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Rationale: Pseudomonas aeruginosa (PA) is an environmental pathogen that commonly infects individuals with cystic fibrosis (CF) and non-CF bronchiectasis, impacting morbidity and mortality. To understand the pathobiology of interactions between the bacterium and host adaptive immunity and to inform rational vaccine design, it is important to understand the adaptive immune correlates of disease. Objectives: To characterize T-cell immunity to the PA antigen outer membrane porin F (OprF) by analyzing immunodominant epitopes in relation to infection status. Methods: Patients with non-CF bronchiectasis were stratified by frequency of PA isolation. T-cell IFN-γ immunity to OprF and its immunodominant epitopes was characterized. Patterns of human leukocyte antigen (HLA) restriction of immunodominant epitopes were defined using HLA class II transgenic mice. Immunity was characterized with respect to cytokine and chemokine secretion, antibody response, and T-cell activation transcripts. Measurements and Main Results: Patients were stratified according to whether PA was never, sometimes (

Studies of the effect of minor H antigen mismatching on the outcome of renal transplantation are scarce and concern mainly single center studies. The International Histocompatibility and Immunogenetics Workshops (IHIW) provide a collaborative platform to execute crucial large studies. In collaboration with 16 laboratories of the IHIW, the role of 15 autosomal, 10 Y-chromosome encoded minor H antigens and 3 CD31 polymorphisms, was investigated in relation to the incidence of renal graft rejection and graft loss in 444 human leukocyte antigens (HLA)-identical sibling renal transplantations. Recipient and donor DNA samples were genotyped for the minor H antigens HA-1, HA-2, HA-3, HA-8, HB-1, ACC-1, ACC-2, SP110, PANE1, UGT2B17, C19Orf48, LB-ECGF-1, CTSH, LRH-1, LB-ADIR and HY. The correlation between minor H antigen mismatch and the primary outcome graft rejection or graft loss was statistically analyzed. The incidence of rejection was very low and no correlation was observed between one or more minor H antigen mismatch(es) and a rejection episode (n = 36), of which only eight resulted in graft loss. In summary, in our study cohort of 444 renal transplants, mismatching for neither autosomal nor HY minor H antigens correlate with rejection episodes or with graft loss.

Natural killer (NK) cells are expanded in chronic myeloid leukemia (CML) patients on tyrosine kinase inhibitors (TKI) and exert cytotoxicity. The inherited repertoire of killer immunoglobulin-like receptors (KIR) may influence response to TKI. We investigated the impact of KIR-genotype on outcome in 166 chronic phase CML patients on first-line imatinib treatment. We validated our findings in an independent patient group. On multivariate analysis, KIR2DS1 genotype (RR=1.51, P=0.03) and Sokal risk score (low-risk RR=1, intermediate-risk RR=1.53, P=0.04, high-risk RR=1.69, P=0.034) were the only independent predictors for failure to achieve complete cytogenetic response (CCyR). Furthermore, KIR2DS1 was the only factor predicting shorter progression-free (PFS) (RR=3.1, P=0.03) and overall survival (OS) (RR=2.6, P=0.04). The association between KIR2DS1 and CCyR, PFS and OS was validated by KIR genotyping in 174 CML patients on first-line imatinib in the UK multi-center SPIRIT-1 trial; in this cohort, KIR2DS1(+) patients had significantly lower 2-year probabilities of achieving CCyR (76.9 vs 87.9%, P=0.003), PFS (85.3 vs 98.1%, P=0.007) and OS (94.4 vs 100%, P=0.015) than KIR2DS1(-) patients. The impact of KIR2DS1 on CCyR was greatest when the ligand for the corresponding inhibitory receptor, KIR2DL1, was absent (P=0.00006). Our data suggest a novel role for KIR-HLA immunogenetics in CML patients on TKI.

