Surface molecules of cultured human lymphoid cells.

Cell surface molecules of cultured human lymphoid cells were selectively labeled by lactoperoxidase-catalyzed iodination and examined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Two major iodinated species with apparent mol. wts. of 27 000 and 35 000 daltons were detected on autoradiographs of the labeled proteins of human lymphoid cell lines believed to be of thymus-independent (B) cell origin. Neither molecule was detected on putative thymus-dependent (T) lymphoid cell lines. Metabolic labeling studies showed that both molecules are glycoproteins. Rabbit antisera against cultured B lymphoid cells made specific by absorption for B cells reacted with several labeled species from iodinated B cells including the 27 000 and 35 000 mol. wt. glycoproteins. These molecules were also detected on tonsillar lymphocytes but not on peripheral blood lymphocytes. Reciprocal absorption with B cells of rabbit antisera against cultured T cells gave antisera specifically cytotoxic for T cells. However, these sera did not precipitate iodinated proteins from Nonidet P-40 lysates of T cells.     

Anomalous reactivity of anti-T cell sera on spleen cells from T-cell-deprived mice and on mitogen-stimulated nude mice spleen cells

Recent results have shown that various alloantisera which react in the expected fashion on lymphocyte populations can react anomalously against cultured tumor cell populations because of the presence of contaminating antibodies against murine leukemia virus (MLV) antigens. Since it is now known that activation of MLV can occur in certain types of dividing lymphocyte populations, anti-T cell sera were tested on lymphoid cell populations in which T cells were absent or greatly reduced in numbers, but where activation of MLV in the B cell population would be expected. Whereas normal, freshly harvested, nude mice spleen cells were unreactive, in vitro:stimulation of these cells with the B cell mitogen, lipopolysaccharide, led to a high degree of sensitivity to the cytotoxic effects of anti-T alloantisera or heteroantisera. Spleen cells from adult thymectomized, lethally-irradiated, bone marrow-reconstituted mice also showed unexpected reactivity with the anti-T cell sera. In both cases, reactivity was noted only if absorbed rabbit serum was used as a complement source in the cytotoxic assays. The anomalous anti-B cell activity of the anti-T cell sera could be removed by absorption with relatively small numbers of cells from Thy-negative cultured tumor cell lines, including fibrosarcomas, but not by absorption with thymocytes. Hence, activated or stimulated B cells may react strongly with allo-or hetero- anti-T cell sera under certain conditions, and this anomalous reactivity appears unrelated to the presence of the anti-T cell antibodies in the sera.     

Screening and Use of High Titered Anti–HLA–DR Sera in PHA-Blast Complement Fixation and B-Lymphocytotoxicity Techniques

Screening for anti-HLA-DR sera was performed by complement fixation on PHA stimulated peripheral blood lymphocytes (PHA-CF), or cultured B lymphoid cell lines. Out of 1,350 sera from multiparous women, multitransfused patients, and patients transfused during extra-corporal circulation (ECC), 219 contained anti-HLA-DR activity (16.2%). Anti-HLA-DR antibodies developed after ECC were often high titered (1:10 to 1:100). In half of these sera anti-HLA-A, B antibodies were weak or absent, making it possible to use them as anti-HLA-DR reagents without platelet absorption. Of the 219 positive sera 51 contained defined anti-DR antibodies (20 monospecific and 31 bi-or multispecific). The 13 best sera recognized DR1 to DR7 specificities with r values from 0.83 to 1.
Twenty-four sera selected by CF were also studied by lymphocytotoxicity technique against peripheral blood B lymphocytes (B-LCT). Both PHA-CF and B-LCT techniques gave similar results, detecting the same specificities and showing comparable sensitivity.
The advantages of CF are: easy storage of target cells at −80°C or +4°C, and fast reading. For these reasons PHA-CF or CF on cultured B lymphoid cell lines can be proposed for large scale screening of anti-HLA-DR sera. The sera thus screened can be used for HLA-DR typing either by PHA-CF or B-LCT.     

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.     

Characterization of antigens on murine tumor cells reacting with alloantisera against foreign H-2 specificities: analysis by absorption with purified murine leukemia virus and normal lymphoid cells of different H-2 haplotypes.

