Permanent Lymphoid Lines from Genetically Marked Lymphocytes: Success with Lymphocytes Recovered from Frozen Storage

Permanent human lymphoid cell lines were established successfully from peripheral blood lymphocytes which had been separated for HL-A typing and stored in liquid nitrogen for two years. Frozen lymphocytes were chosen from two siblings who were homozygous at the LA and FOUR HL-A loci. Thawed lymphocytes were transformed with EB virus produced by the marmoset lymphoid line B95-8. No chromosome abnormalities were seen on karyotypes prepared on cells from the established human lymphoid lines using G and Q banding techniques. HL-A typing showed the expected HL-A antigens plus a considerable number of additional reactions. Separation of lymphocytes and freezing them for possible future use requires a relatively small investment. This method of preserving cells can be applied to patients with interesting genetic disorders or other biochemical markers to provide cells which can be transformed and propagated years later.     

Antigenic Expression and Cross Reactions in HL-A Variants of Lymphoid Cell Lines

Immunogenetic studies were carried out on an HL-A2/HL-A3 heterozygous lymphoid cell line, 8866, and on two HL-A2 negative variant sublines of 8866 which had arisen independently of one another. The parent 8866 line reacted positively with some W28 sera: these cross reactions were absent in both HL-A2 "loss" variant sublines. Other serum reactions, apparently characteristic of lymphoid cell lines, were present in 8866 and were retained in the variants. HL-A3 was expressed in hemizygous rather than homozygous dosage in the variants, ruling out mitotic crossing over as the mechanism producing HL-A2 loss. HL-A2 was undetectable either in the disrupted membranes or in the cytosol of the variant cells.     

Tissue Typing Antisera from Immunization by Pregnancy

Anti-HL-A antibodies developing during pregnancy were investigated and identified in 1400 pregnancy sera. Of these 169 (12 %) were observed to be strongly reactive with at least 1 out of 20 lymphocytes. A panel of 33 lymphocyte donors phenotyped for 22 HL-A antigens was used for the determination of antibody specificity in the reactive sera. During this investigation 11 sera showing a perfect or almost perfect correlation with one known HL-A specificity were identified. Fifteen other useful sera showed correlation with more than one HL-A specificity. Eight additional sera reacted with patterns that did not fit a defined group but suggested the possibility of reactions with as yet unidentified HL-A antigens. The results of this study demonstrate that the development of tissue typing reagents from pregnancy sera in a clinical tissue typing laboratory can be highly productive.     

A New Alloantigenic System on Human Lymphocytes

Certain otherwise well-defined HL—A typing sera contain extra antibodies which do not appear to be closely related to known HL-A specificities and which react in the cytotoxic test with chronic lymphatic leukemia lymphocytes but not with normal lymphocytes. Despite nonreactivity in the cytotoxic test, absorption studies show that the antigenic factors are well represented on normal lymphocytes. Based on cross-absorption experiments using both normal and leukemic cells, followed by back-testing with the cytotoxic test against a leukemic cell panel, evidence is presented suggesting the existence of a complex, probably multiple allelic system on human lymphoid cells which appears distinct from the HL-A system, and is definable by serologic techniques.     

Sensitivity of Various HL-A Typing Techniques

It has become apparent that the crossmatch procedures for the HL-A typing of organs, tissues and leukocytes for transplant and transfusion recipients are assuming a more prognostic role than conventional HL-A typing. Recent data has shown that regardless of the grade of the HL-A match, a positive crossmatch invariably indicates a troublesome clinical course for the patient. The antiglobulin lymphocytotoxicity technique is a relatively new HL-A crossmatch procedure. This technique was evaluated against the NIH standard lymphocytotoxicity test, with standardized HL-A typing sera. Pre- and post-transplant sera from several patients were compared using the two test systems in an attempt to show correlations with clinical courses. In addition, sera from patients who exhibited febrile reactions, which were not attributable to red cell incompatibilities were evaluated for crossmatch and conventional HL-A incompatibilities.     

