Tissue Typing of Cells in Culture. III. HLA Antigens of Established Human Cell Lines. Attempts at Typing by the Mixed Hemadsorption Technique

Thirty established cell lines from solid human tumors were typed by the mixed hemadsorption technique with respect to the HLA-A and HLA-B and partly also the HLA-C series. All cultures exhibited unique antigenic patterns. The discriminatory potential was further increased very much taking into consideration one as yet undefined antigen series tentatively named Ek-1 to Ek-11. Sixteen cell lines not designated as HeLa cells but on various non-immunological indications suspected to be HeLa cell contaminants, all gave, with our present test, the same antigenic composition as HeLa cells or clones thereof. The mixed hemadsorption reaction is a highly sensitive and convenient method for immunological typing of cells in monolayer cultures. Some important implications of this are discussed.     

The interaction of normal lymphocytes and cells from lymphoid cell lines: IV. HL-A typing of the cell line cells

Cells from thirty-three human lymphoid cell lines and sublines have been typed for HL-A antigens by a microcytotoxicity test. Similar patterns of HL-A antigens were found for the cell line cells and for the fresh lymphocytes of the donor of the line (eleven cases). However, certain typing sera gave positive reactions with the cell line cells which were not found with the fresh lymphocytes. No correlation was noted between the pattern of these `extra' reactions and the HL-A typing of the cells. These same typing sera often gave positive reactions with blood lymphocytes cultured for several days in conventional media. These positive reactions were quantitatively more pronounced and sometimes quantitatively different if the cells had been stimulated. A range of normal sera failed to react with cell line cells suggesting that the HL-A typing sera giving `extra' reactions are detecting antigens in some way related to a histocompatibility system. Absorption studies performed with two of the lines confirmed the HL-A typing by the direct cytotoxicity test. Two sera giving `extra' reactions were also tested in the absorption experiments. The results indicated that antibodies other than those of the HL-A specificity designated for these sera were responsible for the `extra' reactions. It is suggested that `extra' reactions indicate a change in the apparent antigenic expression of lymphoid cells reflecting altered membrane characteristics as they adapt to a culture environment.     

Genetically Appropriate Expression of HLA and DR (IA) Alloantigens on Human Melanoma Cell Lines

Studies of the expression of HLA and DR alloantigens on cultured human melanoma cells in comparison with those expressed on peripheral lymphocytes and B-cells derived from the same patients have shown that the HLA-A,B, and C locus antigens expressed on the cultured tumor cells were consistent with those expected from the typing of peripheral blood lymphocytes. One melanoma cell line failed to express all the HLA antigens expected from donor typing. All five of the lines tested also expressed DR antigens and in three instances these could be demonstrated to have genetically consistent allotypes. However, in preliminary studies stimulation of allogeneic lymphocytes by the DR positive melanoma cells could not be demonstrated.     

HLA-DR and HLA-A, B, C typing of human fetal tissue

In anticipation of clinical trials of fetal pancreas transplantation we have investigated the feasibility of performing HLA-DR and HLA-A, B, C typing on fetal lymphoid cells other than PBL. Using the standard NIH microcytotoxicity test modified for HLA-DR typing it was possible to demonstrate HLA-DR antigens on subpopulations of bone marrow cells and splenocytes but not on thymocytes or hepatocytes. In contrast, HLA-A, B, C antigens could be detected on all four tissues. Excellent HLA-DR typing, confirmed by maternal typing, was obtained for 19 fetuses (14 to 23 weeks old) using bone marrow cells isolated by two-fold purification on discontinuous Percoll buoyant density gradients. Similar purification of splenocytes resulted in weak reactions with anti-DR sera; however, adherent splenocytes recovered from nylon wool columns proved to be primarily DR-bearing and also provided excellent DR typing. As a corollary to these results, non-adhering splenocytes depleted of DR-bearing cells were ideal for HLA-A, B, C typing since spurious reactions due to DR antigens were greatly diminished, whereas strong specific reactions were obtained with anti-HLA-A, B, C sera. Despite weaker reactions with HLA-A, B, C antisera obtained for thymocytes, reliable HLA-A, B, C typing could be obtained when results from thymocytes were evaluated together with typing from bone marrow cells or splenocytes. The possible benefits of fetal HLA typing for fetal pancreas transplantation are discussed.     

