 ambiguous tested individuals. Population frequencies were estimated using data from the local bone marrow donor database. Phenotype and genotype frequency of DRB1*1454 and DRB1*1401 were 5.592%, 0.2828, and 0.773%, 0.00391, respectively. The corresponding figures for DRB1*1404 were 0.238% and 0.00119. Other DRB1*14 alleles were very rare. The most frequent haplotype was DRB1*1454-DRB3*0202-DQB1*0503. Although DRB1*1454 is almost exclusively associated with DRB3*0202, DRB1*1401 is linked with either DRB3*0201 or DRB3*0202. HLA-DRB1*1454 is the most common DRB1*14 allele among German Caucasians and should be considered as a preferred allele over DRB1*1401.

The disruption of disulfide bridges can decrease or abolish the cell surface expression of HLA class I molecules. Such disulfide bridges are formed by cysteine residues between amino acid (aa) positions 101/164 (alpha(2) domain) and 203/259 (alpha(3) domain). Sequence alterations in codons 101, 164, 203 and 259 have been observed in eleven HLA-A molecules. All of these variants except of A*3014L and A*3211Q have been reported to result in null expression alleles. In the case of HLA-A*3014L, a transversion at nucleotide position 563 replaces cysteine by serine at position 164 of the mature polypeptide. HLA-A*3014L is not detectable by standard microlymphocytotoxicity assay. To verify low or non-expression of this allele, we cloned soluble HLA-A*3014L and the reference allele HLA-A*3001 into a eukaryotic expression vector and transfected K562, C1R and HEK293 cells. Expression of soluble HLA-A*3014L and HLA-A*3001 was measured in the supernatants of transfected and untransfected cells incubated with or without IFN-gamma and/or TNF-alpha using a W6/32 and anti-beta(2)-microglobulin-based sandwich ELISA. Expression of mRNA transcripts of both alleles was determined by real-time RT-PCR. HLA-A*3014L was not detected in the supernatant of unstimulated transfectants. Stimulation with IFN-gamma and/or TNF-alpha led to an increase of HLA-A*3014L secretion to a detectable level and increased HLA-A*3001 expression up to 8-fold, but did not show any difference in the increase of mRNA levels between HLA-A*3014L and A*3001. Because of this lack of any difference in the mRNA transcription, the protein expression defect is most likely caused by the missing disulfide bond formation in the alpha2 domain. Thus, exposing the cells to cytokine stress allows to distinguish between low- and non-expressed alleles and to classify alleles with a questionable expression pattern (Q alleles). Classifying HLA alleles in expressed and non-expressed variants is essential for matching assessments. Additionally, this discrimination between cytokine inducible and non-inducible defect alleles may be important in allotransplant settings in which a cytokine storm usually occurs following pre-transplant myeloablative conditioning or post-transplant immunosuppressive therapy.

Cytokines are important immunomodulatory molecules involved in immune responses against microorganisms; they also have an important role in the setting of immune system disorders. Cytokine single nucleotide polymorphisms have been extensively studied in different, normal populations as well as in association with disease. Cytokine gene polymorphisms are potentially important as genetic predictors of disease susceptibility, clinical outcome, and as a tool for anthropological studies. In this study, samples have been collected from 455 healthy individuals located in different regions of Iran (Tehran, Yazd, Sistan and Balochistan). Allele and genotype frequencies of cytokine SNP, including: IL-1alpha, IL-1beta, IL-1R, IL-1RA, IL-2, IL-4, IL-4RA, IL-6, IL-10, IL-12, TNF-alpha, TGF-beta and IFN-gamma were investigated, using the PCR-SSP method. Allele frequencies in Tehran and Yazd populations were similar, except for TGF-beta. Allele frequencies in SistaniBaloch populations were similar at all positions, except for IL-1beta at position of -511 and IFN-gamma genes at position UTR5644; there were some differences in allele frequencies comparing these populations with the Yazd population, including: IL-4, IL-6, IL-10, TGF-beta and TNF-alpha. Although some significant differences were observed for some cytokines, it seems that the cytokine gene polymorphism profile of the Iranian population is similar to that of Caucasians, particularly the Italian population.

