HLA class I homologous transcripts in the human embryonal carcinoma cell line Tera-2

We have used the human teratocarcinoma-derived embryonal carcinoma cell line Tera-2 cl. 13 to explore the putative expression of novel HLA class I(-like) genes. Serological analyses revealed that Tera-2 cells do not express polymorphic HLA class I (-A, -B, -C) specificities, but do express HLA class I-like antigens. These phenotypic properties parallel those of certain mouse embryonal carcinoma cells. To study the expression of HLA class I(-like) genes in the Tera-2 cells two different approaches were used. Screening of a Tera-2 cDNA library with a full-length HLA class I cDNA probe under conditions that would allow for the identification of relatively distinct HLA class I-like sequences yielded 27 positive clones, all of which were of the regular HLA-A, -B, -C type. Reverse northern hybridizations of the restriction enzyme-digested Tlab region comprising cosmids with Tera-2 cDNA as the probe resulted in the identification of several putative human genes whose equivalents map within the mouse Tla region. However, none of these genes appeared to be structurally related to HLA class I. A putative H3.3 histone gene was identified in the proximal Tla region of the C57BL/10 mouse. It is concluded that no structural homologues of mouse Qa/Tla genes are expressed in the human developmental cell line Tera-2.     

Comparison of HLA class I gene sequences. Derivation of locus-specific oligonucleotide probes specific for HLA-A, HLA-B, and HLA-C genes

The major histocompatibility complex in man contains at least 20 class I genes. Included within this family are three closely linked loci with 11-47 codominant alleles that encode the classical transplantation antigens HLA-A, -B, and -C. The study of individual HLA-A, -B, and -C genes is complicated both by the high degree of sequence homology among all members of the class I gene family and by the high degree of polymorphism exhibited by HLA-A, -B, and -C genes. Identification of potential locus-specific regions suitable for use as unique probes has been limited by the small number of nucleotide sequences available for comparison. In the present study, the nucleotide sequences of two cDNA clones, designated HLA-4 and HLA-10, that encode previously unsequenced alleles of HLA-C and HLA-A genes, respectively, are compared with those of other class I genes. From these intergenic and interallelic comparisons, it was deduced that the nucleotide sequence encoding amino acids 291-299 of the transmembrane region showed sufficient divergence between loci and similarity between alleles, to be suitable for the generation of locus-specific probes. Synthetic oligonucleotides were generated and shown to be highly locus-specific in hybridization. These probes were used successfully for the quantitation of the relative amounts of mRNA transcribed in human liver from HLA-A, -B, and -C genes; they should greatly simplify future studies of restriction fragment length polymorphisms of HLA-A, -B, and -C alleles as genetic markers of disease susceptibility.     

Recognition of nonclassical HLA class I antigens by gamma delta T cells during pregnancy

The healthy trophoblast does not express classical HLA-A and HLA-B products; therefore, an MHC-restricted recognition of trophoblast-presented Ags is unlikely. In the decidua and also in peripheral blood of healthy pregnant women, gammadelta T cells significantly increase in number. We investigated the possible role of gammadelta T cells in recognition of trophoblast-presented Ags. PBL and isolated gammadelta T cells from healthy pregnant women as well as from those at risk for premature pregnancy termination were conjugated to choriocarcinoma cells (JAR) transfected with nonclassical HLA Ags (HLA-E, HLA-G). To investigate the involvement of killer-inhibitory/killer-activatory receptors in trophoblast recognition, we tested the effect of CD94 block on cytotoxic activity of Vdelta2(+) enriched gammadelta T cells to HLA-E- and/or HLA-G-transfected targets. Lymphocytes from healthy pregnant women preferentially recognized HLA(-) choriocarcinoma cells, whereas those from pathologically pregnant patients did not discriminate between HLA(+) and HLA(-) cells. Normal pregnancy Vdelta2(+) T cells conjugated at a significantly increased rate to HLA-E transfectants, whereas Vdelta2(+) lymphocytes from pathologically pregnant women did not show a difference between those and HLA(-) cells. Blocking of the CD94 molecule of Vdelta2(+) lymphocytes from healthy pregnant women resulted in an increased cytotoxic activity to HLA-E-transfected target cells. These data indicate that Vdelta2(+) lymphocytes of healthy pregnant women recognize HLA-E on the trophoblast, whereas Vdelta1 cells react with other than HLA Ags. In contrast to Vdelta2(+) lymphocytes from healthy pregnant women, those from women with pathological pregnancies do not recognize HLA-E via their killer-inhibitory receptors and this might account for their high cytotoxic activity.     