Survival after volunteer unrelated donor (VUD) stem cell transplantation (SCT) is influenced by matching for human leucocyte antigens (HLA). We analysed the effects of serological and molecular typing at HLA-A, -B, -C and -DRB1 in 100 patient/VUD pairs from a single transplant centre. Patients received SCT for good risk [chronic myeloid leukaemia in first chronic phase (CML-CP1), n = 55] or poor risk ( n = 45) diseases after myeloablative conditioning and T-cell depletion with alemtuzumab. By serological typing, 70 pairs were fully matched, whereas molecular typing revealed 10 pairs with additional mismatches. The day 100 transplant related mortality was 15%. Acute graft versus host disease (GvHD) grades III–IV occurred in 11%, whilst extensive chronic GvHD in 13% of evaluable patients. There was no statistical difference in GvHD rates between patients who received grafts from fully matched or from mismatched donors. In univariate analysis the disease risk group and CMV seronegativity of recipient and donor were the only significant predictors for survival, with 3-year survival probabilities of 71.2% for CML-CP1 and 28% for poor risk diseases. In the poor risk group, HLA mismatches had a negative impact on survival ( p = 0.003) and progression free survival ( p = 0.009) contrary to CML-CP1 patients, in whom HLA mismatches at molecular or serological level did not have any impact.

Background Epidemiological studies describe a latitude gradient for increased MS prevalence and a preponderance of disease in Caucasian individuals. However, individuals from other ethnic backgrounds and low-risk regions can acquire a raised risk through migration. Nearly a fifth of the London population is of Asian/Asian-British origin and a significant proportion of referrals are from this group. Methods We investigated whether there were differences in timing, presentation, severity, and immunology of disease (with respect to CD4 myelin epitope recognition) between individuals in London with MS who were either of S. Asian or Caucasian origin. Individuals of S. Asian origin with MS were compared with healthy S. Asian controls, individuals with MS and of Caucasian origin and Caucasian controls. Results Age at MS onset is significantly lower in the S. Asian group, attributable to earlier onset specifically in UK-born individuals, though clinical presentation is similar. Analysis of CD4 autoimmunity to myelin antigens shows disease in S. Asian individuals to encompass recognition of novel epitopes; immunity to MBP116-130 in S. Asian individuals was highly disease-specific. Conclusions These findings emphasize the need to define disease profiles across ethnicities and identify environmental triggers conferring acquired risk. Such findings must inform choices for immunotherapeutic interventions suitable for all, across ethnicities. Electronic supplementary material The online version of this article (doi:10.1186/s12883-015-0324-2) contains supplementary material, which is available to authorized users.

Annotation of top-ranked DMPs using epigenomic reference datasets. The genomic locus on chromosome 14q24.2 (position = 70,261,006–70,349,114; genome build = hg19) harboring the top-ranked DMR is shown using the WashU Epigenome Browser v40.0.0 ( http://epigenomegateway.wustl.edu/browser/ ). The top-ranked DMR containing CpG classifiers of aGVHD severity (Table 2) is located at a CpG island (position = 70,316,847–70,317,240; indicated with an orange arrow). RefSeq and Gencode v17 genes, as well as CpG islands, are shown in the bottom panel of the figure. A total of 50 epigenomic reference tracks provided by the NIH Roadmap Epigenomics Project are displayed. Specifically, we show both the primary and imputed chromatin state maps in 22 primary hematopoietic cell types. The highlighted DMR overlaps with an active transcription start site (red) or poised promoter (pink) in G-CSF-mobilized CD34+ hematopoietic stem cells, and a Polycomb-repressed region in CD3+ T cells of peripheral blood. (PDF 960 kb)

Ontology annotation of genes flanking DMRs using GREAT. We report the ontology of genes flanking the 453 identified DMRs. Specifically, we indicate all enriched terms in the binomial test over genomic regions at an FDR of less than 5Â %. (PDF 107 kb)

Quality assessment of the Illumina Infinium HumanMethylation450 assay. The quality of the 450K array data was assessed after normalization, probe filtering, and batch correction (for details see Methods). (a) Density of DNA methylation β-values. (b) Multidimensional scaling (MDS) indicates the similarities and differences of samples by calculating the Euclidean distances between samples based on all CpG sites, and then projecting these distances into 2D coordinates. We found Dimension 4 to associate with aGVHD severity, accounting for 3 % of the total variance. HSCT donors matched to healthy recipients and those matched to recipients diagnosed with mild aGVHD could not be stratified using MDS. Therefore, these two sample groups were combined for subsequent analysis. (c) Singular value decomposition (SVD) determines the nature of the largest components of variation (Teschendorff AE et al. PLoS One. 2009;4:e8274). We assessed the first six principal components (PCs), and correlated these to phenotypic factors of donors (e.g., sex, age at transplant, and CMV serostatus), phenotypic factors of recipients (e.g., aGVHD status and severity), factors related to the experimental setup (e.g., Sentrix ID, sample plate, and sample well), as well as internal control parameters (e.g., bisulfite conversion efficiency). DNA methylation age (‘DNAm age’) was predicted based on the raw DNA methylation data using the DNA Methylation Age Calculator ( https://dnamage.genetics.ucla.edu/ ), as described by Horvath (Horvath S. Genome Biol. 2013;14:R115). The phenotypic factor ‘transplant date’ denotes the year of the day of the graft transplant (day 0). We found PC4 and PC5 to most strongly correlate with aGVHD severity, achieving a significance level of P

