Human leukocyte antigen HLA-C, HLA-G, HLA-F, and HLA-E placental profiles are altered in early severe preeclampsia and preterm birth with chorioamnionitis.

BACKGROUND: The extravillous trophoblast expresses each of the nonclassical major histocompatibility complex class I antigens-human leukocyte antigens E, F, and G-and a single classical class I antigen, human leukocyte antigen C. We recently demonstrated dynamic expression patterns of human leukocyte antigens C, G, and F during early extravillous trophoblast invasion and placentation. OBJECTIVE: This study aimed to investigate the hypothesis that the immune inflammatory mediated complications of pregnancy such as early preeclampsia and preterm labor may show altered expression profiles of nonclassical human leukocyte antigens. STUDY DESIGN: Real-time quantitative polymerase chain reaction, western blot, and immunohistochemistry were performed on placental villous tissues and basal plate sections from term nonlaboring deliveries, preterm deliveries, and severe early-onset preeclampsia, both with and without small-for-gestational-age neonates. RESULTS: Human leukocyte antigen G is strongly and exclusively expressed by the extravillous trophoblast within the placental basal plate, and its levels increase in pregnancies complicated by severe early-onset preeclampsia with small-for-gestational-age neonates relative to those of healthy term controls. Human leukocyte antigen C shows a similar profile in the extravillous trophoblast of preeclamptic pregnancies, but significantly decreases in the villous placenta. Human leukocyte antigen F protein levels are decreased in both extravillous trophoblast and villous placenta of severe early-onset preeclamptic pregnancies, both with and without small-for-gestational-age neonates, compared with those found in term and preterm birth deliveries. Human leukocyte antigen E decreases in blood vessels in placentas from preeclamptic pregnancies relative to its levels in term and preterm birth deliveries. Placental levels of human leukocyte antigens F and C are increased in cases of preterm birth with chorioamnionitis relative to those of cases of idiopathic preterm birth. CONCLUSION: Dysregulation of placental human leukocyte antigen expression at the maternal-fetal interface may contribute to compromised maternal tolerance in preterm birth with chorioamnionitis and excessive maternal systemic inflammation associated with severe early-onset preeclampsia.

High-resolution HLA class II sequencing of Swedish multiple sclerosis patients.

Multiple sclerosis (MS) is a chronic neurological disease believed to be caused by autoimmune pathogenesis. The aetiology is likely explained by a complex interplay between inherited and environmental factors. Genetic investigations into MS have been conducted for over 50 years, yielding >100 associations to date. Globally, the strongest linkage is with the human leukocyte antigen (HLA) HLA-DRB5*01:01:01-DRB1*15:01:01-DQA1*01:02:01-DQB1*06:02:01 haplotype. Here, high-resolution sequencing of HLA was used to determine the alleles of DRB3, DRB4, DRB5, DRB1, DQA1, DQB1, DPA1 and DPB1 as well as their extended haplotypes and genotypes in 100 Swedish MS patients. Results were compared to 636 population controls. The heterogeneity in HLA associations with MS was demonstrated; among 100 patients, 69 extended HLA-DR-DQ genotypes were found. Three extended HLA-DR-DQ genotypes were found to be correlated to MS; HLA-DRB5*01:01:01-DRB1*15:01:01-DQA1*01:02:01-DQB1*06:02:01 haplotype together with (A) HLA-DRB4*01:01:01//DRB4*01:01:01:01-DRB1*07:01:01-DQA1*02:01//02:01:01-DQB1*02:02:01, (B) HLA-DRBX*null-DRB1*08:01:01-DQA1*04:01:01-DQB1*04:02:01, and (C) HLA-DRB3*01:01:02-DRB1*03:01:01-DQA1*05:01:01-DQB1*02:01:01. At the allelic level, HLA-DRB3*01:01:02 was considered protective against MS. However, when combined with HLA-DRB3*01:01:02-DRB1*03:01:01-DQA1*05:01:01-DQB1*02:01:01, this extended haplotype was considered a predisposing risk factor. This highlights the limitations as included with investigations of single alleles relative to those of extended haplotypes/genotypes. In conclusion, with 69 genotypes presented among 100 patients, high-resolution sequencing was conducted to underscore the wide polymorphisms present among MS patients. Additional studies in larger cohorts will be of importance to define MS among the patient group not associated with HLA-DRB5*01:01:01-DRB1*15:01:01-DQA1*01:02:01-DQB1*06:02:01.

HLA-F on Autologous HIV-Infected Cells Activates Primary NK Cells Expressing the Activating Killer Immunoglobulin-Like Receptor KIR3DS1.