The effect of NK-KIR alloreactivity on HSCT outcome, although established for myeloid malignancies, has not been confirmed for lymphoid leukemic disorders. Lower expression of KIR ligands on lymphoid malignant cells may account for the differences observed between the two disease groups as to a possible “KIR effect”. The aim of this study was to investigate the role of HLA-E as non-KIR NK-ligand in an ALL HSCT context. 143 ALL patients (P) undergone 10/10 HLA matched unrelated HSCT (MUT) and their donors (D) were HLA-E genotyped by sequence based typing. Two alleles (01:01, 01:03) and three genotypes (01:01/01:01, 01:01/01:03 and 01:03/01:03) were identified and assigned accordingly. The effect of P and D HLA-E genotype as well as of P–D HLA-E match grade was assessed using univariate Kaplan–Meier (KM), multivariate Cox regression and competing risks analyses. OS (overall survival), DFS (disease free survival), RI (relapse incidence) and TRM (transplantation related mortality) were set as endpoints and statistical significance was set to a p p = 0.017) in the HLA-E MM compared to the HLA-E M pairs. Multivariate Cox regression analysis confirmed these results for both OS (HR = 0.48, CI = 0.27–0.86, p = 0.015) and DFS (HR = 0.6, CI = 0.37–0.99, p = 0.047). RI was also lower in HLA-E MM pairs (25% vs 30%) but did not reach statistical significance. P as well as D HLA-E genotypes showed no effect on the study endpoints. Our results indicate an association between HLA-E MM and prognosis of ALL patients after MUT. A plausible explanation could be a potentially enhanced GVL effect mediated both by NK and NK-CTLs, however, in vitro studies are required in order to define the underlying biological mechanisms. In conclusion, our study is the first so far to provide information regarding the role of HLA-E matching in ALL patients undergoing HSCT. These initial findings, however, have to be confirmed by larger cohort studies before further conclusions can be drawn.

Twenty-four common single nucleotide polymorphisms (SNPs) in 10 cytokine and chemokine genes were defined in 579 North Americans at high risk of HIV-1 infection due to sexual behavior and injection drug use. Among the 3 major ethnic (African-American, Hispanic/Latino, and other) groups involved, HIV-1-seropositive individuals differed significantly from ethnically matched HIV-1-seronegative individuals (odds ratios = 2.13-4.82; P = 0.003-0.05) for several SNPs and haplotypes defined at the IL4, IL4R, IL6, IL10, CCL5 (RANTES), and CXCL12 (SDF1) loci. In addition, the homozygous IL4-590T/T genotype was associated with higher (+87-131 cells/microL) CD4 T-cell counts in HIV-1-infected and AIDS-free adolescents not receiving antiretroviral therapy (adjusted P = 0.004). No SNPs at IFNG, IL2, IL12B, TNF, or CCL2 (MCP1) showed any association with HIV-related outcomes. Additional typing for IL1A, IL1B, IL1R1, IL1RN, and TGFB1 SNPs also failed to demonstrate any influence on HIV-1 infection or virologic/immunologic control in more selected patient groups. Coupled with previous findings, our data suggest that heritable IL4 and IL10 variations may contribute to the acquisition or progression of HIV infection and that the effects of other targeted loci in the cytokine and chemokine system cannot be established unequivocally in the study populations.