Box-and-whisker plots of DNA methylation values in graft donors in the discovery cohort assessed using MethyLight and 450K arrays. Only HSCT donors of the discovery cohort that were profiled on both assay platforms are shown. We identified a DNA hypomethylation phenotype at the top-ranked DMP cg20475486 in graft donors matched to recipients with severe aGVHD. HSCT donors matched to healthy recipients and those matched to recipients diagnosed with mild aGVHD could not be discriminated. (PDF 256 kb)

Detailed characterization of the protective T-cell response in salmonellosis is a pressing unmet need in light of the global burden of human Salmonella infections and the likely contribution of CD4 T cells to immunity against this intracellular infection. In previous studies screening patient sera against antigen arrays, SseB was noteworthy as a serodominant target of adaptive immunity, inducing significantly raised antibody responses in HIV-seronegative compared with seropositive patients. SseB is a secreted protein, part of the Espa superfamily, localized to the bacterial surface and forming part of the translocon of the type III secretion system (T3SS) encoded by Salmonella pathogenicity island 2. We demonstrate here that SseB is also a target of CD4 T-cell immunity, generating a substantial response after experimental infection in human volunteers, with around 0·1% of the peripheral repertoire responding to it. HLA-DR/peptide binding studies indicate that this protein encompasses a number of peptides with ability to bind to several different HLA-DR alleles. Of these, peptide 11 (p11) was shown in priming of both HLA-DR1 and HLA-DR4 transgenic mice to contain an immunodominant CD4 epitope. Analysis of responses in human donors showed immunity focused on p11 and another epitope in peptide 2. The high frequency of SseB-reactive CD4 T cells and the broad applicability to diverse HLA genotypes coupled with previous observations of serodominance and protective vaccination in mouse challenge experiments, make SseB a plausible candidate for next-generation Salmonella vaccines.

The effect of minor H antigen mismatching on the occurrence of graft-versus-host disease (GvHD) and graft-versus-leukemia (GvL) after HLA-matched hematopoietic stem cell transplantation (HSCT) has mainly been demonstrated in single-center studies. Yet, the International Histocompatibility and Immunogenetics Workshops (IHIW) provide a collaborative platform to execute crucial large studies. In collaboration with 20 laboratories of the IHIW, the roles of 10 autosomal and 10 Y chromosome-encoded minor H antigens were investigated on GvHD and relapse incidence in 639 HLA-identical related donor (IRD) and 210 HLA-matched unrelated donor (MUD) HSCT recipients. Donor and recipient DNA samples were genotyped for the minor H antigens HA-1, HA-2, HA-3, HA-8, HB-1, ACC-1, ACC-2, SP110, PANE1, UGT2B17, and HY. The correlations with the primary outcomes GvHD (acute or chronic GvHD), survival, and relapse were statistically analyzed. The results of these multicenter analyses show that none of the HLA class I-restricted HY antigens were found to be associated with any of the primary outcomes. Interestingly, of the HLA class II-restricted HY antigens analyzed, HLA-DQ5 positive recipients showed a significantly increased GvHD-free survival in female-to-male HSCT compared with male-to-female HSCT (P= .013). Yet, analysis of the overall gender effect, thus independent of the known HY antigens, between the gender groups demonstrated an increased GvHD incidence in the female-to-male transplantations (P < .005) and a decreased GvHD-free survival in the female-to-male transplantations (P < .001). Of all autosomally encoded minor H antigens, only mismatching for the broadly expressed minor H antigen HA-8 increased the GvHD incidence in IRD HSCT (Hazard ratio [HR]= 5.28, P < .005), but not in MUD HSCT. Most striking was the influence of hematopoietic restricted minor H antigens on GvL as mismatching for hematopoietic minor H antigens correlated with lower relapse rates (P= .078), higher relapse-free survival (P= .029), and higher overall survival (P= .032) in recipients with GvHD, but not in those without GvHD. In conclusion, the significant GvHD effect of the broadly expressed minor H antigen HA-8 favors matching for HA-8 in IRD, but not in MUD, patient/donor pairs. The GvHD-GvL association demonstrating a significant lower relapse in hematopoietic minor H antigen mismatched patient/donor pairs underlines their clinical applicability for adoptive immunotherapy, enhancing the GvL effect in a GvHD controllable manner. © 2013 American Society for Blood and Marrow Transplantation.

