Functionally distinct ERAP1 and ERAP2 are a hallmark of HLA-A29-(Birdshot) Uveitis.

Birdshot Uveitis (Birdshot) is a rare eye condition that affects HLA-A29-positive individuals and could be considered a prototypic member of the recently proposed 'MHC-I (major histocompatibility complex class I)-opathy' family. Genetic studies have pinpointed the endoplasmic reticulum aminopeptidase (ERAP1) and (ERAP2) genes as shared associations across MHC-I-opathies, which suggests ERAP dysfunction may be a root cause for MHC-I-opathies. We mapped the ERAP1 and ERAP2 haplotypes in 84 Dutch cases and 890 controls. We identified association at variant rs10044354, which mediated a marked increase in ERAP2 expression. We also identified and cloned an independently associated ERAP1 haplotype (tagged by rs2287987) present in more than half of the cases; this ERAP1 haplotype is also the primary risk and protective haplotype for other MHC-I-opathies. We show that the risk ERAP1 haplotype conferred significantly altered expression of ERAP1 isoforms in transcriptomic data (n = 360), resulting in lowered protein expression and distinct enzymatic activity. Both the association for rs10044354 (meta-analysis: odds ratio (OR) [95% CI]=2.07[1.58-2.71], P = 1.24 × 10(-7)) and rs2287987 (OR[95% CI]: =2.01[1.51-2.67], P = 1.41 × 10(-6)) replicated and showed consistent direction of effect in an independent Spanish cohort of 46 cases and 2103 controls. In both cohorts, the combined rs2287987-rs10044354 haplotype associated with Birdshot more strongly than either variant alone [meta-analysis: P=3.9 × 10(-9)]. Finally, we observed that ERAP2 protein expression is dependent on the ERAP1 background across three European populations (n = 3353). In conclusion, a functionally distinct combination of ERAP1 and ERAP2 are a hallmark of Birdshot and provide rationale for strategies designed to correct ERAP function for treatment of Birdshot and MHC-I-opathies more broadly.

CTL clones isolated from an HLA-Cw-mismatched bone marrow transplant recipient with acute graft-versus-host disease.

HLA-Cw disparity in a donor increases the risk of acute graft-vs-host disease (GVHD) after bone marrow transplantation. Acute GVHD is mediated by donor CTLs. However, mismatched HLA-Cw-specific CTLs generated in posttransplant recipients who developed acute GVHD have not been characterized in detail. In this study, CTL clones isolated from a recipient at the onset of acute GVHD who was transplanted from an HLA-A, -B, and -DRB1-matched, HLA-Cw-mismatched (recipient, Cw*0303/Cw*0702; donor, Cw*0801/Cw*0702), unrelated donor were characterized. The seven isolated CTLs, including CD4(+), CD8(+), and CD4(+)CD8(+) T lymphocytes, lysed recipient cells, HLA-Cw*0303-transfected 721.211 cells, and HLA-Cw*0303-transfected donor cells, but not untransfected 721.211 cells or donor cells. Thus, all CTLs recognized the mismatched Cw*0303 molecule as an alloantigen. The sequences of Cw*0303 and Cw*0801 differ by 16 aas. Stimulation of CTLs by COS cells transfected with Cw*0303 cDNA constructs demonstrated that Cw*0303 mutants in which individual amino acids constituting peptide-binding pockets were substituted with the corresponding Cw*0801 amino acids significantly decreased IFN-gamma production by all CTLs, whereas Cw*0303 mutants bearing Cw*0801 amino acids outside the positions constituting peptide-binding pockets stimulated all CTLs to the same degree as the wild-type Cw*0303 construct. These data suggest that all CTLs recognized the Cw molecule in a peptide-dependent manner. ELISPOT revealed that Cw*0303-reactive T cells accounted for one-half of the total of alloreactive T cells in the blood during GVHD. Taken together, non-self Cw-specific CTL clones with a variety of phenotypes and peptide specificities can be generated in posttransplant recipients with acute GVHD.

Toward targeting B cell cancers with CD4+ CTLs: identification of a CD19-encoded minor histocompatibility antigen using a novel genome-wide analysis.