Expression of class I HLA genes by trophoblast cells. Analysis by in situ hybridization

Evaluation of trophoblast cells by immunohistology has shown that subpopulations of trophoblast cells express class I HLA differently from one another and differently from embryonic and adult cells. Placental syncytial trophoblast does not express detectable levels of class I HLA; chorion membrane cytotrophoblasts bind one mAb to monomorphic determinants of class I Ag, W6/32, but not a second, 61D2. In the present study, sections of normal term placentae and matching extraplacental membranes were evaluated by in situ hybridization procedures for cells containing class I HLA mRNA using pHLA1.1, which is complementary to HLA-B. Class I Ag expression was identified by immunohistology using two mAb to class I HLA (W6/32, 61D2) and the mAb 4E to identify HLA-B. Placental syncytial trophoblast contained low to undetectable levels of class I mRNA and failed to bind all three mAb. Chorion membrane cytotrophoblast cells contained moderate levels of class I HLA mRNA and were positive with the mAb W6/32 but were negative with 61D2 and 4E. In adjacent tissues, fetal mesenchymal cells and maternal decidual cells contained high levels of class I mRNA and were positive with all three mAb. The results suggest that syncytial trophoblast may not express class I HLA because of low steady-state levels of class I HLA mRNA. In contrast, chorionic cytotrophoblast cells may express truncated versions of class I HLA or nonclassical HLA-A,B,C-like Ag. Regulation of the expression of class I HLA gene products may be essential to the development of a satisfactory immunologic relationship between the mother and her semiallogeneic fetus during pregnancy.     

Association and differentiation of MHC class I and II polymorphic Alu insertions and HLA-A, -B, -C and -DRB1 alleles in the Chinese Han population

In order to investigate the polymorphism of Alu insertions (POALINs) in the HLA region, we genotyped ten Alu loci (AluMICB, AluTF, AluHJ, AluHG, AluHF in the HLA class I region and AluDPB2, AluDQA2, AluDQA1, AluDRB1, AluORF10 in the HLA class II region) to determine their allele frequencies and associations with the HLA-A, HLA-B, HLA-C and HLA-DRB1 genes in the Chinese Han population. Our results showed the ten-loci POALINs varied in frequency between 0.003 and 0.425. By comparing the data of the ten-loci POALIN in Chinese Han with Japanese and Caucasian data, marked differences were observed between the three ethnic groups at the allelic or haplotypic levels. Each POALIN was in significant linkage disequilibrium with a variety of HLA-A, -B, -C and -DRB1 alleles, and was associated with a variety of HLA-A, -B, -C and -DRB1 allele in Chinese Han. This comparative study of multilocus POALINs in the HLA class I and II regions of the Chinese Han population shows that POALINs alone or as haplotypes together with the HLA class I and II alleles are informative genetic markers for the identification of HLA class I and II allele and variations, such as crossing over events within the same and/or different populations.     

The <Emphasis Type="Italic">HLA-G*0105N</Emphasis> null allele induces cell surface expression of HLA-E molecule and promotes CD94/NKG2A-mediated recognition in JAR choriocarcinoma cell line

HLA-G is a non-classical HLA class Ib molecule primarily expressed in trophoblast cells, and is thought to play a key role in the induction of materno-fetal tolerance during pregnancy. In addition, the HLA-G gene provides a suitable leader sequence peptide capable of binding to HLA-E. However, the existence of placentas homozygous for the HLA-G*0105N null allele suggests that HLA-G1 might not be essential for fetal survival. To investigate whether expression of the HLA-G*0105N allele supports HLA-E cell surface expression, we transfected the HLA-G*0105N gene into JAR trophoblast cells. Flow cytometry analysis showed that HLA-G*0105N-transfected cells express surface HLA-E to a similar extent as the unmutated HLA-G gene, whereas HLA-G1 cell surface expression was undetectable. Using the NKL cell line in a standard ⁵¹Cr release assay, the HLA-E molecules were found to inhibit natural killer lysis, through a mechanism partially dependent on CD94/NKG2A-mediated recognition.F.G. Sala and P-M. Del Moral contributed equally to this work.     

Rhesus macaque KIR bind human MHC class I with broad specificity and recognize HLA-C more effectively than HLA-A and HLA-B

Human killer cell immunoglobulin-like receptors (KIR) recognize A3/11, Bw4, C1, and C2 epitopes carried by mutually exclusive subsets of human leukocyte antigen (HLA)-A, -B, and -C allotypes. Chimpanzee and orangutan have counterparts to HLA-A, -B, and -C, and KIR that recognize the A3/11, Bw4, C1, and C2 epitopes, either individually or in combination. Because rhesus macaque has counterparts of HLA-A and -B, but not HLA-C, we expected that rhesus KIR would better recognize HLA-A and -B, than HLA-C. Comparison of the interactions of nine rhesus KIR3D with 95 HLA isoforms, showed the KIR have broad specificity for HLA-A, -B, and -C, but vary in avidity. Considering both the strength and breadth of reaction, HLA-C was the major target for rhesus KIR, followed by HLA-B, then HLA-A. Strong reactions with HLA-A were restricted to the minority of allotypes carrying the Bw4 epitope, whereas strong reactions with HLA-B partitioned between allotypes having and lacking Bw4. Contrasting to HLA-A and -B, every HLA-C allotype bound to the nine rhesus KIR. Sequence comparison of high- and low-binding HLA allotypes revealed the importance of polymorphism in the helix of the α₁ domain and the peptide-binding pockets. At peptide position 9, nonpolar residues favor binding to rhesus KIR, whereas charged residues do not. Contrary to expectation, rhesus KIR bind more effectively to HLA-C, than to HLA-A and -B. This property is consistent with major histocompatibility complex (MHC)-C having evolved in hominids to be a generally superior ligand for KIR than MHC-A and MHC-B.     