Although the feasibility of using HLA-mismatched unrelated donors as an alternate graft source for haematopoietic SCT (HSCT) has been shown, little is known about the safety of HLA-mismatched DLI for the treatment of relapse. We examined the outcome of 58 consecutive leukaemia patients who received escalating-dose DLI for treatment of relapse after alemtuzumab-conditioned myeloablative unrelated donor HSCT at our institution. High-resolution HLA typing on stored DNA samples revealed mismatches in 28/58 patients who were considered HLA-matched at the time of transplantation. Following DLI from HLA-matched (10/10) (n=30) or -mismatched (7-9/10) (n=28) unrelated donors, we found no significant difference in the incidence of acute GVHD (17.2% versus 23.1%, P=0.59), probability of remission at 3 years (62.1% versus 63.9%, P=0.89) or 5-year OS (89.8% versus 77.7%, P=0.22). We conclude that escalating-dose DLI can be safely given to HLA-mismatched recipients following T-depleted myeloablative HSCT.

The importance of human leukocyte antigen (HLA) matching in renal transplantation is well recognized, with HLA-DR compatibility having the greatest influence. De novo DQ donor-specific antibodies (DSAbs) are the predominant HLA class II DSAb after transplantation. The aim of this study was to establish the incidence and outcomes after the development of DQ DSAbs along with the impact of class II HLA mismatch on their development.We retrospectively analyzed 505 patients who received a renal-alone transplant between 2005 and 2010. We excluded patients who received an ABO- and HLA-incompatible allograft, which we defined as those with a positive crossmatch or preformed DSAbs detected by single-antigen beads only.Of 505 patients, 92 (18.2%) developed DSAbs, with 50 (54.3%) of these 92 patients having DQ DSAbs. Patients who developed DQ DSAbs were at significant risk for antibody-mediated rejection, transplant glomerulopathy, and allograft loss (P0.0001). Of 505 patients, 108 (21.4%) were matched at both the DR and DQ loci, 284 (56.2%) were mismatched at both loci, 38 (7.5%) were matched at DR alone, and 75 (14.9%) were matched at DQ alone. Patients mismatched at both DR and DQ were at risk for developing class II DSAbs when compared with those mismatched at either DR or DQ alone, P=0.001, and were at risk for antibody-mediated rejection, P=0.001.DQ DSAbs are associated with inferior allograft outcomes. This study shows the importance of establishing the DQ match before transplantation to define immunologic risk.

Given that it is possible to extract DNA from the urine of kidney transplant donors and recipients we studied whether the donor HLA type can be determined from recipient urine. This would be useful especially when there is limited information on donors or when the transplant was performed long ago when tissue typing was less precise. We extracted and purified DNA from fresh urine and used the standard HLA class I and class II PCR-SSP assays comparing the findings to those obtained from peripheral blood of donor and recipient HLA types. Using the urine of 31 renal transplant recipients we assayed for the 140 known mismatches, and all were detected in technically successful assays with only a single false positive. This shows that urine samples of transplant recipients can be used to generate historical HLA typing information of the donor thereby aiding post-transplant immunologic monitoring.