HIV-exposed seronegative KIR3DS1 homozygotes have a reduced risk of HIV infection. HLA-F is the ligand for the activating NK cell receptor (NKR) KIR3DS1. HLA-F is expressed on HIV-infected CD4 T cells. Coculture of sorted, HIV-infected CD4- (siCD4-) T cells with NK cells activated a higher frequency of KIR3DS1+ than KIR3DS1- NK cells from KIR3DS1 homozygotes to elicit anti-HIV functions such as CCL4, gamma interferon (IFN-γ), and CD107a expression. This was the case whether KIR3DS1+/- NK cells were analyzed inclusively or exclusively by gating out NK cells coexpressing the NKRs, KIR2DL1/L2/L3, 3DL2, KIR2DS1/S2/S3/S5, NKG2A, and ILT2. Blocking the interaction of HLA-F on siCD4- cells with KIR3DS1 on exclusively gated KIR3DS1+ NK cells with KIR3DS1-Fc chimeric protein or an HLA-F-specific monoclonal antibody reduced the frequency of activated KIR3DS1+ cells compared to that under control conditions. KIR3DS1+ NK cell activation by HIV-infected CD4+ cells may underlie the reduced risk of KIR3DS1 homozygotes to HIV infection.IMPORTANCE This study investigated a mechanism that may underly epidemiological studies showing that carriage of the KIR3DS1 homozygous genotype is more frequent among HIV-exposed seronegative subjects than among HIV-susceptible individuals. Carriage of this genotype is associated with a reduced risk of HIV infection. The protective mechanism involves the interaction of HLA-F on CD4+ cells infected with replication-competent HIV with the activating NK receptor, KIR3DS1. This interaction leads to the activation of KIR3DS1+ NK cells for secretion of cytokines and chemokines with anti-HIV activity. Among these is CCL4, which binds and blocks CCR5, the coreceptor for HIV entry of HIV into new target cells. In the setting of an exposure to HIV, incoming HIV-infected cells expressing HLA-F rapidly activate KIR3DS1+ NK cells to elicit anti-HIV activity. Exclusive gating strategies and blocking experiments support the notion that the HLA-F/KIR3DS1 interaction is sufficient to activate NK cell functions.

The Education of NK Cells Determines Their Responsiveness to Autologous HIV-Infected CD4 T Cells.

Several studies support a role for specific killer immunoglobulin-like receptor (KIR)-HLA combinations in protection from HIV infection and slower progression to AIDS. Natural killer (NK) cells acquire effector functions through education, a process that requires the interaction of inhibitory NK cell receptors with their major histocompatibility complex (MHC) class I (or HLA class I [HLA-I]) ligands. HLA-C allotypes are ligands for the inhibitory KIRs (iKIRs) KIR2DL1, KIR2DL2, and KIR2DL3, whereas the ligand for KIR3DL1 is HLA-Bw4. HIV infection reduces the expression of HLA-A, -B, and -C on the surfaces of infected CD4 (iCD4) T cells. Here we investigated whether education through iKIR-HLA interactions influenced NK cell responses to autologous iCD4 cells. Enriched NK cells were stimulated with autologous iCD4 cells or with uninfected CD4 cells as controls. The capacities of single-positive (sp) KIR2DL1, KIR2DL2, KIR2DL3, and KIR3DL1 NK cells to produce CCL4, gamma interferon (IFN-γ), and/or CD107a were assessed by flow cytometry. Overall, we observed that the potency of NK cell education was directly related to the frequency of each spiKIR+ NK cell's ability to respond to the reduction of its cognate HLA ligand on autologous iCD4 cells, as measured by the frequency of production by spiKIR+ NK cells of CCL4, IFN-γ, and/or CD107a. Both NK cell education and HIV-mediated changes in HLA expression influenced NK cell responses to iCD4 cells.IMPORTANCE Epidemiological studies show that natural killer (NK) cells have anti-HIV activity: they are able to reduce the risk of HIV infection and/or slow HIV disease progression. How NK cells contribute to these outcomes is not fully characterized. We used primary NK cells and autologous HIV-infected cells to examine the role of education through four inhibitory killer immunoglobulin-like receptors (iKIRs) from persons with HLA types that are able to educate NK cells bearing one of these iKIRs. HIV-infected cells activated NK cells through missing-self mechanisms due to the downmodulation of cell surface HLA expression mediated by HIV Nef and Vpu. A higher frequency of educated than uneducated NK cells expressing each of these iKIRs responded to autologous HIV-infected cells by producing CCL4, IFN-γ, and CD107a. Since NK cells were from non-HIV-infected individuals, they model the consequences of healthy NK cell-HIV-infected cell interactions occurring in the HIV eclipse phase, when new infections are susceptible to extinction.

