MHC Haplotype Research Articles

High Resolution Donor/Recipient HLA Matching Level in Unrelated Hematopoietic Stem Cell Transplantation and Impact on the Transplant Outcome: The Italian Experience on Behalf of GITMO, IBMDR and Aibt

Abstract Introduction: HLA molecules play an important role for immunoreactivity in allogeneic hematopoietic stem cell transplantation (HSCT). To elucidate the effect and the potential identification of "permissive" and "non permissive" I and II class HLA mismatching (mm) loci on the early and long term transplant outcome, we conducted a retrospective/prospective observational analysis on 1789 patients transplanted with unmanipulated haematopoietic stem cells from a volunteer unrelated donor (URD). Methods Between January 2012 to December 2015, 1789 adult patients with a median age of 49 years (18-70) affected by haematological malignant diseases, performed an unrelated HSCT, coordinated by the Italian Bone Marrow Donor Registry (IBMDR). All patients have been typed by high resolution (HR) HLA typing for HLA-A/B/C/DRB1/DQB1 loci, at the start of the donor's search process. Patient and donor characteristics are shown in Table 1. As conditioning regimen and GVHD prophylaxis, 71% of patients received a myeloablative conditioning and 76% a combination of anti-Thymoglobuline, Cyclosporine and Metotrexate short course. Total Body Irradiation was part of conditioning regimen in 14% of cases and PBSC was used as stem cell source in 80% of transplants. Median follow for survivors was 38 months (1-76). Regarding to the allelic compatibility, 890 (50%) of donor/recipient (D/R) pairs were 10/10 HLA matched, 677 (38%) showed 1 mm for A, B, C, DRB1 or DQB1 in 249, 141, 173, 2 and 112 cases, respectively and 222 (12%) received HSCT from a 8/10 or less HLA matched donor. Results: Overall 90% and 79% of patients achieved PMN and PLTS engraftment within 30 and 90 days, respectively. Probabilities of 3-y Overall Survival (3-yr OS), Progression Free Survival (3-yr PFS), and Graft Relapse Free Survival (3-yr GRFS) were 52%, 42%, and 30%, respectively. The 3-y CI of Transplant Related Mortality (TRM) was 26%, with a 100-days CI of acute GVHD ≥2 of 26%, whereas the 3-yr CI of chronic GVHD was 30%, of which 10% extensive. Cox multivariate analysis showed that, compared to 10/10 HLA-matched HSCT, 9/10 and ≤8/10 HLA-matched HSCT were associated with worse outcomes in terms of OS (HR 1.16, p=0.04 and HR 1.3, p=0.007, respectively), GRFS (HR 1.2, p=0.005 and HR 1.2, p=0.07, respectively), TRM (HR 1.3, p=0.007 and HR 1.6, p<0.0001, respectively), grade 3-4 aGVHD (HR 1.8, p=0.0001 and HR 1.8, p=0.01, respectively) and cGvHD (HR 1.3, p=0.005 and HR 1.1, p=0.35, respectively). Notably, no significant differences occurred through the comparison between ≤8/10 and 9/10 matching. Univariate comparisons are shown in Figure 1. Moreover, in order to identify permissive and non permissive allelic mismatching, we analyzed the donor/recipient pairs with a single HLA mm with a frequency > 5%: the presence of A02:01 in the patient's HLA, after adjustment for HLA matching at the other loci and other clinical variables known to affect HSCT outcome, was associated with significant higher risk of TRM (HR 1.9, p= 0.03) and worst OS (HR 1.7, p=0.04). Patient's age > 49 years (p<0.0001), advanced disease stage (p<0.0001), presence of 1 or more co- morbidity according to the Sorror Hematopoietic Cell Transplant-Comorbidity Index (p=0.01) were associated with a hazard risk of 1.4, 2, 1.2 for OS and 1.6, 1.75, 1.4 for TRM. Moreover, the Italian origin of recipient and donor resulted in reduced grade 2-4 acute (HR=0.6, p=0.001) and chronic GVHD (p=0.002, HR=0.4). Finally, the Transplant Program expertise (>10 HSCT/year) is associated with reduced TRM (HR 0.8, p=0.0001), HSCT from female donor to male recipient was associated with higher risk of extensive cGvHD (HR 1.4, p=0.03), and CMV negative/negative status versus other combinations had protective effect on development of grade 3-4 aGVHD (HR 0.56, p=0.04). Conclusions: Our large cohort data of homogeneously treated 1789 URD transplants, show that 10/10 HLA matching remains a significantly favorable prognostic factor for OS, TRM, GRFS and acute/chronic GVHD, whereas there are no significant differences between 8/10 and 9/10 matching transplants. Moreover, the HLA A02:01 as single mm seems to play a "non permissive" role. Finally the Italian origin of recipient and donor is related to a reduced development of GVHD probably due to the matching of the extended MHC haplotypes in individuals of the same geographic origin. Disclosures Rambaldi: Amgen Inc.: Consultancy; Pfizer: Consultancy; Celgene: Consultancy; Omeros: Consultancy; Roche: Consultancy; Novartis: Consultancy; Italfarmaco: Consultancy. Vago:Moderna TX: Research Funding; GENDX: Research Funding. Patriarca:Janssen: Other: Advisory role; Jazz: Other: Travel, accommodations, expenses; Medac: Other: Travel, accommodations, expenses; Celgene: Other: Advisory Role; Travel, accommodations, expenses; MSD Italy: Other: Advisory Role.