Some minor histocompatibility antigens (mHags) are expressed exclusively on patient hematopoietic and malignant cells, and this unique set of antigens enables specific targeting of hematological malignancies after human histocompatability leucocyte antigen (HLA)-matched allogeneic stem cell transplantation (allo-SCT). We report the first hematopoietic mHag presented by HLA class II (HLA-DQA1*05/B1*02) molecules to CD4(+) T cells. This antigen is encoded by a single-nucleotide polymorphism (SNP) in the B cell lineage-specific CD19 gene, which is an important target antigen for immunotherapy of most B cell malignancies. The CD19(L)-encoded antigen was identified using a novel and powerful genetic strategy in which zygosity-genotype correlation scanning was used as the key step for fine mapping the genetic locus defined by pairwise linkage analysis. This strategy was also applicable for genome-wide identification of a wide range of mHags. CD19(L)-specific CD4(+) T cells provided antigen-specific help for maturation of dendritic cells and for expansion of CD8(+) mHag-specific T cells. They also lysed CD19(L)-positive malignant cells, illustrating the potential therapeutic advantages of targeting this novel CD19(L)-derived HLA class II-restricted mHag. The currently available immunotherapy strategies enable the exploitation of these therapeutic effects within and beyond allo-SCT settings.

Modo-UG, a marsupial nonclassical MHC class I locus.

Modo-UG is a class I gene located in the MHC of the marsupial Monodelphis domestica, the gray, short-tailed opossum. Modo-UG is expressed as three alternatively spliced mRNA forms, all of which encode a transmembrane form with a short cytoplasmic tail that lacks phosphorylation sites typically found in classical class I molecules. The three alternative mRNAs would encode a full-length form, an isoform lacking the alpha2 domain, and one lacking both alpha2 and alpha3 domains. Genotyping both captive-bred and wild M. domestica from different geographic regions revealed no variation in the residues that make up Modo-UG's peptide-binding groove. Modo-UG's low polymorphism is contrasting to that of a nearby class I locus, Modo-UA1, which has a highly polymorphic peptide-binding region. Absence of functional polymorphism in Modo-UG is therefore not a general feature of opossum class I genes but the result of negative selection. Modo-UG is the first MHC linked marsupial class I to be described that appears to clearly have nonclassical features.

Identification of two novel female-specific non-major histocompatibility complex loci regulating collagen-induced arthritis severity and chronicity, and evidence of epistasis.

OBJECTIVE: To identify additional sex-specific and epistatic quantitative trait loci (QTL) regulating collagen-induced arthritis (CIA) severity overall, as well as within different stages during the disease course, in an intercross between major histocompatibility complex-identical inbred rat strains DA/Bkl (susceptible) and ACI/Hsd (resistant). METHODS: Arthritic male (DA x ACI)F2 intercross offspring (n = 143) were analyzed separately from the females (n = 184). Phenotypic extremes (maximum arthritis scores [MAS]) were genotyped and used for QTL analysis. All 327 rats were genotyped with the simple sequence-length polymorphism (SSLP) markers closest to the peak of Cia7 and Cia10, the major loci previously identified in this intercross, and with SSLPs covering chromosomes 12 and 18. Phenotypes studied were disease onset, arthritis severity scores on days 14-39, MAS, mean and cumulative arthritis scores, delayed-type hypersensitivity, and antibody responses to rat type II collagen. RESULTS: A new female-specific arthritis-severity recessive locus was identified on rat chromosome 12 (Cia25), with a maximum effect observed on day 28 (logarithm of odds [LOD] 4.7). The homozygous DA genotype at Cia25 was associated with a 45% higher median arthritis score in females. Sequencing analyses of the Cia25 candidate gene Ncf1 revealed polymorphisms between DA and ACI. The previously identified locus, Cia10, was found to be male-specific. A 2-locus interaction model analysis identified a novel recessive chromosome 18 QTL, Cia26, which was dependent on Cia7, with its maximum effect observed at later stages during the disease course (peak LOD score of 3.6 for arthritis scores on day 39). CONCLUSION: This study identified 2 novel female-specific loci, and 1 male-specific locus. Cia25 regulates MAS and disease severity during the mid-to-late stages of the disease course and may be accounted for by Ncf1 polymorphisms. Cia26 is in epistasis with Cia7 and regulates later stages of disease, suggesting an involvement in disease perpetuation and/or chronicity.

Enhanced tumor responses to dendritic cells in the absence of CD8-positive cells.

Wild-type mice immunized with MART-1 melanoma Ag-engineered dendritic cells (DC) generate strong Ag-specific immunity that has an absolute requirement for both CD8(+) and CD4(+) T cells. DC administration to CD8 alpha knockout mice displayed unexpectedly enhanced levels of protection to tumor challenge despite this deficiency in CD8(+) T cells and the inability to mount MHC class I-restricted immune responses. This model has the following features: 1) antitumor protection is Ag independent; 2) had an absolute requirement for CD4(+) and NK1.1(+) cells; 3) CD4(+) splenocytes are responsible for cytokine production; 4) lytic cells in microcytotoxicity assays express NK, but lack T cell markers (NK1.1(+) alpha beta TCR(-) CD3(-)); and 5) the lytic phenotype can be transferred to naive CD8 alpha knockout mice by NK1.1(+) splenocytes. Elucidation of the signaling events that activate these effective cytotoxic cells and the putative suppressive mechanisms in a wild-type environment may provide means to enhance the clinical activity of DC-based approaches.