Fc Gamma Receptor Polymorphisms Modulated the Vaccine Effect on HIV-1 Risk in the HVTN 505 HIV Vaccine Trial.

HIV Vaccine Trials Network (HVTN) 505 was a phase 2b efficacy trial of a DNA/recombinant adenovirus 5 (rAd5) HIV vaccine regimen. Although the trial was stopped early for lack of overall efficacy, later correlates of risk and sieve analyses generated the hypothesis that the DNA/rAd5 vaccine regimen protected some vaccinees from HIV infection yet enhanced HIV infection risk for others. Here, we assessed whether and how host Fc gamma receptor (FcγR) genetic variations influenced the DNA/rAd5 vaccine regimen's effect on HIV infection risk. We found that vaccine receipt significantly increased HIV acquisition compared with placebo receipt among participants carrying the FCGR2C-TATA haplotype (comprising minor alleles of four FCGR2C single-nucleotide polymorphism [SNP] sites) (hazard ratio [HR] = 9.79, P = 0.035) but not among participants without the haplotype (HR = 0.86, P = 0.67); the interaction of vaccine and haplotype effect was significant (P = 0.034). Similarly, vaccine receipt increased HIV acquisition compared with placebo receipt among participants carrying the FCGR3B-AGA haplotype (comprising minor alleles of the 3 FCGR3B SNPs) (HR = 2.78, P = 0.058) but not among participants without the haplotype (HR = 0.73, P = 0.44); again, the interaction of vaccine and haplotype was significant (P = 0.047). The FCGR3B-AGA haplotype also influenced whether a combined Env-specific CD8+ T-cell polyfunctionality score and IgG response correlated significantly with HIV risk; an FCGR2A SNP and two FCGR2B SNPs influenced whether anti-gp140 antibody-dependent cellular phagocytosis correlated significantly with HIV risk. These results provide further evidence that Fc gamma receptor genetic variations may modulate HIV vaccine effects and immune function after HIV vaccination.IMPORTANCE By analyzing data from the HVTN 505 efficacy trial of a DNA/recombinant adenovirus 5 (rAd5) vaccine regimen, we found that host genetics, specifically Fc gamma receptor genetic variations, influenced whether receiving the DNA/rAd5 regimen was beneficial, neutral, or detrimental to an individual with respect to HIV-1 acquisition risk. Moreover, Fc gamma receptor genetic variations influenced immune responses to the DNA/rAd5 vaccine regimen. Thus, Fc gamma receptor genetic variations should be considered in the analysis of future HIV vaccine trials and the development of HIV vaccines.

Expansion and CD2/CD3/CD28 stimulation enhance Th2 cytokine secretion of human invariant NKT cells with retained anti-tumor cytotoxicity.

BACKGROUND AIMS: Key obstacles in human iNKT cell translational research and immunotherapy include the lack of robust protocols for dependable expansion of human iNKT cells and the paucity of data on phenotypes in post-expanded cells. METHODS: We delineate expansion methods using interleukin (IL)-2, IL-7 and allogeneic feeder cells and anti-CD2/CD3/CD28 stimulation by which to dependably augment Th2 polarization and direct cytotoxicity of human peripheral blood CD3+Vα24+Vß11+ iNKT cells. RESULTS: Gene and protein expression profiling demonstrated augmented Th2 cytokine secretion (IL-4, IL-5, IL-13) in expanded iNKT cells stimulated with anti-CD2/CD3/CD28 antibodies. Cytotoxic effector molecules including granzyme B were increased in expanded iNKT cells after CD2/CD3/CD28 stimulation. Direct cytotoxicity assays using unstimulated expanded iNKT cell effectors revealed α-galactosyl ceramide (α-GalCer)-dependent killing of the T-ALL cell line Jurkat. Moreover, CD2/CD3/CD28 stimulation of expanded iNKT cells augmented their (α-GalCer-independent) killing of Jurkat cells. Co-culture of expanded iNKT cells with stimulated responder cells confirmed contact-dependent inhibition of activated CD4+ and CD8+ responder T cells. DISCUSSION: These data establish a robust protocol to expand and novel pathways to enhance Th2 cytokine secretion and direct cytotoxicity in human iNKT cells, findings with direct implications for autoimmunity, vaccine augmentation and anti-infective immunity, cancer immunotherapy and transplantation.