Serologic Specificity and Crossreactivity of Soluble HL-A Alloantigens

The serologic activity of soluble human histocompatibility (HL-A) antigens extracted from four human cultured lymphoid cell lines by the 3M KCI method has been evaluated by their ability to inhibit specifically the cytotoxicity of 38 operationally monospecific alloantisera recognizing 18 HL-A specificities. Several HL-A alloantisera directed against the same HL-A specificity, although used at functionally comparable levels of activity, varied in their susceptibility to inhibition by the same soluble HL-A alloantigen preparation. Similarly, lymphocytes from a number of subjects, when used as target cells for the same alloantiserum, detected quantitatively different activities of a given soluble HL-A alloantigen preparations in the inhibition test. Soluble HL-A alloantigens can block the cytotoxicity of antisera directed against HL-A specificities either crossreacting with or present on the cultured lymphoid cells used as source for extraction of soluble HL-A alloantigens. Crossreactivity was exhibited only by some of the soluble alloantigens and alloantisera with a given HL-A specificity tested. Quantitative studies indicate that the amount of soluble HL-A antigens necessary to inhibit the cytotoxicity of operationally monospecific alloantisera (used at the highest dilution producing 95% lysis) directed against crossreacting specificities is significantly greater than that necessary to inhibit alloantisera directed against HL-A determinants present on the soluble HL-A antigens. Since the alloantisera are used at relatively high dilutions, these data suggest that the crossreactivity in the HL-A system is not caused by contamination of operationally monospecific alloantisera with antibodies of low avidity. On the contrary, these data substantiate the hypothesis that crossreactivity is caused by structural similarities among different HL-A antigenic determinants.     

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.     

Serologic Specificity and Crossreactivity of Soluble HL-A Alloantigens

The serologic activity of soluble human histocompatibility (HL-A) antigens extracted from four human cultured lymphoid cell lines by the 3M KCI method has been evaluated by their ability to inhibit specifically the cytotoxicity of 38 operationally monospecific alloantisera recognizing 18 HL-A specificities. Several HL-A alloantisera directed against the same HL-A specificity, although used at functionally comparable levels of activity, varied in their susceptibility to inhibition by the same soluble HL-A alloantigen preparation. Similarly, lymphocytes from a number of subjects, when used as target cells for the same alloantiserum, detected quantitatively different activities of a given soluble HL-A alloantigen preparations in the inhibition test. Soluble HL-A alloantigens can block the cytotoxicity of antisera directed against HL-A specificities either crossreacting with or present on the cultured lymphoid cells used as source for extraction of soluble HL-A alloantigens. Crossreactivity was exhibited only by some of the soluble alloantigens and alloantisera with a given HL-A specificity tested. Quantitative studies indicate that the amount of soluble HL-A antigens necessary to inhibit the cytotoxicity of operationally monospecific alloantisera (used at the highest dilution producing 95% lysis) directed against crossreacting specificities is significantly greater than that necessary to inhibit alloantisera directed against HL-A determinants present on the soluble HL-A antigens. Since the alloantisera are used at relatively high dilutions, these data suggest that the crossreactivity in the HL-A system is not caused by contamination of operationally monospecific alloantisera with antibodies of low avidity. On the contrary, these data substantiate the hypothesis that crossreactivity is caused by structural similarities among different HL-A antigenic determinants.     

Antibodies to Surface Antigens of Lymphocytes and Lymphoblastoid Cells in Cold-Agglutinin-Positive Sera from Patients with Mycoplasma pneutnoniae Infection

IgM antibodies reacting in the cold with surface antigens of normal human blood lymphocytes were demonstrated by indirect immunofluorescence (IFL) in cold-agglutinin-positive sera from 21 patients with Mycoplasma pneumonias (MP) infection. Twenty to fifty per cent of the lymphocytes were stained. MP sera reacted also with 75%-100% of cells from two lymphoblastoid cell lines and one Burkitt lymphoma line and with about 80% of peripheral blood lymphocytes from one patient with chronic lymphatic leukaemia. The IFL reaction was negative with cells from a leukaemic lymphoid T-cell line (Molt-4). Lymphocyte fractionation experiments gave no evidence of a selective reactivity of MP sera with B or T lymphocytes of peripheral blond. Removal of cold agglutinins from the MP sera by absorption with human O erythrocytes significantly reduced the IFL reactivity with lymphocytes and lymphoblastoid cells, indicating the presence of I antigen on these cells. Furthermore, lymphoblastoid cells but not Molt lymphoid cells absorbed out most of the cold erythroagglutinins.     

HL-A Typing of Cultured Peripheral Lymphoblastoid Cells

The HL-A antigens of a number of cultured cell lines established from peripheral blood leucocytes have been determined over a period of months, and changes in the expression of these antigens noted. The HL-A profile of some of the original donors of the cells could be studied and compared to those of the cultured cells: HL-A antigens appeared and disappeared in culture. An additional property of the lymphoblastoid cells was noted in the cytotoxic effect of a number of HL-A typing sera on such cells, irrespective of their HL-A phenotype.     