The H4b minor histocompatibility antigen is caused by a combination of genetically determined and posttranslational modifications.

Minor histocompatibility (H) Ag disparities result in graft-vs-host disease and chronic solid allograft rejection in MHC-identical donor-recipient combinations. Minor H Ags are self protein-derived peptides presented by MHC class I molecules. Most arise as a consequence of allelic variation in the bound peptide (p) that results in TCR recognizing the p/MHC as foreign. We used a combinational peptide screening approach to identify the immune dominant H2K(b)-restricted epitope defining the mouse H4(b) minor H Ag. H4(b) is a consequence of a P3 threonine to isoleucine change in the MHC-bound peptide derived from epithelial membrane protein-3. This allelic variation also leads to phosphorylation of the H4(b) but not the H4(a) epitope. Further, ex vivo CD8(+) T lymphocytes bind phosphorylated Ag tetramers with high efficiency. Although we document the above process in the minor H Ag system, posttranslational modifications made possible by subtle amino acid changes could also contribute to immunogenicity and immune dominance in tumor immunotherapeutic settings.

Biochemical and immunogenetic analysis of an immunodominant peptide (B6dom1) encoded by the classical H7 minor histocompatibility locus.

Of the many minor histocompatibility (H) Ags that have been detected in mice, the ability to induce graft vs host disease (GVHD) after bone marrow transplantation is restricted to a limited number of immunodominant Ags. One such murine Ag, B6dom1, is presented by the H2-Db MHC class I molecule. We present biochemical evidence that the natural B6dom1 peptide is indistinguishable from AAPDNRETF, and we show that this peptide can be isolated from a wide array of tissues, with highest levels from the lymphoid organs and lung. Moreover, we employ a novel, somatic cell selection technique involving CTL-mediated immunoselection coupled with classical genetics, to show that B6dom1 is encoded by the H7 minor H locus originally discovered approximately 40 years ago. These studies provide a molecular genetic framework for understanding B6dom1, and exemplify the fact that mouse minor H loci that encode immunodominant CTL epitopes can correspond to classical H loci originally identified by their ability to confer strong resistance to tumor transplantation. Additionally, these studies demonstrate the utility of somatic cell selection approaches toward resolving H Ag immunogenetics.

Linked suppression of skin graft rejection can operate through indirect recognition.

Adult mice can be rendered immunologically tolerant of allogeneic tissues if transplanted under cover of mAbs to CD4 and CD8. Tolerance generated in this manner is characterized by the presence of regulatory CD4+ T cells that can recruit naive T cells to become tolerant also through "infectious tolerance." Regulatory CD4+ T cells can also suppress rejection of third party transplant Ags provided they are expressed on the same graft as the tolerated Ags. This process of linked suppression can act across whole MHC barriers and represents a powerful mechanism with therapeutic potential. Tolerance can also be induced to reprocessed minor transplantation Ags presented through host APCs (indirect recognition). We here demonstrate that linked suppression can also be induced through the indirect pathway. This finding may be important in the development of transplantation tolerance in the clinic.

Positional cloning and molecular characterization of an immunodominant cytotoxic determinant of the mouse H3 minor histocompatibility complex.

Immune responses to minor histocompatibility antigens are poorly understood and present substantial barriers to successful solid tissue and bone marrow transplantation among MHC-matched individuals. We exploited a unique positional cloning approach relying on the potent negative selection capability of cytotoxic T cells to identify the H3a gene responsible for immunodominant H2-Db-restricted determinants of the classically defined mouse autosomal H3 complex. The allelic basis for reciprocal H3a antigens is two amino acid changes within a single nonamer H2-Db-binding peptide. The H3a gene, now called Zfp106, encodes a 1888-amino acid protein with three zinc fingers and a beta-transducin domain consistent with DNA/protein binding. A region of ZFP106 is identical to a 600-amino acid sequence implicated in the insulin receptor signaling pathway.

On histocompatibility barriers, Th1 to Th2 immune deviation, and the nature of the allograft responses.