HLA-F on HLA-Null 721.221 Cells Activates Primary NK Cells Expressing the Activating Killer Ig-like Receptor KIR3DS1.

NK cells elicit important responses against transformed and virally infected cells. Carriage of the gene encoding the activating killer Ig-like receptor KIR3DS1 is associated with slower time to AIDS and protection from HIV infection. Recently, open conformers of the nonclassical MHC class Ib Ag HLA-F were identified as KIR3DS1 ligands. In this study, we investigated whether the interaction of KIR3DS1 on primary NK cells with HLA-F on the HLA-null cell line 721.221 (221) stimulated KIR3DS1+ NK cells. We used a panel of Abs to detect KIR3DS1+CD56dim NK cells that coexpressed the inhibitory NK cell receptors KIR2DL1/L2/L3, 3DL2, NKG2A, and ILT2; the activating NK cell receptors KIR2DS1/S2/S3/S5; and CCL4, IFN-γ, and CD107a functions. We showed that both untreated and acid-pulsed 221 cells induced a similar frequency of KIR3DS1+ cells to secrete CCL4/IFN-γ and express CD107a with a similar intensity. A higher percentage of KIR3DS1+ than KIR3DS1- NK cells responded to 221 cells when either inclusive or exclusive (i.e., coexpressing none of the other inhibitory NK cell receptors and activating NK cell receptors detected by the Ab panel) gating strategies were employed to identify these NK cell populations. Blocking the interaction of HLA-F on 221 cells with KIR3DS1-Fc chimeric protein or anti-HLA-F Abs on exclusively gated KIR3DS1+ cells reduced the frequency of functional cells compared with that of unblocked conditions for stimulated KIR3DS1+ NK cells. Thus, ligation of KIR3DS1 activates primary NK cells for several antiviral functions.

Genetic risk factors for sclerotic graft-versus-host disease.

Sclerotic graft-versus-host disease (GVHD) is a distinctive phenotype of chronic GVHD after allogeneic hematopoietic cell transplantation, characterized by fibrosis of skin or fascia. Sclerotic GVHD has clinical and histopathological similarities with systemic sclerosis, an autoimmune disease whose risk is influenced by genetic polymorphisms. We examined 13 candidate single-nucleotide polymorphisms (SNPs) that have a well-documented association with systemic sclerosis to determine whether these SNPs are also associated with the risk of sclerotic GVHD. The study cohort included 847 consecutive patients who were diagnosed with chronic GVHD. Genotyping was performed using microarrays, followed by imputation of unobserved SNPs. The donor rs10516487 (BANK1: B-cell scaffold protein with ankyrin repeats 1) TT genotype was associated with lower risk of sclerotic GVHD (hazard ratio [HR], 0.43; 95% confidence interval [CI], 0.21-0.87; P = .02). Donor and recipient rs2056626 (CD247: T-cell receptor ζ subunit) GG or GT genotypes were associated with higher risk of sclerotic GVHD (HR, 1.57; 95% CI, 1.13-2.18; P = .007 and HR, 1.66; 95% CI, 1.19-2.32; P = .003, respectively). Donor and recipient rs987870 (5'-flanking region of HLA-DPA1) CC genotypes were associated with higher risk of sclerotic GVHD (HR, 2.50; 95% CI, 1.22-5.11; P = .01 and HR, 2.13; 95% CI, 1.00-4.54; P = .05, respectively). In further analyses, the recipient DPA1*01:03∼DPB1*04:01 haplotype and certain amino acid substitutions in the recipient P1 peptide-binding pocket of the HLA-DP heterodimer were associated with risk of sclerotic GVHD. Genetic components associated with systemic sclerosis are also associated with sclerotic GVHD. HLA-DP-mediated antigen presentation, T-cell response, and B-cell activation have important roles in the pathogenic mechanisms of both diseases.

An Object-Oriented Regression for Building Disease Predictive Models with Multiallelic HLA Genes.

Recent genome-wide association studies confirm that human leukocyte antigen (HLA) genes have the strongest associations with several autoimmune diseases, including type 1 diabetes (T1D), providing an impetus to reduce this genetic association to practice through an HLA-based disease predictive model. However, conventional model-building methods tend to be suboptimal when predictors are highly polymorphic with many rare alleles combined with complex patterns of sequence homology within and between genes. To circumvent this challenge, we describe an alternative methodology; treating complex genotypes of HLA genes as "objects" or "exemplars," one focuses on systemic associations of disease phenotype with "objects" via similarity measurements. Conceptually, this approach assigns disease risks base on complex genotype profiles instead of specific disease-associated genotypes or alleles. Effectively, it transforms large, discrete, and sparse HLA genotypes into a matrix of similarity-based covariates. By the Kernel representative theorem and machine learning techniques, it uses a penalized likelihood method to select disease-associated exemplars in building predictive models. To illustrate this methodology, we apply it to a T1D study with eight HLA genes (HLA-DRB1, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DQA1, HLA-DQB1, HLA-DPA1, and HLA-DPB1) to build a predictive model. The resulted predictive model has an area under curve of 0.92 in the training set, and 0.89 in the validating set, indicating that this methodology is useful to build predictive models with complex HLA genotypes.