To the editor: Most chronic myeloid leukemia (CML) patients achieve complete cytogenetic response (CCyR) with tyrosine kinase inhibitors (TKI).[1][1] However, many relapse on therapy discontinuation.[2][2] The curative effect of allogeneic stem cell transplantation (allo-SCT) in CML is believed to

Abstract 888 Natural killer (NK) cells are expanded in patients with chronic myeloid leukemia (CML) on tyrosine kinase inhibitors (TKI) and exert cytotoxicity against CML cells. NK cytotoxicity is determined by the balance between inhibitory and activating signals from cell surface killer immunoglobulin-like receptors (KIRs). The inherited repertoire of KIR genes may therefore influence susceptibility to treatment and prognosis in CML patients treated with TKI. To determine if innate immunity is associated with CML susceptibility and response to TKI, we investigated the impact of KIR genotype on the outcome of 166 consecutive patients with Philadelphia+ CML in chronic phase (CP) who received imatinib as first line treatment in our institution. The median follow up was 36 months and 75% of the patients were followed for a minimum of 24 months. One hundred and nineteen patients (71.7%) achieved CCyR, 10 (6.0%) progressed to advanced phase disease and 7 (4.2%) died. The 2-year cumulative incidence of CCyR was 72.2% and the 2 year probability of PFS and OS were 95.7% and 97.3% respectively. The presence of 2 KIR genes, namely K2DL5A and KIR2DS1, was associated with a significantly lower 2 year probability of CCyR (78.8% vs 63.2%, p=0.04 and 82.3% and 65.6%, p=0.03) respectively, PFS (98.9% vs 91.5%, p=0.02 and 98.4% vs 91.0%, p=0.01 respectively) and OS (100% vs 93.9%, p=0.02 and 100% vs 92.6% p=0.03 respectively). On multivariate analysis, the presence of KIR2DS1 (RR=0.66, p=0.03) and Sokal risk score (low risk RR=1, intermediate risk RR= 0.65, p= 0.04 and high risk RR= 0.59, p=0.034) were the only independent predictors for the achievement of CCyR. Furthermore, on multivariate analysis, the presence of KIR2DS1 was the only independent predictor for both PFS (P=0.02) and OS (P=0.03). In order to validate our results we explored the influence of the KIR genotype on achievement of CCyR and PFS and OS in a second independent patient group: KIR genotype was determined in 174 patients with CML-CP treated with first line imatinib within the multi-center STI571 Prospective International Randomised Trial (SPIRIT). The 65 patients who were KIR2DL5A positive had a significantly lower 2 year probability of achieving CCyR, PFS and OS than the 106 patients who were negative for this gene, namely 80.4% vs 86.8 (p=0.04), 85.7% vs 98.1% (p=0.01) and 94.3% vs 100% (p=0.02) respectively. Similarly, the 66 patients who were KIR2DS1 positive had a lower probability of achieving CCyR and lower PFS and OS than the 106 patients who did not have the gene (76.9% vs 87.9%, p=0.004, 85.3% vs 98.1% p=0.01 and 94.4% vs 100%, p=0.02 respectively). On multivariate analysis of this patient cohort KIR2DS1 remained the only independent predictor for the three outcomes. When patients were categorized based on their Sokal risk score and KIR2DS1 genotype, the most significant difference in CCyR, PFS and OS was observed in the 77 patients who had a high Sokal risk score and those with an intermediate Sokal risk score who were KIR2DS1 positive compared to the 89 patients who had a low Sokal risk score and those with an intermediate Sokal risk score who were KIR2DS1 negative; namely, 82.4% vs 59.3% (p=0.002), 92.1% vs 98.8% (p=0.03) and 94.2% vs 100% (p=0.03) and respectively (Figure 1). The mechanism by which KIR2DS1 predicts for a sub-optimal response to therapy is unknown. KIR2DS1+ve NK cells have been shown to secrete transforming growth factor-beta (TGF-beta) upon interaction with their ligand. TGF-b was recently shown to inhibit Akt signalling, a suppressor of the forkhead O transcription factor, FOXO3a, in the CML leukaemia-initiating cells (LICs) and may represent an important mechanism for the CML LIC to survive imatinib. Alternatively, KIR2DS1 may be simply a surrogate marker for another neighboring gene that is directly involved in pathogenesis of CML. In conclusion, our data demonstrate that KIR immunogenetics represent a novel prognostic tool for patients with CML-CP on TKI, and that KIR2DS1 positivity may predict response to imatinib and identify patients at greater risk of treatment failure. Functional and phenotypic studies to determine the expression of KIR2DS1 on the surface of NK cells and to assess the role of the cytokine milieu and the NK phenotype on outcome are currently underway. Figure 1 CCyR and OS according to the expression of K2DS1 genotype and Sokal score Figure 1. CCyR and OS according to the expression of K2DS1 genotype and Sokal score Disclosures: No relevant conflicts of interest to declare.