In the present study, we have sought to determine the basis for the frequent failure of Th1 to Th2 immune deviation to blunt the severity of allograft rejection, as such immune deviation has proven highly effective in the treatment of several T cell-dependent autoimmune states. Our study demonstrates that treating islet allograft recipient mice with anti-IL-12 mAb is highly effective in producing Th1 to Th2 immune deviation in several model systems (i.e., fully MHC, partially MHC, or multiple minor Ag barriers). Nevertheless, anti-IL-12 failed to prolong the engraftment of fully MHC-mismatched islet allografts. However, anti-IL-12-treated recipients carrying MHC-matched but multiple minor Ag-mismatched allografts experienced prolonged engraftment; allograft tolerance was frequently achieved in the DBA/2J (H-2d) to BALB/c (H-2d) strain combination. In another model, in which the host response was dominated by CD4+ T cells responding to donor allopeptides presented upon host APCs in the context of self MHC class II molecules, anti-IL-12 treatment proved to be extremely potent. Thus, Th1 to Th2 immune deviation produces prolonged engraftment as compared with recipients of MHC-mismatched allografts when rejection is dependent upon indirectly presented allogeneic peptides and a reduced mass of responding alloreactive T cells.

Expression screening of a yeast artificial chromosome contig refines the location of the mouse H3a minor histocompatibility antigen gene.

The H3 complex, on mouse Chromosome 2, is an important model locus for understanding mechanisms underlying non-self Ag recognition during tissue transplantation rejection between MHC-matched mouse strains. H3a is a minor histocompatibility Ag gene, located within H3, that encodes a polymorphic peptide alloantigen recognized by cytolytic T cells. Other genes within the complex include beta2-microglobulin and H3b. A yeast artificial chromosome (YAC) contig is described that spans the interval between D2Mit444 and D2Mit17, a region known to contain H3a. This contig refines the position of many genes and anonymous loci. In addition, 23 new sequence-tagged sites are described that further increase the genetic resolution surrounding H3a. A novel assay was developed to determine the location of H3a within the contig. Representative YACs were modified by retrofitting with a mammalian selectable marker, and then introduced by spheroplast fusion into mouse L cells. YAC-containing L cells were screened for the expression of the YAC-encoded H3a(a) Ag by using them as targets in a cell-mediated lympholysis assay with H3a(a)-specific CTLs. A single YAC carrying H3a was identified. Based on the location of this YAC within the contig, many candidate genes can be eliminated. The data position H3a between Tyro3 and Epb4.2, in close proximity to Capn3. These studies illustrate how genetic and genomic information can be exploited toward identifying genes encoding not only histocompatibility Ags, but also any autoantigen recognized by T cells.

A genetic linkage map of mouse chromosome 2 extending from thrombospondin to paired box gene 1, including the H3 minor histocompatibility complex.

The classical minor histocompatibility 3 (H3) locus was originally defined by the phenotype of skin graft rejection, which is a complex genetic trait. H3 is now known to be a gene complex comprised of a minimum of two functionally interdependent alloantigen-encoding loci, H3a and H3b. H3a encodes a peptide recognized by cytotoxic T cells, and H3b encodes a peptide that stimulates helper T cells. The H3 complex also contains the beta2-microglobulin gene (B2m), and polymorphisms in B2m contribute to the tissue rejection phenotype. We describe a high-density genetic linkage map of a 16-cM region of mouse Chromosome 2 from thrombospondin (Thbs1) to paired box gene 1 (Pax1). This genetic map includes H3a, H3b, and B2m. Other genes and anonymous loci have also been placed on the map. H3a maps between D2Mit444 and B2m in close vicinity to several known genes. H3b maps 12 cM distal to H3a, and the proprotein convertase subtilisin/kexin type 2 gene (Pcsk2; formerly Nec2) cosegregates with H3b in a high-resolution backcross panel. The H3 complex spans a region that shows conserved synteny to human chromosomes 15q, 2q, and 20p.

Expression of alpha and beta subunit isoforms of Na,K-ATPase in the mouse inner ear and changes with mutations at the Wv or Sld loci.