Building and validating a prediction model for paediatric type 1 diabetes risk using next generation targeted sequencing of class II HLA genes.

AIM: It is of interest to predict possible lifetime risk of type 1 diabetes (T1D) in young children for recruiting high-risk subjects into longitudinal studies of effective prevention strategies. METHODS: Utilizing a case-control study in Sweden, we applied a recently developed next generation targeted sequencing technology to genotype class II genes and applied an object-oriented regression to build and validate a prediction model for T1D. RESULTS: In the training set, estimated risk scores were significantly different between patients and controls (P = 8.12 × 10-92 ), and the area under the curve (AUC) from the receiver operating characteristic (ROC) analysis was 0.917. Using the validation data set, we validated the result with AUC of 0.886. Combining both training and validation data resulted in a predictive model with AUC of 0.903. Further, we performed a "biological validation" by correlating risk scores with 6 islet autoantibodies, and found that the risk score was significantly correlated with IA-2A (Z-score = 3.628, P < 0.001). When applying this prediction model to the Swedish population, where the lifetime T1D risk ranges from 0.5% to 2%, we anticipate identifying approximately 20 000 high-risk subjects after testing all newborns, and this calculation would identify approximately 80% of all patients expected to develop T1D in their lifetime. CONCLUSION: Through both empirical and biological validation, we have established a prediction model for estimating lifetime T1D risk, using class II HLA. This prediction model should prove useful for future investigations to identify high-risk subjects for prevention research in high-risk populations.

Analysis of HLA A*02 association with vaccine efficacy in the RV144 HIV-1 vaccine trial.

UNLABELLED: The RV144 HIV-1 vaccine trial demonstrated partial efficacy of 31% against HIV-1 infection. Studies into possible correlates of protection found that antibodies specific to the V1 and V2 (V1/V2) region of envelope correlated inversely with infection risk and that viruses isolated from trial participants contained genetic signatures of vaccine-induced pressure in the V1/V2 region. We explored the hypothesis that the genetic signatures in V1 and V2 could be partly attributed to selection by vaccine-primed T cells. We performed a T-cell-based sieve analysis of breakthrough viruses in the RV144 trial and found evidence of predicted HLA binding escape that was greater in vaccine versus placebo recipients. The predicted escape depended on class I HLA A*02- and A*11-restricted epitopes in the MN strain rgp120 vaccine immunogen. Though we hypothesized that this was indicative of postacquisition selection pressure, we also found that vaccine efficacy (VE) was greater in A*02-positive (A*02(+)) participants than in A*02(-) participants (VE = 54% versus 3%, P = 0.05). Vaccine efficacy against viruses with a lysine residue at site 169, important to antibody binding and implicated in vaccine-induced immune pressure, was also greater in A*02(+) participants (VE = 74% versus 15%, P = 0.02). Additionally, a reanalysis of vaccine-induced immune responses that focused on those that were shown to correlate with infection risk suggested that the humoral responses may have differed in A*02(+) participants. These exploratory and hypothesis-generating analyses indicate there may be an association between a class I HLA allele and vaccine efficacy, highlighting the importance of considering HLA alleles and host immune genetics in HIV vaccine trials. IMPORTANCE: The RV144 trial was the first to show efficacy against HIV-1 infection. Subsequently, much effort has been directed toward understanding the mechanisms of protection. Here, we conducted a T-cell-based sieve analysis, which compared the genetic sequences of viruses isolated from infected vaccine and placebo recipients. Though we hypothesized that the observed sieve effect indicated postacquisition T-cell selection, we also found that vaccine efficacy was greater for participants who expressed HLA A*02, an allele implicated in the sieve analysis. Though HLA alleles have been associated with disease progression and viral load in HIV-1 infection, these data are the first to suggest the association of a class I HLA allele and vaccine efficacy. While these statistical analyses do not provide mechanistic evidence of protection in RV144, they generate testable hypotheses for the HIV vaccine community and they highlight the importance of assessing the impact of host immune genetics in vaccine-induced immunity and protection. (This study has been registered at ClinicalTrials.gov under registration no. NCT00223080.).