Rapid whole blood assay using flow cytometry for measuring phagocytic activity of chicken leukocytes

Phagocytic activity of leukocytes in whole blood was assessed as a potential immune competence trait in chickens. A flow cytometry based whole blood phagocytosis (WBP) assay was set up and evaluated using blood from chickens homozygous for four different MHC haplotypes, B12, B15, B19 and B21. Fluorescent latex beads and two serotypes of fluorescently labelled heat-killed bacteria (Salmonella Infantis and Salmonella. Typhimurium) were evaluated as phagocytic targets. In addition, the opsonophagocytic potential (OPp) of individual sera from the birds was included in a phagocytosis assay using the HD11 chicken macrophage cell line. Results showed that both serotypes of bacteria but not the latex beads were effectively phagocytosed by leukocytes in the whole blood cultures. Differences were observed in the phagocytic capacity of monocytes and thrombocyte/lymphocytes, respectively between the different MHC lines. No significant differences on the OPp of serum was identified between MHC lines. In addition, for both phagocytic activity of leukocytes and OPp of serum large variations between individuals were observed within MHC haplotypes. No significant relationships were observed between the phagocytic activity of leukocytes and serum OPp or Salmonella-specific IgY levels.In conclusion, our results suggest that the WBP assay, using a no-lyse no-wash single staining method, is a rapid and convenient method to assess phagocytic functions of different leukocyte populations.

Allelic nomenclature for the duplicated MHC class II DQ genes in sheep.

The principal MHC class II molecules involved in the presentation of peptides to the antigen specific receptors on CD4+ T cells genes in sheep are derived from DR and DQ genes. Allelic nomenclature systems for the DRB1 and its partner DRA loci are available for Ovid's; however, no official nomenclature is available for the DQ genes which creates ambiguity within the research community. Ovine MHC haplotypes include at least two pairs of DQA and DQB genes, termed DQA1, DQB1 and DQA2, DQB2 and both sets are polymorphic and both seem to be functional. In a number of haplotypes, the DQA1 locus appears to be absent (DQA1-null) and is replaced by a second locus termed DQA2-like. Here, we identify families of alleles based on sequence similarity and phylogenetic clustering which correspond to each of the DQA and DQB genes identified in previous genomic and transcript analyses of homozygous animals. Using such criteria to cluster sequences, we have named 82 full-length and partial cDNA transcripts derived from domestic sheep (Ovis aries) which correspond to alleles at the Ovar-DQA1, DQA2, DQA2-like, DQB1, DQB2 and DQB2-like genes and provide associated sequence resources available to the research community through the IPD-MHC Database. This sets the basis for naming and annotation of DQ genes within the ovine MHC and may be used as a template for DQ genes in other ruminant species which will ultimately support research in livestock infectious disease.

Distinct mechanisms survey the structural integrity of HLA-B*27:05 intracellularly and at the surface.

