Melioidosis, caused by Burkholderia pseudomallei, is a potentially lethal infection with no licensed vaccine. There is little understanding of why some exposed individuals have no symptoms, while others rapidly progress to sepsis and death, or why diabetes confers increased susceptibility. We prospectively recruited a cohort of 183 acute melioidosis patients and 21 control subjects from Northeast Thailand and studied immune parameters in the context of survival status and the presence or absence of diabetes. HLA-B*46 (one of the commonest HLA class I alleles in SE Asia) and HLA-C*01 were associated with an increased risk of death (odds ratio 2.8 and 3.1 respectively). Transcriptomic analysis during acute infection in diabetics indicated the importance of interplay between immune pathways including those involved in antigen presentation, chemotaxis, innate and adaptive immunity and their regulation. Survival was associated with enhanced T cell immunity to nine of fifteen immunodominant antigens analysed including AhpC (BPSL2096), BopE (BPSS1525), PilO (BPSS1599), ATP binding protein (BPSS1385) and an uncharacterised protein (BPSL2520). T cell immunity to GroEL (BPSL2697) was specifically impaired in diabetic individuals. This characterization of immunity associated with survival during acute infection offers insights into correlates of protection and a foundation for design of an effective multivalent vaccine.

Killer cell immunoglobulin-like receptor/HLA class I (KIR/HLA-I) combinations are associated with disease risk, implicating functional roles for NK cells (NKCs) or KIR(+) T cells. KIR/HLA-I interactions can act through inhibition of NKC activation by target cells and NKC licensing for greater intrinsic responsiveness. We compared licensing conferred by the weaker, HLA-C group 1/KIR2DL3, and the stronger, HLA-C group 2/KIR2DL1, inhibitory combinations. The "rheostat model" predicts weaker licensing by HLA-C1/KIR2DL3 interactions than HLA-C2/KIR2DL1. We analyzed degranulation in NKC subsets expressing single and multiple receptors for HLA-I. NKG2A had the strongest licensing impact, while KIR2DL3, KIR2DL1, and KIR3DL1 were weaker, and not significantly different to each other. Presence of one or two matched HLA-C allotypes did not alter licensing of KIR2DL3(+) and KIR2DL1(+) NKC. Coexpression of activating KIR2DS1 disarmed KIR2DL3(+) and KIR2DL1(+) NKC to a similar extent. KIR3DL1 and NKG2A combined for more enhanced licensing of double-positive NKC than the combination of KIR2DL3 and KIR2DL1. Thus, KIR2DL3 and KIR2DL1 have similar capacity to license NKC, suggesting that inhibitory signal strength and amount of available HLA-C ligands do not correlate with NKC licensing. Altogether, our results show that the basis for disease associations of HLA-C and KIR2DL likely encompasses factors other than licensing.

Allogeneic hematopoietic stem cell transplantation (HSCT) is a curative treatment for many hematological conditions. Acute graft-versus-host disease (aGVHD) is a prevalent immune-mediated complication following HSCT. Current diagnostic biomarkers that correlate with aGVHD severity, progression, and therapy response in graft recipients are insufficient. Here, we investigated whether epigenetic marks measured in peripheral blood of healthy graft donors stratify aGVHD severity in human leukocyte antigen (HLA)-matched sibling recipients prior to T cell-depleted HSCT.

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 (<50%), or frequently (≥50%) isolated from sputum. Patients with frequent PA sputum-positive isolates were more likely to be infected by mucoid PA, and they showed a narrow T-cell epitope response and a relative reduction in Th1 polarizing transcription factors but enhanced immunity with respect to antibody production, innate cytokines, and chemokines. CONCLUSIONS:We have defined the immunodominant, HLA-restricted T-cell epitopes of OprF. Our observation that chronic infection is associated with a response of narrowed specificity, despite strong innate and antibody immunity, may help to explain susceptibility in these individuals and pave the way for better vaccine design to achieve protective immunity.

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.

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 216 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 FILA 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 [HRI = 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. (C) 2013 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.