Immunocytochemical and ultrastructural characterization of human T-lymphotropic virus type I (HTLV-I)-producing rabbit lymphoid cell lines

Summary Fine structural and immunocytochemical characterization of rabbit lymphoid cell lines transformed by human T-lymphotropic virus type I (HTLV-I) was carried out. All nine cell lines tested were reactive with anti-HTLV-I-positive human, monkey, and rabbit sera and monoclonal antibody to HTLV-Ip 19, but not with anti-HTLV-I-negative sera and monoclonal antibodies to human Ia and pan-T antigens. All cell lines were strongly positive for monoclonal antibodies to rabbit Ia and pan-T antigens. Ultrastructurally, each cell line contained C-type virus particles in varying numbers in the extracellular space. These particles showed replication patterns similar to those in HTLV-I or simian T-lymphotropic virus type I (STLV-I)-producing human or monkey cells. In addition, anti-HTLV-I-positive rabbit serum gave positive immunoreactivity to HTLV-I or STLV-I by indirect immunoferritin method.These results indicate that the ultramorphology and replication patterns of HTLV-I in rabbit cell lines are indistinguishable from those of HTLV-I in human and monkey cell lines, HTLV-I in rabbit cells shares the common surface antigenic determinants with HTLV-I or STLV-I in human or monkey cells, and that these cells are definitely rabbit T cells bearing their own Ia antigens.     

Reactivity of monoclonal antibodies against human leucocyte antigens with lymphocytes of non-human primate origin

The phylogenetic distribution of antigens present on human lymphocytes was investigated by incubating human or simian cells with murine anti-human monoclonal antibodies and then determining the level of reactivity with a radiolabelled anti-murine IgG reagent. The monoclonal antibodies used were specific for a T-cell antigen, lymphoid and lymphoid:myeloid antigens, Ia antigens, and beta 2 microglobulin. The cells examined included B- and T-lymphoblastoid cell lines and fresh peripheral blood lymphocytes separated by sheep erythrocyte rosetting into T-cell and non T-cell fractions. Results of these studies showed that the antibodies gave complete cross-reactivity with gorilla and chimpanzee cells while B-cell lines of orangutan origin had lost lymphoid and beta 2 microglobulin markers. Gibbon cells and cells of Old World and New World monkeys reacted strongly only with monoclonal antibodies against Ia antigenic determinants. These Ia antigens were found on the non T-cell fraction of fresh peripheral lymphocytes, on B-cell lines and on some virus induced T-cell tumour lines. Immunoprecipitation analysis using the anti-Ia antibodies showed a degree of molecular diversity on owl monkey and marmoset cells compared to the Ia antigens associated with human cells.     

Production of Anti-W24 Xenoantisera in Rabbits1

One out of four rabbits injected with partially purified soluble HL-A antigen from serum produced cytotoxic antibodies to human lymphocytes. These antibodies appear to be directed to the specificity W24 as determined by correlation studies with HL-A alloantisera and by absorption-inhibition experiments with human platelets and with serum soluble HL-A antigens. In the sera from the other three immunized rabbits, no antibodies to human lymphoid cells could be detected by the cytotoxic test, blocking of T cell rosette formation, stimulation of DNA synthesis and inhibition of mixed lymphocyte reaction.     

Independence of HL-A antigens and immunoglobulin determinants on the surface of human lymphoid cells

Membrane-bound HL-A antigens and μ-chain determinants were studied on normal and leukemic lymphocytes and on a tissue culture cell line derived from a Burkitt's tumor, using membrane immunofluorescence on living cells. A redistribution of both antigens at the cell surface occurred when cells labeled in the cold by conjugated antisera were incubated at 37 °C. Double labeling experiments showed that HL-A antigens, either of the first or the second sublocus, were unaffected by the redistribution of IgM determinants and vice versa. This result strongly suggests molecular independence of these two systems. When redistribution was induced with a mixture of conjugated sera to HL-A and Ig, the caps showing each of the two specificities were located at the same pole of the cells.     