HLA-B*27:05 is associated with the development of autoimmune spondyloarthropathies, but the precise causal relationship between the MHC haplotype and disease pathogenesis is yet to be elucidated. Studies focusing on the structure and cellular trafficking of HLA-B*27:05 implicate several links between the onset of inflammation and the unusual conformations of the molecule inside and at the surface of antigen presenting cells. Several lines of evidence emphasize the emergence of those unnatural protein conformations under conditions where peptide loading onto B*27:05 is impaired. To understand how cellular factors distinguish between poorly loaded molecules from the optimally loaded ones, we have investigated the intracellular transport, folding, and cell surface expression of this particular B27 subtype. Our findings show that B*27:05 is structurally unstable in the absence of peptide, and that an artificially introduced disulfide bond between residues 84 and 139 conferred enhanced conformational stability to the suboptimally loaded molecules. Empty or suboptimally loaded B*27:05 can escape intracellular retention and arrive at the cell surface leading to the appearance of increased number of β2m-free heavy chains. Our study reveals a general mechanism found in the early secretory pathways of murine and human cells that apply to the quality control of MHC class I molecules, and it highlights the allotype-specific structural features of HLA-B*27:05 that can be associated with aberrant antigen presentation and that might contribute to the etiology of disease.

Increasing antigen presentation on HSV-1-infected cells increases lesion size but does not alter neural infection or latency.

CD8+ T cells have a role in the control of acute herpes simplex virus (HSV) infection and may also be important in the maintenance of latency. In this study we have explored the consequences of boosting the efficacy of CD8+ T cells against HSV by increasing the amount of an MHC I-presented epitope on the surface of infected cells. To do this we used HSVs engineered to express an extra copy of the immunodominant CD8+ T cell epitope in C57Bl/6 mice, namely gB498 (SSIEFARL). Despite greater presentation of gB498 on infected cells, CD8+ T cell responses to these viruses in mice were similar to those elicited by a control virus. Further, the expression of extra gB498 did not significantly alter the extent or stability of latency in our mouse model, and virus loads in skin and sensory ganglia of infected mice were not affected. Surprisingly, mice infected with these viruses developed significantly larger skin lesions than those infected with control viruses and notably, this phenotype was dependent on MHC haplotype. Therefore increasing the visibility of HSV-infected cells to CD8+ T cell attack did not impact neural infection or latency, but rather enhanced pathology in the skin.

Identification of Tumoricidal TCRs from Tumor-Infiltrating Lymphocytes by Single-Cell Analysis.

T-cell receptor (TCR) gene therapy is a promising next-generation antitumor treatment. We previously developed a single-T-cell analysis protocol that allows the rapid capture of paired TCRα and β cDNAs. Here, we applied the protocol to analyze the TCR repertoire of tumor-infiltrating lymphocytes (TIL) of various cancer patients. We found clonally expanded populations of T cells that expressed the same clonotypic TCR in 50% to 70% of CD137+CD8+ TILs, indicating that they responded to certain antigens in the tumor environment. To assess the tumor reactivity of the TCRs derived from those clonally expanded TILs in detail, we then analyzed the CD137+CD8+ TILs from the tumor of B16F10 melanoma cells in six C57BL/6 mice and analyzed their TCR repertoire. We also found clonally expanded T cells in 60% to 90% of CD137+CD8+ TILs. When the tumor reactivity of dominant clonotypic TCRs in each mouse was analyzed, 9 of 13 TCRs induced the secretion of IFNγ in response to, and showed killing of, B16F10 cells in vitro, and 2 of them showed strong antitumor activity in vivo Concerning their antigen specificity, 7 of them reacted to p15E peptide of endogenous murine leukemia virus-derived envelope glycoprotein 70, and the rest reacted to tumor-associated antigens expressed on EL4 lymphoma as well as B16 melanoma cells. These results show that our strategy enables us to simply and rapidly obtain the tumor-specific TCR repertoire with high fidelity in an antigen- and MHC haplotype-independent manner from primary TILs. Cancer Immunol Res; 6(4); 378-88. ©2018 AACR.