It has been suggested that increased monocyte responses might play a role in chronic allograft rejection.We investigated in vitro monokine responses in 112 patients with long-term stable kidney graft function (ST patients; n=80, non-mycophenolate mofetil [MMF]; n=32, MMF) and 25 patients with chronic renal transplant rejection (CR patients; non-MMF). Interleukin 10 and tumor necrosis factor (TNF)-alpha promoter gene polymorphisms were tested by polymerase chain reaction and sequence-specific primers; antigen-presenting capacity (AC) of monocytes was tested by incubation with staphylococcal superantigens (SEA, SEE, SED).Although non-MMF-based immunosuppression in ST patients did not result in compromised AC or lipopolysaccharide (LPS)-stimulated monokine responses compared with healthy controls, we found MMF therapy to be associated with significantly reduced TNF-R1 expression on monocytes (P0.001), suppressed AC (P0.02, SED), and suppressed LPS-stimulated IL-1 beta, IL-10, and TNF-alpha secretion (P0.01). Coinciding with a significantly higher steroid dosage in CR patients, IL-6 receptor and TNF-R1 expression on monocytes were down-regulated (Por =0.02) and AC was suppressed in CR compared with ST (non-MMF) patients (P0.01, SED; P0.05, SEE). However, LPS-stimulated monokine secretion was not decreased or even enhanced (IL-6, granulocyte-macrophage colony-stimulating factor [GM-CSF]; P0.05). Enhanced in vitro IL-10 responses (500 pg/mL) were found predominantly in non-MMF-treated patients with the IL-10 genotype GCC (GCC: 23/62 [37%], non-GCC: 2/27 [7%], P0.005; GCC and non-MMF: 22/47 [47%], GCC and MMF: 1/15 [7%], P0.005].Steroids and azathioprine did not sufficiently suppress monokine responses, whereas MMF treatment might inhibit chronic graft rejection because of suppression of TNF-R1 expression and vigorous inhibition of monokine secretion. MMF treatment may especially be indicated in patients with the IL-10 "high-producer" genotype GCC.

Persistent infection with human papillomavirus (HPV), particularly HPV16, represents the prime risk factor in cervical carcinogenesis. HPV variants ( e.g. , within the E6 gene) together with immunogenetic factors of the host may be responsible either for effective viral clearance, or alternatively, for viral persistence. Peripheral blood from 27 HPV16 positive Swedish women with cervical carcinoma, who had previously been tested for HPV16 E6 variants, was used for human leukocyte antigen (HLA) class I typing. Women with HLA-B*44, HLA-B*51, or HLA-B*57 who were infected with the HPV16 E6 variant L83V had an approximately four- to fivefold increased risk for cancer compared with controls (odds ratio [OR] = 3.5, 95% CI = 1.1–11.1, OR = 4.2, 95% CI = 1.19–14.69, or OR = 4.67, 95% CI = 1.2–18.6, respectively). Epitope predictive algorithm with SYFPEITHI revealed that the variant at amino acid 83 affects the binding affinity in association with HLA-B*44. Interestingly, the HLA-B*15 allele seems protective because it was absent in HPV16 positive cancer. It is concluded that specific HLA class I alleles, combined with certain HPV16 E6 variants, may be crucial for immune surveillance in cervical carcinogenesis. The evaluation of associations of HLA alleles with HPV variants may be helpful in defining prognostic markers and in designing vaccines capable of mediating immune protection against HPV infection.

Polymerase chain reaction with sequence specific primers subtyping of a DRB1*15 allele in a male 11-year-old German Caucasian and his father revealed a reaction pattern that did not agree with any known specificity, thus suggesting the existence of a novel allele. Sequence-based typing after allelic separation revealed the new allele human leukocyte antigen (HLA)-DRB1*15014. The sequence pattern of HLA-DRB1*15014 might have been generated as a result of two independent recombinations, most likely over several generations.

In a previous study, DNA typing revealed that 25% of serological HLA-DR typings of kidney transplants were incorrect. In the current study, we analyzed whether this error rate had improved in recent years, and whether there were differences according to geographical region. From 1988 to 1991 the error rate of serological typing improved slightly in Western Europe from 19% to 16%, and in North America, from 21% to 16%. In Eastern Europe, the error rate decreased from 49% to 33% in 1991, whereas the rate remained high in South America at 60% in 1988 and 72% in 1991. The high error rates in South America and Eastern Europe reflected a lack of good quality serological typing reagents. The 16% typing errors in Western Europe and North America demonstrated the current limit of serological techniques for cadaver donor typing and underlined the need for prospective DNA typing.