HLA-F and MHC class I open conformers are ligands for NK cell Ig-like receptors.

Killer Ig-like receptors (KIRs) are innate immune receptors expressed by NK and T cells classically associated with the detection of missing self through loss of their respective MHC ligand. Some KIR specificities for allelic classical class I MHC (MHC-I) have been described, whereas other KIR receptor-ligand relationships, including those associated with nonclassical MHC-I, have yet to be clearly defined. We report in this article that KIR3DL2 and KIR2DS4 and the nonclassical Ag HLA-F, expressed as a free form devoid of peptide, physically and functionally interact. These interactions extend to include classical MHC-I open conformers as ligands, defining new relationships between KIR receptors and MHC-I. The data collectively suggest a broader, previously unrecognized interaction between MHC-I open conformers--including prototypical HLA-F--and KIR receptors, acting in an immunoregulatory capacity centered on the inflammatory response.

HLA-F and MHC-I open conformers cooperate in a MHC-I antigen cross-presentation pathway.

Peptides that are presented by MHC class I (MHC-I) are processed from two potential sources, as follows: newly synthesized endogenous proteins for direct presentation on the surface of most nucleated cells and exogenous proteins for cross-presentation typically by professional APCs. In this study, we present data that implicate the nonclassical HLA-F and open conformers of MHC-I expressed on activated cells in a pathway for the presentation of exogenous proteins by MHC-I. This pathway is distinguished from the conventional endogenous pathway by its independence from TAP and tapasin and its sensitivity to inhibitors of lysosomal enzymes, and further distinguished by its dependence on MHC-I allotype-specific epitope recognition for Ag uptake. Thus, our data from in vitro experiments collectively support a previously unrecognized model of Ag cross-presentation mediated by HLA-F and MHC-I open conformers on activated lymphocytes and monocytes, which may significantly contribute to the regulation of immune system functions and the immune defense.

Vaccine-induced gag-specific T cells are associated with reduced viremia after HIV-1 infection.

The contribution of host T-cell immunity and HLA class I alleles to the control of human immunodeficiency virus (HIV-1) replication in natural infection is widely recognized. We assessed whether vaccine-induced T-cell immunity, or expression of certain HLA alleles, impacted HIV-1 control after infection in the Step MRKAd5/HIV-1 gag/pol/nef study. Vaccine-induced T cells were associated with reduced plasma viremia, with subjects targeting ≥3 gag peptides presenting with half-log lower mean viral loads than subjects without Gag responses. This effect was stronger in participants infected proximal to vaccination and was independent of our observed association of HLA-B*27, -B*57 and -B*58:01 alleles with lower HIV-1 viremia. These findings support the ability of vaccine-induced T-cell responses to influence postinfection outcome and provide a rationale for the generation of T-cell responses by vaccination to reduce viremia if protection from acquisition is not achieved. Clinical trials identifier: NCT00095576.

HLA Class II (DR, DQ, DP) Genes Were Separately Associated With the Progression From Seroconversion to Onset of Type 1 Diabetes Among Participants in Two Diabetes Prevention Trials (DPT-1 and TN07).

OBJECTIVE: To explore associations of HLA class II genes (HLAII) with the progression of islet autoimmunity from asymptomatic to symptomatic type 1 diabetes (T1D). RESEARCH DESIGN AND METHODS: Next-generation targeted sequencing was used to genotype eight HLAII genes (DQA1, DQB1, DRB1, DRB3, DRB4, DRB5, DPA1, DPB1) in 1,216 participants from the Diabetes Prevention Trial-1 and Randomized Diabetes Prevention Trial with Oral Insulin sponsored by TrialNet. By the linkage disequilibrium, DQA1 and DQB1 are haplotyped to form DQ haplotypes; DP and DR haplotypes are similarly constructed. Together with available clinical covariables, we applied the Cox regression model to assess HLAII immunogenic associations with the disease progression. RESULTS: First, the current investigation updated the previously reported genetic associations of DQA1*03:01-DQB1*03:02 (hazard ratio [HR] = 1.25, P = 3.50*10-3) and DQA1*03:03-DQB1*03:01 (HR = 0.56, P = 1.16*10-3), and also uncovered a risk association with DQA1*05:01-DQB1*02:01 (HR = 1.19, P = 0.041). Second, after adjusting for DQ, DPA1*02:01-DPB1*11:01 and DPA1*01:03-DPB1*03:01 were found to have opposite associations with progression (HR = 1.98 and 0.70, P = 0.021 and 6.16*10-3, respectively). Third, DRB1*03:01-DRB3*01:01 and DRB1*03:01-DRB3*02:02, sharing the DRB1*03:01, had opposite associations (HR = 0.73 and 1.44, P = 0.04 and 0.019, respectively), indicating a role of DRB3. Meanwhile, DRB1*12:01-DRB3*02:02 and DRB1*01:03 alone were found to associate with progression (HR = 2.6 and 2.32, P = 0.018 and 0.039, respectively). Fourth, through enumerating all heterodimers, it was found that both DQ and DP could exhibit associations with disease progression. CONCLUSIONS: These results suggest that HLAII polymorphisms influence progression from islet autoimmunity to T1D among at-risk subjects with islet autoantibodies.