HLA-inferred extended haplotype disparity level is more relevant than the level of HLA mismatch alone for the patients survival and GvHD in T cell-replate hematopoietic stem cell transplantation from unrelated donor

Serious risks in unrelated hematopoietic stem cell transplantation (HSCT) including graft versus host disease (GvHD) and mortality are associated with HLA disparity between donor and recipient. The increased risks might be dependent on disparity in not-routinely-tested multiple polymorphisms in genetically dense MHC region, being organized in combinations of two extended MHC haplotypes (Ehp). We assessed the clinical role of donor-recipient Ehp disparity levels in N = 889 patients by the population-based detection of HLA allele phase mismatch. We found increased GvHD incidences and mortality rates with increasing Ehp mismatch level even with the same HLA mismatch level. In multivariate analysis HLA mismatch levels were excluded from models and Ehp disparity level remained independent prognostic factor for high grade acute GvHD (p = 0.000037, HR = 10.68, 95%CI 5.50–32.5) and extended chronic GvHD (p < 0.000001, HR = 15.51, CI95% 5.36–44.8). In group with single HLA mismatch, patients with double Ehp disparity had worse 5-year overall survival (45% vs. 56%, p = 0.00065, HR = 4.05, CI95% 1.69–9.71) and non-relapse mortality (40% vs. 31%, p = 0.00037, HR = 5.63, CI95% 2.04–15.5) than patients with single Ehp disparity. We conclude that Ehp-linked factors contribute to the high morbidity and mortality in recipients given HLA-mismatched unrelated transplant and Ehp matching should be considered in clinical HSCT.

Novel and Haplotype Specific MicroRNAs Encoded by the Major Histocompatibility Complex

The MHC is recognized for its importance in human health and disease. However, many disease-associated variants throughout the region remain of unknown significance, residing predominantly within non-coding regions of the MHC. The characterization of non-coding RNA transcripts throughout the MHC is thus central to understanding the genetic contribution of these variants. Therefore, we characterize novel miRNA transcripts throughout the MHC by performing deep RNA sequencing of two B lymphoblastoid cell lines with completely characterized MHC haplotypes. Our analysis identifies 89 novel miRNA transcripts, 48 of which undergo Dicer-dependent biogenesis and are loaded onto the Argonaute silencing complex. Several of the identified mature miRNA and pre-miRNA transcripts are unique to specific MHC haplotypes and overlap common SNPs. Furthermore, 43 of the 89 identified novel miRNA transcripts lie within linkage disequilibrium blocks that contain a disease-associated SNP. These disease associated SNPs are associated with 65 unique disease phenotypes, suggesting that these transcripts may play a role in the etiology of numerous diseases associated with the MHC. Additional in silico analysis reveals the potential for thousands of putative pre-miRNA encoding loci within the MHC that may be expressed by different cell types and at different developmental stages.

MHC-mismatched mixed chimerism restores peripheral tolerance of noncross-reactive autoreactive T cells in NOD mice

Autoimmune type 1 diabetes (T1D) and other autoimmune diseases are associated with particular MHC haplotypes and expansion of autoreactive T cells. Induction of MHC-mismatched but not -matched mixed chimerism by hematopoietic cell transplantation effectively reverses autoimmunity in diabetic nonobese diabetic (NOD) mice, even those with established diabetes. As expected, MHC-mismatched mixed chimerism mediates deletion in the thymus of host-type autoreactive T cells that have T-cell receptor (TCR) recognizing (cross-reacting with) donor-type antigen presenting cells (APCs), which have come to reside in the thymus. However, how MHC-mismatched mixed chimerism tolerizes host autoreactive T cells that recognize only self-MHC-peptide complexes remains unknown. Here, using NOD.Rag1-/-BDC2.5 or NOD.Rag1-/-BDC12-4.1 mice that have only noncross-reactive transgenic autoreactive T cells, we show that induction of MHC-mismatched but not -matched mixed chimerism restores immune tolerance of peripheral noncross-reactive autoreactive T cells. MHC-mismatched mixed chimerism results in increased percentages of both donor- and host-type Foxp3+ Treg cells and up-regulated expression of programmed death-ligand 1 (PD-L1) by host-type plasmacytoid dendritic cells (pDCs). Furthermore, adoptive transfer experiments showed that engraftment of donor-type dendritic cells (DCs) and expansion of donor-type Treg cells are required for tolerizing the noncross-reactive autoreactive T cells in the periphery, which are in association with up-regulation of host-type DC expression of PD-L1 and increased percentage of host-type Treg cells. Thus, induction of MHC-mismatched mixed chimerism may establish a peripheral tolerogenic DC and Treg network that actively tolerizes autoreactive T cells, even those with no TCR recognition of the donor APCs.

