KIR2DL3 and KIR2DL1 show similar impact on licensing of human NK cells.

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.

Chronic Infection by Mucoid Pseudomonas aeruginosa Associated with Dysregulation in T-Cell Immunity to Outer Membrane Porin F.

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.

MS in South Asians in England: early disease onset and novel pattern of myelin autoimmunity.

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.

The serodominant secreted effector protein of Salmonella, SseB, is a strong CD4 antigen containing an immunodominant epitope presented by diverse HLA class II alleles.

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.

Multicenter analyses demonstrate significant clinical effects of minor histocompatibility antigens on GvHD and GvL after HLA-matched related and unrelated hematopoietic stem cell transplantation.

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.

Interaction between KIR3DS1 and HLA-Bw4 predicts for progression-free survival after autologous stem cell transplantation in patients with multiple myeloma.

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.

Phenotype frequencies of autosomal minor histocompatibility antigens display significant differences among populations.

Minor histocompatibility (H) antigens are allogeneic target molecules having significant roles in alloimmune responses after human leukocyte antigen-matched solid organ and stem cell transplantation (SCT). Minor H antigens are instrumental in the processes of transplant rejection, graft-versus-host disease, and in the curative graft-versus-tumor effect of SCT. The latter characteristic enabled the current application of selected minor H antigens in clinical immunotherapeutic SCT protocols. No information exists on the global phenotypic distribution of the currently identified minor H antigens. Therefore, an estimation of their overall impact in human leukocyte antigen-matched solid organ and SCT in the major ethnic populations is still lacking. For the first time, a worldwide phenotype frequency analysis of ten autosomal minor H antigens was executed by 31 laboratories and comprised 2,685 randomly selected individuals from six major ethnic populations. Significant differences in minor H antigen frequencies were observed between the ethnic populations, some of which appeared to be geographically correlated.

Retrospective tissue typing of the kidney donor from recipient urine.

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.

Impact of HLA class I and class II DNA high-resolution HLA typing on clinical outcome in adult unrelated stem cell transplantation after in vivo T-cell depletion with alemtuzumab.

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.

Infection with Burkholderia pseudomallei - immune correlates of survival in acute melioidosis.

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.

A donor-specific epigenetic classifier for acute graft-versus-host disease severity in hematopoietic stem cell transplantation.

BACKGROUND: 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. METHODS: We measured DNA methylation levels genome-wide at single-nucleotide resolution in peripheral blood of 85 HSCT donors, matched to recipients with various transplant outcomes, with Illumina Infinium HumanMethylation450 BeadChips. RESULTS: Using genome-wide DNA methylation profiling, we showed that epigenetic signatures underlying aGVHD severity in recipients correspond to immune pathways relevant to aGVHD etiology. We discovered 31 DNA methylation marks in donors that associated with aGVHD severity status in recipients, and demonstrated strong predictive performance of these markers in internal cross-validation experiments (AUC = 0.98, 95% CI = 0.96-0.99). We replicated the top-ranked CpG classifier using an alternative, clinical DNA methylation assay (P = 0.039). In an independent cohort of 32 HSCT donors, we demonstrated the utility of the epigenetic classifier in the context of a T cell-replete conditioning regimen (P = 0.050). CONCLUSIONS: Our findings suggest that epigenetic typing of HSCT donors in a clinical setting may be used in conjunction with HLA genotyping to inform both donor selection and transplantation strategy, with the ultimate aim of improving patient outcome.

De novo DQ donor-specific antibodies are associated with a significant risk of antibody-mediated rejection and transplant glomerulopathy.

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.

Development of a cross-platform biomarker signature to detect renal transplant tolerance in humans.

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.

A T-cell epitope encoded by a subset of HLA-DPB1 alleles determines nonpermissive mismatches for hematologic stem cell transplantation.

The importance of HLA-DPB1 matching for the outcome of allogeneic hematologic stem cell (HSC) transplantation is controversial. We have previously identified HLA-DPB1*0901 as a target of cytotoxic T cells mediating in vivo rejection of an HSC allograft. Here we show that HLA-DPB1*0901 encodes a T-cell epitope shared by a subset of DPB1 alleles that determines nonpermissive mismatches for HSC transplantation. Several T-cell clones obtained from the patient at the time of rejection showed HLA-DP restricted recognition of allogeneic targets expressing HLA-DPB1*0901, *1001, *1701, *0301, *1401, and *4501, but not other alleles. Based on these findings, we developed an algorithm for prediction of nonpermissive HLA-DPB1 mismatches. Retrospective evaluation of 118 transplantations showed that the presence of nonpermissive HLA-DPB1 mismatches was correlated with significantly increased hazards of acute grade II to IV graft-versus-host disease (HR = 1.87, P =.046) and transplantation-related mortality (HR = 2.69, P =.027) but not relapse (HR = 0.98, P =.939), as compared with the permissive group. There was also a marked but statistically not significant increase in the hazards of overall mortality (HR = 1.64, P =.1). These data suggest that biologic characterization of in vivo alloreactivity can be a tool for definition of clinically relevant nonpermissive HLA mismatches for unrelated HSC transplantation.