Subtypes of HLA-A1 defined on the basis of CTL precursor frequencies

Recently we have shown that limiting dilution analysis can be used to detect cytotoxic T-cell precursor frequencies directed against individual HLA class I antigens. Using the same protocol, we have been able to define two subtypes of HLA-A1, which are indistinguishable by conventional typing sera as well as by cell-mediated lympholysis. One-dimensional isoelectric focusing analysis of the variants did not show any overall charge differences. However, family studies indicated that these HLA-A1 subtypes are genetically determined and can be distinguished on the bases of T-cell precursor frequencies in HLA-A1-negative blood donors.     

Serological Identification of la Antigens: Report of a British Region la Workshop

In preparation for the 7th International Histocompatibility Workshop 13 laboratories in the British Region participated in a local workshop. One hundred and twenty-three sera which had been previously shown to have activity on either normal B cells, CLL cells or B cell lymphoid lines in the absence of HLA-A, B or C activity were exchanged between the laboratories. These sera were tested on a total of 212 B cells, 101 CLL cells, 76 T cells and 76 lymphoid cell lines. The data was collected and analyzed in Oxford. The analysis showed that six groups of sera could be distinguished. When these groups were compared with the D locus typing of some of the lymphoid lines which were derived from individuals used as MLC typing cells, they were seen to have significant associations with D locus antigens. The serological groups defined were therefore given numbers corresponding to the D locus numbers they associate with, i.e. UK1 is associated with DW1 and so on for UK2, 3, 4, 5 and 7. Comparison of typing techniques showed that long incubation both with antiserum and then with complement, 1 hour + 2 hours gave the best and most reproducible reactions on normal B cells. Residual anti-HLA-A, B or C activity in some of the sera even after platelet absorption showed the importance of adequate checking on T cells after absorption.     

A double determinant immunoassay for HLA class I typing using serum as an antigen source

We have applied a double determinant immunoassay (DDIA) to HLA-A2,A28, and B13 typing, using serum as an antigen source. The results obtained show a correlation of 96% (B13) and 89.1% (A2,A28) with the results obtained by conventional HLA typing. Furthermore, the results obtained were highly reproducible, since testing of 18 sera on two occasions gave concordant results with all samples tested. The variation in the content of HLA-A2 antigens in sera taken at different times from a given donor was less than 5%. A sevenfold variation was found in the serum level of HLA-A2,A28 antigens: the highest level was found in the sera from HLA-A2,A28 donors and in decreasing order in HLA-A2 homozygous, HLA-A28 homozygous, HLA-A2 heterozygous, and HLA-A28 heterozygous donors. The results of this study indicate that the DDIA is a sensitive, simple, and reproducible procedure for HLA class I typing. The DDIA offers the following advantages in comparison with the conventional lymphocytotoxic assay: it provides information not only about the expression of a given alloantigen, but also about its level; it does not require viable cells, thus facilitating retrospective studies and typing of leucopenic patients; it eliminates variability of results caused by abnormal susceptibility of target cells to complement-dependent lysis.     

体外培养肿瘤细胞系MHC分型及定量检测

为了对肿瘤细胞进行HLA分型,首先我们应用经肿瘤细胞吸收后的抗血清进行间接淋巴细胞毒试验,对五种人肝癌细胞进行了HLA-A位点常见等位基因A2和A11的检测.结果表明,该方法具有一定的敏感性,对高表达的HLA-A2具有较好的可靠性,但对A11位点检测不明确.使用可获得的抗血清,采用流式细胞免疫荧光法(FACS)进行分析,结果不仅证实了上述结果,而且发现A2和A11分子的表达量普遍低于正常人外周血淋巴细胞.在此基础上,进一步应用该法分析了人肝癌细胞SMMC7721经IFN-γ基因修饰后的HLA-A2表达.     