Thymic-Specific Serine Protease Limits Central Tolerance and Exacerbates Experimental Autoimmune Encephalomyelitis.

The genetic predisposition to multiple sclerosis (MS) is most strongly conveyed by MHC class II haplotypes, possibly by shaping the autoimmune CD4 T cell repertoire. Whether Ag-processing enzymes contribute to MS susceptibility by editing the peptide repertoire presented by these MHC haplotypes is unclear. Thymus-specific serine protease (TSSP) is expressed by thymic epithelial cells and thymic dendritic cells (DCs) and, in these two stromal compartments, TSSP edits the peptide repertoire presented by class II molecules. We show in this article that TSSP increases experimental autoimmune encephalomyelitis severity by limiting central tolerance to myelin oligodendrocyte glycoprotein. The effect on experimental autoimmune encephalomyelitis severity was MHC class II allele dependent, because the lack of TSSP expression conferred protection in NOD mice but not in C57BL/6 mice. Importantly, although human thymic DCs express TSSP, individuals segregate into two groups having a high or 10-fold lower level of expression. Therefore, the level of TSSP expression by thymic DCs may modify the risk factors for MS conferred by some MHC class II haplotypes.

Discovery of gorilla MHC-C expressing C1 ligand for KIR

In comparison to humans and chimpanzees, gorillas show low diversity at MHC class I genes (Gogo), as reflected by an overall reduced level of allelic variation as well as the absence of a functionally important sequence motif that interacts with killer cell immunoglobulin-like receptors (KIR). Here, we use recently generated large-scale genomic sequence data for a reassessment of allelic diversity at Gogo-C, the gorilla orthologue of HLA-C. Through the combination of long-range amplifications and long-read sequencing technology, we obtained, among the 35 gorillas reanalyzed, three novel full-length genomic sequences including a coding region sequence that has not been previously described. The newly identified Gogo-C*03:01 allele has a divergent recombinant structure that sets it apart from other Gogo-C alleles. Domain-by-domain phylogenetic analysis shows that Gogo-C*03:01 has segments in common with Gogo-B*07, the additional B-like gene that is present on some gorilla MHC haplotypes. Identified in ~ 50% of the gorillas analyzed, the Gogo-C*03:01 allele exclusively encodes the C1 epitope among Gogo-C allotypes, indicating its important function in controlling natural killer cell (NK cell) responses via KIR. We further explored the hypothesis whether gorillas experienced a selective sweep which may have resulted in a general reduction of the gorilla MHC class I repertoire. Our results provide little support for a selective sweep but rather suggest that the overall low Gogo class I diversity can be best explained by drastic demographic changes gorillas experienced in the ancient and recent past.

Quantification and phenotypic characterisation of peripheral IFN-γ producing leucocytes in chickens vaccinated against Newcastle disease

The aim of this study was to optimise and evaluate an intracellular cytokine staining (ICS) assay for assessment of T cell IFN-γ responses in chickens vaccinated against Newcastle disease (ND). We aimed to validate currently available antibodies to chicken IFN-γ using transfected CHO cells. Moreover, this ICS assay was evaluated for use to detect mitogen and antigen induced IFN-γ production in chicken peripheral blood leucocytes.Chickens from an inbred white leghorn line containing two MHC haplotypes, B19 and B21, were divided into three experimental groups; one group was kept as naive controls, one group was vaccinated intramuscularly twice with a commercial inactivated ND virus (NDV) vaccine, and the last group was vaccinated orally twice with a commercial live attenuated NDV vaccine. PBMC were ex vivo stimulated with ConA or with NDV antigen. The ICS assay was used to determine the phenotype and frequency of IFN-γ positive cells. ConA stimulation induced extensive IFN-γ production in both CD3+TCRγδ+ (γδ T cells) cells and CD3+TCRγδ− cells (αβ T cells), but no significant differences were observed between the experimental groups. Furthermore, a large proportion of the IFN-γ producing cells were CD3− indicating that other cells than classic T cells, secreted this cytokine. NDV antigen stimulation induced IFN-γ production but to a lower extent than ConA and with a large variation between individuals. The CD3+TCR1γδ−CD8α+ (CTL) population produced the highest NDV specific IFN-γ responses, with significantly elevated levels of IFN-γ producing cells in the B19 chickens vaccinated orally with live attenuated NDV vaccine. This was not the case in the B21 animals, indicating a haplotype restricted variation. In contrast, the CD3+TCR1γδ−CD4+ (Th) population did not show a significant increase in IFN-γ production in NDV stimulated samples which was in part due to a high number of IFN-γ producing cells after incubation with medium alone. In conclusion, an ICS assay for phenotyping of IFN-γ producing chicken leukocytes was set up that proved useful in identifying cytokine producing cells upon either mitogen or antigen-specific stimulation.