Detection of HLA antigens on lymphoblastoid and epithelial cell lines and cross-reactivity of HLA-Cw5 and HLA-Cw8

Antibody-dependent cell-mediated cytotoxicity was used to detect HLA antigens on tissue cultured lymphoblastoid cells (phytohemagglutin blasts and Epstein-Barr virus lines) and transitional cell carcinomas. The results agreed with those obtained on fresh peripheral blood lymphocytes by conventional HLA typing. The same HLA antigens were detected on cells from an individual irrespective of their tissue origin or length of time in vitro. Antibody-dependent cell-mediated cytotoxicity (ADCC) showed that HLA-Cw5 and HLA-Cw8 were cross-reactive. An HLA-Cw5 antiserum that was negative for HLA-Cw8 positive cells in complement-mediated lymphocytotoxicity reacted strongly with HLA-Cw8 donor cells in ADCC. Similarly HLA-Cw8 antibodies were detected in HLA-B14 antisera, which reacted on all HLA-Cw5-positive donor cells. Absorption of sera with HLA-Cw5-positive lymphoid cells removed HLA-Cw5 and HLA-Cw8 specificities but spared HLA-B14. Absorption of HLA-B14 antisera with HLA-B14/Cw8-positive cells removed HLA-Cw5, HLA-Cw8, and HLA-B14 reactivities. Sequential immune precipitation and gel electrophoresis confirmed that HLA-Cw5 and HLA-Cw8 were cross-reactive and that HLA-B14 was physically separable from HLA-Cw8.     

Continuous in vitro production of IgA by a human colostral immortalized cell line

Breast milk samples from 8 postpartum mothers were collected and incubated with Epstein-Barr virus, immortalizing their B-lymphocytes and giving rise to lymphoblastoid cell lines. Three samples showed no growing lymphocytes and five samples gave rise to transformed cell lines. IgA positive cells were selected from one such cell line by rosetting with anti-IgA-coated sheep erythrocytes. The emerging cell line was similarly reselected giving rise to an IgA-surface positive lymphoblastoid cell line. Monoclonal IgA-producing cell lines were obtained by cloning in the soft agar method. IgA is continuously secreted by the cell lines which have been growing in vitro for more than 24 mth.     

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.     

Serological studies of HL-A on continuous lymphoblastoid cell lines (CLC) and the definition of an antigen on CLC determined by a heat-labile antibody

Continuous lymphoblastoid cell lines (CLC) are more reactive with HL-A antisera in a complement-dependent cytotoxic test than are peripheral blood lymphocytes (PBL). This additional reactivity leads to assignment to a given CLC of more than four HL-A antigens, the maximum allowable under the two locus concept of the genetic control of HL-A. However, absorption of antisera by CLC shows that no more than four HL-A antigens exist on any of the CLC used in this laboratory. The additional reactivity of these cells lines can be explained in three ways. Firstly, it may be due to the presence of sublytic amounts of HL-A antibody in operationally monospecific antisera. Secondly, it may be due to cross-reactivity between HL-A antigens. Both these findings can be explained on the basis of the increased quantity of HL-A antigens on CLC compared to PBL. Thirdly, it may be due to the presence of a heat-labile (56° for 30 min) complement-dependent cytotoxic antibody which is present in 90% of normal human sera, and detects an antigen group tentatively labelled `D'.     

HL-A antigens in juvenile rheumatoid arthritis.

HL-A antigens were examined in sixty-two patients, thirty-one boys and thirty-one girls, diagnosed as having JRA. HL-A 27 was the only antigen with a significantly increased frequency. This increase concerned predominantly male patients in whom the disease developed at the beginning of puberty. In this group of sixteen boys, the polyarticular form was more frequent and tended to be associated with sacroliitis, while the rheumatoid factor was negative in all but one. The frequency of HL-A 27 in this group was 65.5%. The patients were also examined by the lymphotoxic test with anti H-2 alloimmune sera known to exhibit a high degree of association with some HL-A antigens. On JRA lymphocytes these associations were confirmed for HL-A 2 and anti-H-2f and for HL-A 27 and anti-H-2p. The strongest reactions were observed with lymphocytes from male patients around the age of puberty. These data indicate that steroid hormones influence the expressivity of some HL-A associated cell plasma membrane structures.     

Transmission of human T-cell leukemia virus type I to an S+L- cat kidney cell line

The S+L- cat kidney cell line CCC was cocultivated with lethally irradiated human lymphoid cell lines that were producing human T-cell leukemia virus type I (HTLV-I). Eight of nine S+L- CCC sublines that had been cocultivated with nine different HTLV-producing T-cell lines gave positive reactions for HTLV antigens by indirect immunofluorescence assay. One subline CCC/2M was cloned. The percentages of fluorescent cells differed markedly in different sublines and clones. Southern blot hybridization with HTLV probes and electrophoresis of immunoprecipitates indicated that defective HTLVs were often transmitted into S+L- cat cells. S+L- CCC cells were permissive for HTLV and the properties of HTLV-infected cat cells were heterogeneous.