Oral Insulin Delay of Stage 3 Type 1 Diabetes Revisited in HLA DR4-DQ8 Participants in the TrialNet Oral Insulin Prevention Trial (TN07).

OBJECTIVE: To explore if oral insulin could delay onset of stage 3 type 1 diabetes (T1D) among patients with stage 1/2 who carry HLA DR4-DQ8 and/or have elevated levels of IA-2 autoantibodies (IA-2As). RESEARCH AND METHODS: Next-generation targeted sequencing technology was used to genotype eight HLA class II genes (DQA1, DQB1, DRB1, DRB3, DRB4, DRB5, DPA1, and DPB1) in 546 participants in the TrialNet oral insulin preventative trial (TN07). Baseline levels of autoantibodies against insulin (IAA), GAD65 (GADA), and IA-2A were determined prior to treatment assignment. Available clinical and demographic covariables from TN07 were used in this post hoc analysis with the Cox regression model to quantify the preventive efficacy of oral insulin. RESULTS: Oral insulin reduced the frequency of T1D onset among participants with elevated IA-2A levels (HR 0.62; P = 0.012) but had no preventive effect among those with low IA-2A levels (HR 1.03; P = 0.91). High IA-2A levels were positively associated with the HLA DR4-DQ8 haplotype (OR 1.63; P = 6.37 × 10-6) and negatively associated with the HLA DR7-containing DRB1*07:01-DRB4*01:01-DQA1*02:01-DQB1*02:02 extended haplotype (OR 0.49; P = 0.037). Among DR4-DQ8 carriers, oral insulin delayed the progression toward stage 3 T1D onset (HR 0.59; P = 0.027), especially if participants also had high IA-2A level (HR 0.50; P = 0.028). CONCLUSIONS: These results suggest the presence of a T1D endotype characterized by HLA DR4-DQ8 and/or elevated IA-2A levels; for those patients with stage 1/2 disease with such an endotype, oral insulin delays the clinical T1D onset.

Recombinant structures expand and contract inter and intragenic diversification at the KIR locus.

BACKGROUND: The human KIR genes are arranged in at least six major gene-content haplotypes, all of which are combinations of four centromeric and two telomeric motifs. Several less frequent or minor haplotypes also exist, including insertions, deletions, and hybridization of KIR genes derived from the major haplotypes. These haplotype structures and their concomitant linkage disequilibrium among KIR genes suggest that more meaningful correlative data from studies of KIR genetics and complex disease may be achieved by measuring haplotypes of the KIR region in total. RESULTS: Towards that end, we developed a KIR haplotyping method that reports unambiguous combinations of KIR gene-content haplotypes, including both phase and copy number for each KIR. A total of 37 different gene content haplotypes were detected from 4,512 individuals and new sequence data was derived from haplotypes where the detailed structure was not previously available. CONCLUSIONS: These new structures suggest a number of specific recombinant events during the course of KIR evolution, and add to an expanding diversity of potential new KIR haplotypes derived from gene duplication, deletion, and hybridization.

NKp46 and DNAM-1 NK-cell receptors drive the response to human cytomegalovirus-infected myeloid dendritic cells overcoming viral immune evasion strategies.