OR20 De novo generation of MHC haplotypes using single molecule sequencing

Aim Utilizing a previously developed long-fragment DNA capture methodology, PacBio single molecule real-time (SMRT) sequencing and a custom de novo assembly analysis pipeline, we aim to resolve the long-range, fully phased MHC haplotypes for a single heterozygous individual. Methods Long fragment DNA enrichment for the MHC was performed on fresh blood obtained from a heterozygous individual using our previously described method, RSE. Captured DNA was sequenced on the PacBio RSII sequencing platform. Raw reads were selected to include only those reads that map to annotated MHC haplotype sequences (n = 8). De novo assembly was performed using the CANU pipeline with tuned algorithm parameters to optimize continuity and accuracy of haplotigs (phased contigs) as compared to phased SNP genotyping data. Results Following de novo assembly, PacBio reads were assembled into 27 contiguous fragments (contigs) covering 97% of the 4Mbp MHC (as aligned to the COX MHC haplotype). Phased contigs (haplotigs) were found to agree with phased SNP genotyping data at ∼78% of interrogated positions (n = 4,779 probes) indicating that the majority of haplotigs are properly phased. Phased SNP genotyping data was also used to phase across breaks in the assembly, resulting in the generation of two distinct MHC haplotypes for the sequenced sample. Conclusions Our results demonstrate the capacity to generate de novo assembled, fully phased MHC haplotypes from PacBio single molecule sequencing reads. Although phasing across contig breaks caused by coverage gaps remains a challenge, we hope to resolve this issue using BioNano genomics optical maps, so as not to rely on phased SNP genotyping data. In essence, this method has the capacity to resolve full-length MHC haplotype sequences without the need for any reference genome or familial trio analysis.

P106 Whole gene, whole haplotype and whole genome sequencing in human

Aim Recent advances in sequencing, sample and library preparation technologies made it possible to sequence entire genes, haplotypes and genome in human. In this study, we used four approaches; amplicon based whole gene sequencing, capture probe based MHC haplotype sequencing, PacBio Sequel system based whole genome sequencing and whole genome mapping based on Bionanogenomics’ Technology to study one individual’s MHC. We wanted to compare the information that we have obtained from each technology and confirm the haplotype predictions. Methods We performed whole gene HLA Class I gene sequencing as described earlier (Cereb et al., Hum Immunol. 2015 Dec;76(12):963–74). We performed MHC region target sequence capture using Roche-NimbleGen SeqCap EZ Library. Whole genome sequencing We prepared 40 to 50 kb libraries with 15 to 20 kb cut off size selection and followed manufacturer’s protocol thereafter. Sequencing performed on PacBio Sequel® platform using v2 chemistry and v4 software. Movie time for these large fragments were set to 600 min and secondary analysis is done with SMRTLink’s (v4.0.0) SMRT Analysis Application: de novo Assembly (HGAP 4). To get 85× coverage, we have sequenced the same library in 62 different SMRT® Cell 1 M v2. Whole genome de novo assembly of PacBio sequencing is being performed by DNANexus. BioNano genome mapping was performed by Bio Nano genomics. Results Haplotype determination of the individual tested with whole gene amplicon sequencing performed based on the family studies and is being compared to haplotypes determined by other methods. Conclusions Now thanks to novel technologies we can decipher the complex genetic structure of MHC and other immune response genes with higher precision. This in turn will increase our ability to understand the genetic basis of the complex diseases.