Antisera Recognising HLA-A, -B and DRW Antigens Raised in Rhesus Monkeys

Rhesus monkeys were immunized with partially purified HLA-A, -B, -C and DR antigens. The resulting sera were shown to have activity against species-specific determinants on both HLA-A, -B, -C chains and beta 2 microglobulin by the use of somatic cell hybrids. When this was removed by absorption, the sera showed activity against three of the four HLA-A and -B antigens in the immunogen when tested on a panel of peripheral blood lymphocytes and T cells. Antibodies recognizing HLA-DR antigens were detected by testing platelet absorbed sera on a panel of typed lymphoblastoid cell lines. After absorption to remove activity against species-specific determinants on the HLA-DR antigens, two cross reacting specificities were defined. One consisted of a determinant in common between HLA-DRw1, 2 and 6 and the other a putative determinant in common between HLA-DRw4, and 5. The nature and significance of these cross-reacting groups of HLA-DR antigens is discussed in the light of current HLA-DR serology and the nature of HLA antigens in general.     

HLA typing of Epstein-Barr virus transformed lymphoblastoid cell lines (LCL)

The application of standard tissue typing techniques to cells other than peripheral blood lymphocytes has been accompanied by the problem of extra reactions. This applies as well to Epstein-Barr virus transformed lymphoblastoid cell lines (LCL) as to leukemic cells and human spleen cells. These extra reactions are attributable to additional antibodies in the typing sera which are not apparent under standard conditions with PBLs. Two types are described: Type 1 extras, which becomes apparent after longer incubation times and are attributed to weak antibodies and type 2 extras which are apparent after shorter incubation times and are attributed to subpopulation specific or differentiation antigens. Technical modifications are proposed by which these extras can be circumvented. They include: Only start typing when cells have been cultured for 2 to 3 days. Remove dead cells by spinning over standard ficoll-hypaque or 11% triosil. Use shorter incubation times. Avoid using sera that give too many type 2 extras. In this way phenotypes can be accurately identified on LCL's obtained from kidney transplant donors and recipients. When LCL's were compared with their matching PBL, HLA phenotypes were concordant in 87% of cases for HLA-A, 90% for HLA-B, 81% for HLA-C and 70% for HLA-DR.     

HLA-D and -DR antigens on human amniotic fluid cells. II. Heterogeneous expression of HLA-DR and other cell surface markers

To evaluate further the feasibility of HLA typing for prenatal diagnosis, we tested human amniotic fluid cells (AFC), known to express HLA-A, -B, and -C antigens, for the presence of HLA-DR antigens using type-specific antisera in the microcytotoxicity assay and a monoclonal antibody directed against the common HLA-DR structure (cDR) in indirect immunofluorescence. Prenatal typing of HLA-DR on AFC in the microcytotoxicity test was possible in only one out of eight families studied. The detected DR2 antigen was confirmed by postnatal typings of cord blood lymphocytes. Thereafter, 23 different AFC cultures were tested with monoclonal antibodies in indirect immunofluorescence. Only six cultures were partially positive (23-35% fluorescent cells) with the monoclonal cDR antibody while all AFC cultures demonstrated strong positive fluorescence (68-100%) with a monoclonal antibody against the common HLA-A, -B, and -C structure (cHLA). These data suggest that only a small subpopulation of AFC expresses class II (HLA-DR) antigens in contrast to the nearly ubiquitous expression of class I (HLA-A, -B, and -C) antigens. Furthermore, the heterogeneous expression of cell surface antigens within the various AFC cultures was substantiated with monoclonal antibodies directed toward cell surface antigens of the OKT, OKM, and Lyt series that have been found to be characteristic for subpopulations of lymphoid and hemapoetic cells. Thus, at present, HLA-DR typing is not reliable for prenatal diagnosis.     

Complement-Fixing Antigens in Lymphoid Cell Lines of American Origin

Lymphoid cell lines from Americans with infectious mononucleosis (Choate, EH IV, and OP), and from a normal American (Cassio), were tested for presence of herpes-like virus by electron microscopy (EM) and immunofluorescence (IF), and for complement-fixing (CF) antigens, using both American and African sera. Whereas earlier tests of seven African (Burkitt) lymphoma cell lines showed an absolute correlation between presence of herpes-like virus and CF reactivity with either African or American sera, the same was not true of the American cell lines. Herpes-like particles were found in the Cassio line by both EM and IF, and in a few cells of the OP line by IF, but not by EM. The virus was not found in the Choate or EH IV lines by either EM or IF. African sera from either normal individuals or patients with Burkitt lymphoma contained CF antibodies to extracts of Cassio and OP cells. Normal American sera contained CF antibodies to these extracts as well as to extracts of Choate cells. The EH IV cell line did not produce CF antigens detectable with either African or American sera. The data indicate that the CF antigens of the herpes virus-negative Choate cell line were serologically distinct from those in Burkitt lines. However, it is possible that the antigens from both sources are related to the presence of the genome of the herpes-like virus.     