To the editor: An earlier report by Bucher et al[1][1] indicated a better stem cell transplant (SCT) outcome with younger sibling donors (YSD). Transmaternal cell flow during pregnancy[2][2] might explain this observation. Notably, cord blood comprises minor H antigen experienced T cells that are

Background 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. Methods 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. Results 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 (P<0.0001). 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. Conclusions DQ DSAbs are associated with inferior allograft outcomes. This study shows the importance of establishing the DQ match before transplantation to define immunologic risk.

Natural killer (NK) cells exert antimyeloma cytotoxicity. The balance between inhibition and activation of NK-cells played by the inherited repertoire of killer immunoglobulin-like receptor (KIR) genes therefore may influence prognosis. One hundred eighty-two patients with multiple myeloma (MM) were analyzed for KIR repertoire. Multivariate analysis showed that progression-free survival (PFS) after autologous stem cell transplantation (ASCT) was significantly shorter for patients who are KIR3DS1(+) (P = .01). This was most evident for patients in complete or partial remission (good risk; GR) at ASCT. The relative risk (RR) of progression or death for patients with KIR3DS1(+) compared with KIR3DS1(-) was 1.9 (95% CI, 1.3-3.1; P = .002). The most significant difference in PFS was observed in patients with GR KIR3DS1(+) in whom HLA-Bw4, the ligand for the corresponding inhibitory receptor KIR3DL1, was missing. Patients with KIR3DS1(+) KIR3DL1(+) HLA-Bw4(-) had a significantly shorter PFS than patients who were KIR3DS1(-), translating to a difference in median PFS of 12 months (12.2 vs 24 months; P = .002). Our data show that KIR-human leukocyte antigen immunogenetics represent a novel prognostic tool for patients with myeloma, shown here in the context of ASCT, and that KIR3DS1 positivity may identify patients at greater risk of progression.

Identifying transplant recipients in whom immunological tolerance is established or is developing would allow an individually tailored approach to their posttransplantation management. In this study, we aimed to develop reliable and reproducible in vitro assays capable of detecting tolerance in renal transplant recipients. Several biomarkers and bioassays were screened on a training set that included 11 operationally tolerant renal transplant recipients, recipient groups following different immunosuppressive regimes, recipients undergoing chronic rejection, and healthy controls. Highly predictive assays were repeated on an independent test set that included 24 tolerant renal transplant recipients. Tolerant patients displayed an expansion of peripheral blood B and NK lymphocytes, fewer activated CD4+ T cells, a lack of donor-specific antibodies, donor-specific hyporesponsiveness of CD4+ T cells, and a high ratio of forkhead box P3 to alpha-1,2-mannosidase gene expression. Microarray analysis further revealed in tolerant recipients a bias toward differential expression of B cell-related genes and their associated molecular pathways. By combining these indices of tolerance as a cross-platform biomarker signature, we were able to identify tolerant recipients in both the training set and the test set. This study provides an immunological profile of the tolerant state that, with further validation, should inform and shape drug-weaning protocols in renal transplant recipients.

Abstract 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.

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.

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.

No cell surface expression was observed in most tumor cell lines, although specifi c HLA-E RNA was detected in all cell lines by RT-PCR with specifi c primers. Surface HLA-E expression was detected at low levels in few cell lines including osteosarcoma and neuroblastoma cell lines whereas it was detectable at high levels in the HLA-E 721.221 AEH transfectant, in 721.221 transfectants expressing different HLA class I alleles (A3, B46) and HLA-G. Protein expression was also analysed by Western blot using the MEM-E02 mAb. Peripheral blood mononuclear cells and K562 cell line were used as membrane HLA-E-positive and -negative controls respectively. We also investigated whether HLA-E expression could be correlated with HLA-class I expression (W6/32) and HLA-G expression (MEM-G/9). To this regard, the few HLA-E positive cell lines observed were indeed HLA-class I positive but HLA-G negative, supporting previous reports suggesting that HLA-E expression is mainly dependent on HLAclass I heavy chain expression. 278