Aim: Outcome of unrelated donor stem cell transplantation (SCT) is influenced by matching for HLA class I and II. We investigated the effects of HLA typing on acute and chronic graft-versus-host disease (aGvHD and cGvHD) and overall survival (OS) in adult patients/donors typed for HLA-A, B, Cw, DRB1, DPB1 at high resolution (HR) and low resolution (LR). Patients: 100 patients (pts.)(median age 32 y, 17 – 54) were treated with allogenic SCT for diseases: 55 Ph-positive chronic myeloid leukaemia in first chronic phase (CML CP1), 21 CML in advanced phase (CML AP) and 24 acute leukaemias (AL). Pre-transplant conditioning consisted in 98 pts of total body irradiation (TBI) and cyclophosphamide (91) or etoposide (7) and in 2 pts of cyclophosphamide and busulphan. In all pts. GvHD prophylaxis was provided by cyclosporin A and short-term methotrexate with in vivo T-cell depletion (TCD) by CAMPATH 1H (Anti CD52). Results: With LR typing 68 pts./donors were fully matched but 32 had class I (31) or class II (1) mismatches (MisM). HR typing revealed 9 pts with additional class I (7) or II (7) MisM, thus 59 pts./donors were fully matched. AGvHD grade III–IV occured in 11 pts (11%) whilst extensive cGvHD was seen in 12 pts (14% out of 83). There was no statistical difference in the occurrence of aGvHD or cGvHD between fully matched and mismatched pts./donors at LR or HR. The probability of transplant-related mortality (TRM) of the whole cohort was 15% at day 100 and 23% at day 180 and engraftment failure 1%. The 3-year survival probabilities were significantly different (p=0.004) for pts. with CML CP1 at 63% (good risk group) when compared with CML AP and AL at 23% (poor risk group). In multivariate analysis patients age, diagnosis group and negative CMV status of patient and donor were the only significant predictors for survival. In the entire group the full compatibility at LR or HR for class I or class II or for a single HLA antigen or the KIR ligand mismatch in the GvHD direction did not have an impact on survival. In contrast to the CML CP1 group (n=55) we found that in the poor risk group (n=45) a MisM at HR had a negative impact on survival (p=0.03) and a trend towards worse survival was found for patients with HLA B HR mismatch. Patients with more than one HR MisM had a worse survival when the combination of HLA B and HLA D MisM was present (p=0.008). Conclusion: In 100 pts. treated with unrelated SCT and in vivo TCD with CAMPATH 1H the HR typing did have an impact on survival only in the poor risk group but not in pts with CML CP1. Thus, T-cell depletion with in vivo CAMPATH 1H permits the use of mismatched unrelated donors in patients with CML CP. The overall rates for aGvHD and cGvHD were low compared to published data in non TCD SCT. Further investigations on larger cohorts are needed to elucidate the impact of HR typing in unrelated SCT and the significance of HR MisM at single HLA loci in transplants T-cell depleted with in vivo CAMPATH 1H.

PCR primers and probes used in MethyLight replication experiments. A total of three MethyLight reactions were designed, which targeted top-ranked DMPs associated with aGVHD severity: cg10399005, cg20475486, and cg07280807. Of these reactions, cg20475486 achieved the highest PCR efficiency and was subsequently used in validation experiments (highlighted in gray). Start and end positions of primer and probes are noted in relation to the design start coordinates. Chromosomal positions are reported in genome buildâ =â hg19. (PDF 110 kb)

PCR primers and probes used in MethyLight replication experiments. A total of three MethyLight reactions were designed, which targeted top-ranked DMPs associated with aGVHD severity: cg10399005, cg20475486, and cg07280807. Of these reactions, cg20475486 achieved the highest PCR efficiency and was subsequently used in validation experiments (highlighted in gray). Start and end positions of primer and probes are noted in relation to the design start coordinates. Chromosomal positions are reported in genome buildâ =â hg19. (PDF 110 kb)