HLA-E: strong association with beta2-microglobulin and surface expression in the absence of HLA class I signal sequence-derived peptides

The nonclassical class I HLA-E molecule folds in the presence of peptide ligands donated by the signal sequences of permissive class I HLA alleles, with the aid of TAP and tapasin. To identify HLA-E-specific Abs, four monoclonals of the previously described MEM series were screened by isoelectric focusing (IEF) blot and immunoprecipitation/IEF on >30 single-allele class I transfectants and HLA-homozygous B lymphoid cells coexpressing HLA-E and HLA-A, -B, -C, -F, or -G. Despite their HLA-E-restricted reactivity patterns (MEM-E/02 in IEF blot; MEM-E/07 and MEM-E/08 in immunoprecipitation), all of the MEM Abs unexpectedly reacted with beta(2)-microglobulin (beta(2)m)-free and denatured (but not beta(2)m-associated and folded) HLA-E H chains. Remarkably, other HLA-E-restricted Abs were also reactive with free H chains. Immunodepletion, in vitro assembly, flow cytometry, and three distinct surface-labeling methods, including a modified (conformation-independent) biotin-labeling assay, revealed the coexistence of HLA-E conformers with unusual and drastically antithetic features. MEM-reactive conformers were thermally unstable and poorly surface expressed, as expected, whereas beta(2)m-associated conformers were either unstable and weakly reactive with the prototypic conformational Ab W6/32, or exceptionally stable and strongly reactive with Abs to beta(2)m even in cells lacking permissive alleles (721.221), TAP (T2), or tapasin (721.220). Noncanonical, immature (endoglycosidase H-sensitive) HLA-E glycoforms were surface expressed in these cells, whereas mature glycoforms were exclusively expressed (and at much lower levels) in cells carrying permissive alleles. Thus, HLA-E is a good, and not a poor, beta(2)m assembler, and TAP/tapasin-assisted ligand donation is only one, and possibly not even the major, pathway leading to its stabilization and surface expression.     

Evolutionary relationships of major histocompatibility complex class I genes in simian primates

New World monkeys (NWMs) occupy a critical phylogenetic position in elucidating the evolutionary process of major histocompatibility complex (MHC) class I genes in primates. From three subfamilies of Aotinae, Cebinae, and Atelinae, the 5'-flanking regions of 18 class I genes are obtained and phylogenetically examined in terms of Alu/LINE insertion elements as well as the nucleotide substitutions. Two pairs of genes from Aotinae and Atelinae are clearly orthologous to human leukocyte antigen (HLA) -E and -F genes. Of the remaining 14 genes, 8 belong to the distinct group B, together with HLA-B and -C, to the exclusion of all other HLA class I genes. These NWM genes are classified into four groups, designated as NWM-B1, -B2, -B3, and -B4. Of these, NWM-B2 is orthologous to HLA-B/C. Also, orthologous relationships of NWM-B1, -B2, and -B3 exist among different families of Cebidae and Atelidae, which is in sharp contrast to the genus-specific gene organization within the subfamily Callitrichinae. The other six genes belong to the distinct group G. However, a clade of these NWM genes is almost equally related to HLA-A, -J, -G, and -K, and there is no evidence for their orthologous relationships to HLA-G. It is argued that class I genes in simian primates duplicated extensively in their common ancestral lineage and that subsequent evolution in descendant species has been facilitated mainly by independent loss of genes.     

The chromosome region containing the highly polymorphic HLA class I genes displays limited large scale variability in the human population

The large-scale organization and polymorphism of the HLA class I region was investigated by pulsed field gel (PFG) fractionation of DNA from various HLA-typed cell lines cleaved by different 'rare cutter' restriction enzymes, followed by hybridization with 'general' and locus-specific HLA probes. Results indicate that (i) most HLA class I sequences are contained in a 340 kb MluI DNA fragment which also carries the HLA-A gene; (ii) HLA-A, -B and -C genes are present on different fragments bounded by 'HTF islands' (CpG-rich, unmethylated DNA regions containing multiple sites for 'rare cutter' enzymes) which generally coincide with the 5' regions of expressed genes; and (iii) very little fragment size polymorphism is seen, implying that expansion/contraction events in the HLA class I region due to unequal crossing over (as documented in the mouse class I system) are infrequently found in the human population.