Information on natural killer (NK)-cell receptor-ligand interactions involved in the response to human cytomegalovirus (HCMV) is limited and essentially based on the study of infected fibroblasts. Experimental conditions were set up to characterize the NK response to HCMV-infected myeloid dendritic cells (DCs). Monocyte-derived DCs (moDCs) infected by the TB40/E HCMV strain down-regulated the expression of human leukocyte antigen class I molecules and specifically activated autologous NK-cell populations. NKG2D ligands appeared virtually undetectable in infected moDCs, reflecting the efficiency of immune evasion mechanisms, and explained the lack of antagonistic effects of NKG2D-specific monoclonal antibody. By contrast, DNAM-1 and DNAM-1 ligands (DNAM-1L)-specific monoclonal antibodies inhibited the NK response at 48 hours after infection, although the impact of HCMV-dependent down-regulation of DNAM-1L in infected moDCs was perceived at later stages. moDCs constitutively expressed ligands for NKp46 and NKp30 natural cytotoxicity receptors, which were partially reduced on HCMV infection; yet, only NKp46 appeared involved in the NK response. In contrast to previous reports in fibroblasts, human leukocyte antigen-E expression was not preserved in HCMV-infected moDCs, which triggered CD94/NKG2A(+) NK-cell activation. The results provide an insight on key receptor-ligand interactions involved in the NK-cell response against HCMV-infected moDCs, stressing the importance of the dynamics of viral immune evasion mechanisms.

Using Haplotype-Based Artificial Intelligence to Evaluate SARS-CoV-2 Novel Variants and Mutations.

Importance: Earlier detection of emerging novel SARS-COV-2 variants is important for public health surveillance of potential viral threats and for earlier prevention research. Artificial intelligence may facilitate early detection of SARS-CoV2 emerging novel variants based on variant-specific mutation haplotypes and, in turn, be associated with enhanced implementation of risk-stratified public health prevention strategies. Objective: To develop a haplotype-based artificial intelligence (HAI) model for identifying novel variants, including mixture variants (MVs) of known variants and new variants with novel mutations. Design, Setting, and Participants: This cross-sectional study used serially observed viral genomic sequences globally (prior to March 14, 2022) to train and validate the HAI model and used it to identify variants arising from a prospective set of viruses from March 15 to May 18, 2022. Main Outcomes and Measures: Viral sequences, collection dates, and locations were subjected to statistical learning analysis to estimate variant-specific core mutations and haplotype frequencies, which were then used to construct an HAI model to identify novel variants. Results: Through training on more than 5 million viral sequences, an HAI model was built, and its identification performance was validated on an independent validation set of more than 5 million viruses. Its identification performance was assessed on a prospective set of 344 901 viruses. In addition to achieving an accuracy of 92.8% (95% CI within 0.1%), the HAI model identified 4 Omicron MVs (Omicron-Alpha, Omicron-Delta, Omicron-Epsilon, and Omicron-Zeta), 2 Delta MVs (Delta-Kappa and Delta-Zeta), and 1 Alpha-Epsilon MV, among which Omicron-Epsilon MVs were most frequent (609/657 MVs [92.7%]). Furthermore, the HAI model found that 1699 Omicron viruses had unidentifiable variants given that these variants acquired novel mutations. Lastly, 524 variant-unassigned and variant-unidentifiable viruses carried 16 novel mutations, 8 of which were increasing in prevalence percentages as of May 2022. Conclusions and Relevance: In this cross-sectional study, an HAI model found SARS-COV-2 viruses with MV or novel mutations in the global population, which may require closer examination and monitoring. These results suggest that HAI may complement phylogenic variant assignment, providing additional insights into emerging novel variants in the population.

KIR genotype and haplotype repertoire in Kuwaiti healthy donors, hematopoietic cell transplant recipients and healthy family members.

The gene complex located on chromosome 19q13.4 encodes the Killer-cell Immunoglobulin-like Receptors (KIRs), which exhibit remarkable polymorphism in both gene content and sequences. Further, the repertoire of KIR genes varies within and between populations, creating a diverse pool of KIR genotypes. This study was carried out to characterize KIR genotypes and haplotypes among 379 Arab Kuwaiti individuals including 60 subjects from 20 trio families, 49 hematopoietic cell transplantation (HCT) recipients and 270 healthy Kuwaiti volunteer HCT donors. KIR Genotyping was performed by a combination of reverse sequence specific oligonucleotide probes (rSSO) and/or Real Time PCR. The frequencies of KIR genes in 270 healthy Kuwaiti volunteer donors were compared to previously reported frequencies in other populations. In addition, we compared the differences in KIR repertoire of patients and healthy donors to investigate the reproducibility of previously reported significant differences between patients with hematological malignancies and healthy donors. The observed frequencies in our cohort volunteer HCT donors was comparable to those reported in neighboring Arab populations. The activating genes KIR2DS1, KIR2DS5 and KIR3DS1 and the inhibitory gene KIR2DL5 were significantly more frequent in patients compared to healthy donors, however, none of the previously reported differences were reproducible in our Kuwaiti cohort. This report is the first description of KIR gene carrier frequency and haplotype characterization in a fairly large cohort of the Kuwaiti population, which may have implications in KIR based HCT donor selection strategies.