A spontaneous T cell lymphoma of DBA/2 (H-2-d) mice, SL2, was found to react with anti H-2 typing sera raised against certain foreign haplotypes as well as with anti H-2d sera. The cytotoxic anti-SL2 activity of the anti-foreign H-2 sera was detected in a newly developed microradioassay, not however, in a conventional 51Cr release test. Upon culture in vitro the reactivity of the tumor cells with the anti H-2 sera decreased. The anomalous cytotoxic anti-tumor activity of the anti-foreign H-2 sera appeared to be distinct from anti-murine leukemia virus activity, since it was not removed by absorption with either Friend of AKR leukemia virus. Partial absorption was observed with normal lymphoid cells carrying the respective foreign H-2 antigens, but not with cells of unrelated H-2 haplotypes. In each serum tested, the anti-tumor activity could also be absorbed with syngeneic H-2d lymphoid cells. These results show that the anomalous anti-tumor reactivity of certain anti H-2 typing sera, in particular of sera raised in recipients differing in H-2 from the tumor host strains, is not due to the presence of foreign (derepressed) H-2 molecules on the tumor cells. The differences observed between the tumor cells and normal cells seem to be due to unexpected antibodies in the sera reacting with public H-2 specificities which are better exposed on the tumor cells than on normal cells.     

Identification of four SB antigens by cloned cells: Population studies of Norwegians

By priming in vitro with allogeneic HLA-DR compatible and also HLA-A,B mostly compatible lymphoid cells, PLT cells resulted in recognizing a group of non D/DR allelic antigens provisionally named K, L, M and N. To improve discrimination these bulk primed typing reagents were cloned and expanded. By typing of previously SB typed lymphoblastoid B cell lines (LCL) the provisional specificities could be identified as SB1, 4, 3 and 2, respectively. Typing of 186 unrelated Norwegians gave the following gene frequences: SB1: 0.05, SB2: 0.16, SB3: 0.13, SB4: 0.42 and SB blank: 0.24. No triplets were found, the calculated gene frequencies fit with Hardy-Weinberg equilibrium, and typing of a B-DR recombinant family confirmed that the SB locus is situated centromeric to B. Associations between SB and A, B, DR antigens in the same material were generally weak, the most significant associations found were between SB1-DR3 and SB4-DR2.     

Immunocytochemical staining of T and B lymphocytes in serous effusions

Cell smears from serous effusions containing large numbers of lymphoid cells were stained by the alkaline phosphatase-anti-alkaline phosphatase technique with a panel of monoclonal antibodies, including anti-B and anti-T cell antibodies and anti-HLA-DR. Samples from 17 patients with lymphoproliferative disorders--such as chronic lymphocytic leukaemia and non-Hodgkin's lymphoma--and from 19 patients who had no evidence of lymphoid neoplasia--for example, cases of carcinoma, cardiac failure--were investigated. The majority of lymphoid cells in reactive effusions were T cells, which lacked HLA-DR and showed a marked excess of helper/inducer cells (mean helper to suppressor ratio of 3 X 5). In contrast, lymphoid cells in samples from nine cases of B cell neoplasia were positive for B cell antigen and HLA-DR. In a further four B cell neoplasms most lymphoid cells were reactive T cells. Two cases of T cell lymphoid leukaemia could also be characterised by immunocytochemical staining, both being classified as T helper cell neoplasms. Labelling was performed on routinely prepared, air dried cell smears, which could be stored in the unfixed state for long periods before staining. The technique may therefore be of use in many clinical cytology laboratories for the diagnosis of effusions containing numerous lymphoid cells.     

A Screening Program for Anti-DR Typing Reagents

A total of 694 sera have been tested in a screening program aimed at identifying monospecific reagents for HLA--DR typing. All sera were first tested on a panel of enriched B and T cells without absorption on platelets. Only sera reacting more frequently on B than on T lymphocytes were absorbed on platelets and retested on the panel. This procedure saved a considerable amount of platelets and proved to be reasonably efficient. One-hundred-and-fifty sera were found to contain an anti-B cell antibody. Significantly less frequent B cell reactivity was found when HLA--A, --B, --C antibodies could not be detected. Twenty-five sera were demonstrated to be specific for well-defined DR antigens.     

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.     

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.     