Mice homozygous for mutations at the viable dominant spotting (Wv) and Steel-dickie (Sld) loci exhibit a similar phenotype which includes deafness. The auditory dysfunction derives from failure of the stria vascularis to develop normally and to generate a high positive endocochlear potential (EP). Because strial function is driven by Na,K-ATPase its expression was investigated in inner ears of Wv/Wv and Sld/Sld mice and their wild-type littermates by immunostaining with antisera against four of the enzyme's subunit isoforms. Wild-type mice from two different genetic backgrounds showed an identical distribution of subunit isoforms among inner ear transport cells. Several epithelial cell types coexpressed the alpha 1 and beta 1 subunits. Vestibular dark cells showed no reactivity for beta 1 but expressed abundant beta 2, whereas, strial marginal cells stained strongly for both beta isoforms. The only qualitative difference between mutant and wild-type mice was the absence of beta 1 subunit in marginal cells of the mutant's stria. However, it is unlikely that this difference accounts for failure of mutants to generate a high EP because the beta 1 subunit is not present in the stria vascularis of either rats or gerbils with normal EP values. Strong immunostaining for Na,K-ATPase in lateral wall fibrocytes of normal mice along with diminished immunoreactivity in the mutants supports the concept that these strategically located transport fibrocytes actively resorb K+ leaked across Reissner's membrane into scala vestibuli or effluxed from hair cells and nerves into scala tympani. It is further speculated that the resorbed K+ normally is siphoned down its concentration gradient into the intrastrial space through gap junctions between fibrocytes and strial basal and intermediate cells where it is recycled back to endolymph via marginal cells. Thus, failure of mutants to generate a positive EP could be explained by the absence of intermediate cells which may form the final link in the conduit for moving K+ from perilymph to the intrastrial compartment.

Effect of mismatches for major histocompatibility complex and minor antigens on corneal graft rejection.

The importance of minor histocompatibility genes in corneal graft rejection was investigated using a model that simulates the major histocompatibility complex (MHC) and minor mismatches of the human allograft more accurately than previous animal models. DA(RT1a) x LEW(RT1(1]F1 hybrid rats were backcrossed to LEW, and the backcross generation were used as corneal graft recipients. Female DA(RT1a) strain animals were used as donors throughout. As in humans, the MHC disparity (a to 1) between each donor-recipient pair could be controlled; minor mismatches were variable and unknown. The MHC haplotype of each backcross individual (either homozygous l/l) or heterozygous a/l) was determined. Depending on this haplotype, the transplanted DA cornea was either matched or mismatched with the recipient for MHC antigens. The average proportion of minor disparate loci was 50%, although this was variable and unknown from recipient to recipient. Some animals of each MHC type were sensitized with three subcutaneous DA strain skin grafts at intervals of 2 weeks. Prior sensitization caused more rapid corneal graft rejection in both MHC mismatched (P less than 0.001) and matched (P less than 0.01) animals. All animals in the two MHC-mismatched groups (sensitized, 26; unsensitized, 17) and most in the MHC-matched groups (sensitized, 25 of 27; unsensitized, all 13) rejected their grafts. The MHC matching resulted in a greater range of survival times, although the difference in survival in unsensitized animals between matched and mismatched groups was not significant (unsensitized, P greater than 0.05; sensitized, P less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Complexity at the mouse minor histocompatibility locus H-4.

The fine immunogenetics of the chromosome 7 mouse minor histocompatibility (H) locus H-4 was investigated. Both class I major histocompatibility complex (MHC)-restricted cytotoxic T lymphocytes (CTL) and class II MHC-restricted "helper" T cells (TH) specifically reactive with H-4 antigens were isolated as clones and were used as genetic probes for classical backcross segregation analysis. Results of a four point cross indicated that the H-4 locus was actually comprised of two genes, that have been designated H-46 and H-47. The former encodes antigens recognized by the TH and the latter encodes antigens recognized by the CTL. Moreover, these two genes could be separated from the gene pink-eyed dilution (p) which was found to be "sandwiched" between them. The functional significance of a minor H congenic strain differing by both TH-defined H-46 and CTL-defined H-47 was addressed using F1 complementation tests. Such studies indicated that immune responses against H-46 antigens was required for generation of H-47-specific CTL. Altogether, these results suggest selective presentation of different minor H gene products by class I or class II MHC proteins and that the minor H "locus" H-4 may have necessarily included both TH and CTL-defined genes because of requisite TH-CTL collaboration.

A new cytotoxic lymphocyte-defined antigen coded by a gene closely linked to the H-3 locus.

F1 complementation results indicate that a new gene, putatively controlling a minor histocompatibility antigen, is closely linked to the minor histocompatibility gene, H-3, in the fifth linkage group of chromosome 2 of the mouse. This gene controls a product that was capable of inducing as well as acting as a target for cytotoxic lymphocytes (CTL). The lytic activity of CTL developed in B10.LP-H-3D mice specific for the product of the new gene of B10 was restricted to target cells possessing H-2Db antigens. This contrasts to the H-2Kb-restricted activity of H-3.1 specific CTL.