Assembly and analysis of 100 full MHC haplotypes from the Danish population.

Genes in the major histocompatibility complex (MHC, also known as HLA) play a critical role in the immune response and variation within the extended 4-Mb region shows association with major risks of many diseases. Yet, deciphering the underlying causes of these associations is difficult because the MHC is the most polymorphic region of the genome with a complex linkage disequilibrium structure. Here, we reconstruct full MHC haplotypes from de novo assembled trios without relying on a reference genome and perform evolutionary analyses. We report 100 full MHC haplotypes and call a large set of structural variants in the regions for future use in imputation with GWAS data. We also present the first complete analysis of the recombination landscape in the entire region and show how balancing selection at classical genes have linked effects on the frequency of variants throughout the region.

Neutralizing Antibody Responses to Viral Infections Are Linked to the Non-classical MHC Class II Gene H2-Ob

Select humans and animals control persistent viral infections via adaptive immune responses that include production of neutralizing antibodies. The precise genetic basis for the control remains enigmatic. Here, we report positional cloning of the gene responsible for production of retrovirus-neutralizing antibodies in mice of the I/LnJ strain. It encodes the beta subunit of the non-classical major histocompatibility complex class II (MHC-II)-like molecule H2-O, a negative regulator of antigen presentation. The recessive and functionally null I/LnJ H2-Ob allele supported the production of virus-neutralizing antibodies independently of the classical MHC haplotype. Subsequent bioinformatics and functional analyses of the human H2-Ob homolog, HLA-DOB, revealed both loss- and gain-of-function alleles, which could affect the ability of their carriers to control infections with human hepatitis B (HBV) and C (HCV) viruses. Thus, understanding of the previously unappreciated role of H2-O (HLA-DO) in immunity to infections may suggest new approaches in achieving neutralizing immunity to viruses.

Reactogenicity to major tuberculosis antigens absent in BCG is linked to improved protection against Mycobacterium tuberculosis

MTBVAC is a live-attenuated Mycobacterium tuberculosis vaccine, currently under clinical development, that contains the major antigens ESAT6 and CFP10. These antigens are absent from the current tuberculosis vaccine, BCG. Here we compare the protection induced by BCG and MTBVAC in several mouse strains that naturally express different MHC haplotypes differentially recognizing ESAT6 and CFP10. MTBVAC induces improved protection in C3H mice, the only of the three tested strains reactive to both ESAT6 and CFP10. Deletion of both antigens in MTBVAC reduces its efficacy to BCG levels, supporting a link between greater efficacy and CFP10- and ESAT6-specific reactogenicity. In addition, MTBVAC (but not BCG) triggers a specific response in human vaccinees against ESAT6 and CFP10. Our results warrant further exploration of this response as potential biomarker of protection in MTBVAC clinical trials.