Lymphocytes modulated by β interferon express new non-HLA-A,B,C class I antigens

Peripheral blood lymphocytes were cultured with recombinant β interferon (INF-lymph). Three days in culture and 5 × 10⁴ units/ml produced the best modulatory effect. Platelet absorbed alloantisera previously shown to contain non-HLA-A,B,C class I antibodies were tested with the INF-lymph. The serologic reactivity could be blocked by turkey sera to human B₂-microglobulin but not by turkey anti-Ia-like. Absorption studies with INF-lymph obtained from donors expressing different HLA antigens indicated that the serologic reactivity is not due to HLA. Fourteen sera appeared to cluster in three groups, only one of which was found to be associated with HLA (HLA-A1). Antigenic modulation of INF-lymph obtained from HLA-A1⁺ individuals with HLA-A1 typing alloantisera eliminated subsequent lysis mediated by HLA-A1 alloantibodies and complement but not lysis with platelet absorbed antibodies included in the cluster associated with HLA-A1. These findings suggest independence from HLA-A1 and coexpression of HLA-A1 and A1-associated class I antigens. Family studies indicated that the reactivities segregated with HLA thus mapping it to chromosome VI. Similar to what we have described with PHA, β interferon modulates lymphocytes and induces the expression of new class I differentiation antigens probably analogous to the murine Qa-T1a antigens.     

Identification of HLA-linked antigens by pregnancy-associated non-cytotoxic alloantisera

Non-cytotoxic sera obtained from post-partum primiparous and multiparous women were examined by a rosette inhibition technique for the presence of antibodies mediating blockade of human B lymphocyte Fc receptors. Selective activity was demonstrated against a panel of normal human B lymphocytes and lymphocytes from patients with chronic lymphocytic leukaemia (CLL). A pattern of specific activity was found in sera and in their IgG fractions, which was not accounted for by antibodies directed to known HLA-A, -B or -DR antigens. Several sera were identified with selective activity in this assay. As the results of testing sera in a direct binding assay correlated with those of the EA inhibition assay, and since EA inhibitory activity occurred in F(ab')2 fractions of sera, it is possible that these non-cytotoxic antibodies bind directly to B cell surface antigens. Sera may therefore have been identified which possess antibodies to hitherto undefined HLA antigens.     

A simple and rapid method for the detection of lymphocytotoxic antibodies using cell panels frozen on Terasaki plates

A 40 cell panel of lymphocytes selected for HLA-A,B and DR antigens, frozen in Terasaki microtest trays can be used routinely to identify the presence of specific HLA antibodies within two hours. Blind testing using well defined HLA typing sera showed that specificities could be identified to a high degree of significance using this method. The method has proved successful for screening for T cell, including HLA-A,B and B cell, including HLA-DR antibodies. The method is particularly useful for the routine tissue typing laboratory as the frozen panel can be used without the need for complicated and time-consuming cell washing procedures which have been the downfall of previously published methods.     

基于测序的人类白细胞抗原分型和PCR短串联重复序列技术在人胚胎干细胞鉴定中的应用

目的 探讨基于测序的人类白细胞抗原分型(HLA-sequencing-based typing,HLA-SBT)和PCR短串联重复序列(short tandem repeat,STR)技术在人胚胎干细胞(human embryonic stem cell,hESC)应用前检测中的运用,建立人胚胎干细胞系的基因型档案.方法 人胚胎干细胞系SYSU-I、SYSU-3,分别培养到20代、40代,应用PCR寡核苷酸特异测序探针(sequence specific olignucleotide probe,SSO)技术检测两株细胞系的HLA-A、-B、-DR位点的低分辨分型,再利用HLA-SBT技术检测两株细胞系的HLA-A、-B、-DR位点的高分辨分型.应用PCR-STR技术检测两株细胞系的基因遗传标记.结果 获得两株hESC细胞系的HLA高分辨分型和STR基因型.结论 可以运用HLA-SBT和PCR-STR技术建立人胚胎干细胞应用前的基因型档案.     