FINE-MAPPING OF THE HUMAN LEUKOCYTE ANTIGEN (HLA) LOCUS AS A RISK FACTOR FOR ALZHEIMER'S DISEASE

There is increasing support for the role of neuroinflammation and aberrant immune regulation in the pathophysiology of Alzheimer's disease (AD). The immunoregulatory human leukocyte antigen (HLA) complex has been linked to susceptibility for a number of neurodegenerative diseases including AD, however, studies to date have failed to consistently identify a risk HLA haplotype for AD. Contributing to this difficulty are the complex genetic organization of the HLA region, differences in sequencing and allelic imputation methods, and diversity across ethnic populations. Building on prior work linking the HLA to AD, we used a robust imputation method on two case-control cohorts to examine the relationship between HLA haplotypes and AD risk in 309 individuals from UCSF and 11,381 individuals from the Alzheimer's Disease Genetics Consortium (ADGC), a NIA-funded national data repository. The strongest association with AD risk occurred with MHC haplotype A*03:01∼B*07:02∼DRB1*15:01∼DQA1*01:02∼DQB1*06:02, p=9.6x10-4, odds ratio (95% confidence interval) = 1.2 (1.1–1.4) in the combined UCSF + ADGC cohort. Secondary analysis suggested that this effect may be driven primarily by individuals who are negative for established AD genetic risk factor, APOE E4, and may be of particular relevance in men more than women. Separate analyses of class I and II haplotypes further supported the role of class I haplotype A*03:01∼B*07:02 and class II haplotype DRB1*15:01- DQA1*01:02- DQB1*06:02 (DR15) as risk factors for AD. We also examined cerebrospinal fluid (CSF) biomarker measures and longitudinal cognitive data from the Alzheimer's Disease Neuroimaging Initiative to assess clinical relevance of identified risk haplotypes and found A*03:01∼B*07:02 was associated with higher CSF amyloid levels while DR15 haplotype was associated with greater rates of cognitive decline in a dose-dependent manner. We provide evidence that variation in the HLA—including risk haplotype DR15—contributes to AD risk. DR15 has also been associated with risk for multiple sclerosis, and its component alleles have been implicated in Parkinson's disease and narcolepsy. Our findings thus raise the possibility that DR15-associated mechanisms may contribute to pan-neuronal disease vulnerability. Results must be considered in the context of only representing European-ancestry, clinically diagnosed individuals, and utilizing imputed genotypes for a subset of HLA genes.

MHC class II-peptide complexes displayed on activated T cells guide Treg suppression

Abstract Regulatory T cell (Treg) therapy is a promising strategy for extending allograft survival. Treg suppression requires initial T cell receptor (TCR)/peptide-MHC-II (pMHC-II) contacts, the nature of which remains elusive. As inflammation facilitates transfer of pMHC-II from antigen presenting cells (APCs) to activated T cells, we hypothesize that during transplantation associated inflammation, pMHC-II relocate on alloreactive T effector cells and serve as Treg activation signals. To test this, suppression assays were performed using murine CD4+ CD25− (Teff), CD90 depleted APCs, and CD4+ CD25+ Treg cells of different MHC haplotypes. We also used a transplantation model of skin allografts in RAG1 deficient mice injected with Teff and Treg cells from various MHC backgrounds. Data from more than 150 co-culture experiments show that Treg suppression occurs only when Treg and Teff share the same MHC-II, but independently of Treg matching with APC MHC-II. Further experiments demonstrate that pMHC-II complexes, displayed on activated Teff cells, are crucial to the induction of Treg suppression. In vivo results recapitulate the in vitro data: Treg suppression, leading to graft survival, occurs only when Teff and Treg cells are MHC-II matched. Collectively, these findings suggest that suppression of alloreactivity is directed by Treg TCR recognition of pMHC-II complexes exposed on Teff cells. The nature, origin (host vs. donor), and significance of MHC-II loaded peptides have yet to be established. Future studies to improve targeted Treg therapies for transplantation tolerance will focus on mechanisms coupling Treg function with Teff cell activation.

Immunoregulatory function of kidney CD4−CD8− double negative (DN) αβ T cells is MHC dependent

Abstract Despite strong evidence for the role of immune cells in ischemia reperfusion injury (IRI) in transplant and native kidney, the underlying mechanisms are poorly understood. Furthermore, there is little data regarding unconventional T cells, usually present in small numbers in the kidney. We have recently shown that a subset of alpha/beta T cells that is double negative (DN) for both CD4 and CD8 coreceptors are present in significant numbers in normal kidney of mice and also present in human kidney biopsies. Unlike conventional CD4 and CD8 T cells, DN T cells are highly dividing in the steady and rapidly increases following IRI. In addition, kidney DN T cells secrete IL-10 and IL-27, possess an in vitro regulatory function and ameliorate AKI in mice. Unknown aspects of DN T cells included identification their MHC restriction elements and roles of various types of MHC haplotype in their homeostasis. Our most recent results show that DN T cells are heterogeneous as indicated by the partial effect of lack of β2m and MHC class II on their homeostasis. In addition, lack of β2m significantly reduce activated phenotype that is seen in kidney DN T cells. Conversely, reconstitution of β2m-deficient mice leads to activation and expansion of endogenous DN T cells, suggesting interactions between DN and CD8 T cells. Further dissection of kidney DN T cell biology will help us understand the pathogenesis of IRI and other immune mediated kidney diseases.