基于测序的人类白细胞抗原分型和PCR短串联重复序列技术在人胚胎干细胞鉴定中的应用

目的探讨基于测序的人类白细胞抗原分型(HLA-sequencing-based typing,HLA-SBT)和PCR短串联重复序列(short tandem repeat,STR)技术在人胚胎干细胞(human embryonic stem cell,hESC)应用前检测中的运用,建立人胚胎干细胞系的基因型档案。方法人胚胎干细胞系SYSU-1、SYSU-3,分别培养到20代、40代,应用PCR寡核苷酸特异测序探针(sequence specific olignucleotideprobe,SSO)技术检测两株细胞系的HLA-A、-B、-DR位点的低分辨分型,再利用HLA-SBT技术检测两株细胞系的HLA-A、-B、-DR位点的高分辨分型。应用PCR-STR技术检测两株细胞系的基因遗传标记。结果获得两株hESC细胞系的HLA高分辨分型和STR基因型。结论可以运用HLA-SBT和PCR-STR技术建立人胚胎干细胞应用前的基因型档案。     

Cell Mediated Lympholysis in Man: An Attempt to Type with Cytotoxic Lymphocytes

From approximately 3,000 CML combinations, originally established in order to evaluate the qualitative and quantitative influence of the serologically defined HLA-A, B, and C antigens on cellular, complement independent cytolysis, 12 combinations were selected yielding reproducible positive cytolysis on allogenic target cells, although no HLA-antigenic sharing could be demonstrated between stimulator and target lymphocytes. These 12 CytoToxic Lymphocytes (CTL's) have been tested in parallell as "CML typing combinations" against lymphocytes from a random population sample of 100 unrelated Danes. Based on a pairwise analysis 11 of these CTL's could be classified into two groups of significantly correlated CTL's. These two groups do not define monospecific traits of allelic genetic origin as judged by a mutually positive correlation and a poor fit to Hardy-Weinberg equilibrium. The traits defined by these groups may be either partially identical or governed by closely linked loci. The same groups were identified and the same conclusions reached after exclusion of those individuals in the population sample where HLA-A, B, C, or D antigens may be targets for destruction. Thus, this study gives direct evidence that known HLA antigens are not sole target determinants in CML or that cytotoxic lymphocytes recognize HLA molecules in a different way than lymphocytotoxic antibodies. The studies underline the immunogenetic complexity of CML although this reaction is most probably governed by genes in the HLA region. It is suggested that cytotoxic lymphocytes may recognize "backbone structures" of the HLA molecules.     

New human MHC class I antigens segregating with HLA-A antigens detected on some lymphocyte subpopulations

Recent genetic studies of the murine chromosome 17 have demonstrated that many genes encode class I antigens, most of which are still not detected serologically; most of these genes belong to the Tla region. Five human alloantisera were selected from 383 female sera and were further studied using a panel of peripheral blood lymphocytes (PBL), B lymphocytes (BL), and PHA activated lymphocytes (PHA-L) from the same blood donors. After intensive platelet absorption, the five sera still reacted positively by a complement-dependent cytotoxicity technique with PHA-L, but negatively with PBL and BL. The antigens detected by these antibodies segregated with an HLA-A allele and were assumed to belong to the class I antigen series as they could be blocked by a turkey anti-beta 2 microglobulin serum. They were found on some lymphocyte populations: PHA-L, common acute lymphoblastic leukemia (cALL) cells, and (preliminary results) a small subpopulation of PBL cells (mostly NK cells), but were not found on chronic lymphocytic leukemia (CLL) cells, T and early T acute lymphoblastic as well as myeloblastic leukemia cells. Kinetic studies showed that several hours of culture with PHA were necessary for the antigen to be expressed. These results show that the antigens described do not belong to the classic HLA antigen series but could be considered to belong to the human Qa-like antigens or to be the human counterpart to the second murine H-2K locus antigens.