T and B Cells in Various Lymphoid Tissues in Mice: Membrane Immunofluorescence with Purified Lymphoid Cells

The proportion of T and B cells in several lymphoid tissues of the mouse was determined by membrane immunofluorescence. Lymphoid cell preparations were purified by glass-wool column and Hypoque-Ficoll sedimentation to eliminate the large majority of non-lymphoid cells which might introduce counting errors on fluorescence microscopy. FITC-labelled horse anti-mouse-globulin did not stain thymocytes (T cells) whereas it did stain a proportion of the lymphocytes of other lymphoid tissues (B cells): peripheral blood, 11%; bone marrow, lymph node, 23%; spleen, 24%. These results correlated well with the proportion of cells stained by anti-B cell sera obtained by the complete absorption of anti-lymphocyte serum (ALS) with thymocytes.     

Relation Between HLA-DW and the B-Lymphocyte Specificities

Homozygous DW typing cells were tested for six B-lymphocyte specificities. All four of the second locus specificities of B lymphocytes were strongly associated with the DW specificities. DW1 typing cells were B group 6, DW2 were B4, DW3 were B5, and LD107 were B3. The first B-cell locus antigens 1 and 2 tended to be uniform within the DW groups. From an analysis of the typing responses of a panel of cells to the homozygous typing cells, it has become apparent that the first B-locus specificity present on the homozygous typing cells also plays a role in determining whether a typing response is obtained or not. Thus, the DW3 typing cells were themselves B2 and B5, and cells having B2-B5 were most frequently nonreactive to DW3 in mixed lymphocyte culture. Homozygous typing cells therefore mainly detect the second B-cell locus antigens and, to a lesser degree, the first locus specificities. Stated another way, homozygous typing cells do not define a single specificity, but rather the presence of two B-lymphocyte specificities, even though their responses often reflect matching of only the second B-locus specificity.     

Immunofluorescent detection of surface antigens specific to T and B lymphocytes in the chicken

Rabbit antisera specific for chicken T and B cells as judged by surface immunofluorescent staining have been raised. Specificity was established by the staining of thymus and bursal cell suspension and by the effects of thymectomy and bursectomy on the staining of peripheralized lymphocytes. Furthermore, double labeling experiments showed that anti-T and anti-B sera reacted with different populations of blood lymphocytes. Comparable numbers of cells in blood and spleen stained for B and light chain determinants. No evidence for “null cells” was obtained. There was little change in the percentage of cells staining in the various lymphoid organs from 4 days to 12 months of age. The thymus contained approximately 7 % B cells, although no T cells were demonstrable in the bursa. One antiserum showed only thymocyte specific antibodies not reacting with peripheral T cells. The specific B and T markers seem to be acquired during differentiation within the appropriate central lymphoid organ. Demonstrable surface immunoglobulins appear later in ontogeny than the B antigens. The majority of cells bearing the B marker in bone marrow were large cells lacking surface light chain determinants.     

New monoclonal antibody specific for a 6.5 kDa glycoprotein which presents mainly on a B cell of chronic lymphocytic leukemia (CLL)

Chronic lymphocytic leukaemia (CLL) is a most common form of adult leukemia. No specific marker for CLL has been defined until today. We attempt to produce a specific monoclonal antibody (mAb) to CLL B cells. For this purpose, Balb-C mice were immunised with peripheral blood lymphocytes of a patient with CLL. After the fusion, the immunised mouse spleen cells and SP2/0 myeloma cell line, antibody secreting clones were selected by ELISA and specific antibody was determined by flow cytometry. Leukemic cells from different patients, different cell line and lymphoid tissue were tested with this mAb using flow cytometry and immunoperoxidase methods. Ligand of the mAb on cell surface was identified using epitope analysing method. We have shown that this mAb is specific to a molecule with 6.5 kDa molecular weight, which is present mainly on B CLL cells (63.7+/-16.4%). It has also been shown that this molecule was a glycoprotein. Amongst the different cell lines that were tested, Raji cell, Molt-3 and P3HR-1 cells were expressing this molecule. We, therefore, suggest that it is a novel molecule with unknown function and is mainly present on the B cells of CLL.     

Tissue Typing of Cells in Culture. IV. Cell Surface Antigens of Tumor Cell Lines, not Obviously Belonging to the HLA-System

Studies on the HLA pattern of several human tumor cell lines by the mixed hemadsorption test and also studies on the discriminative patterns formed by a collection of sera on a number of cell lines and diploid cells have been reported elsewhere. It was noted during these studies that some sera reacted in a fashion indicating that they did not represent any of the HLA-A or -B series and probably not the HLA-C series either. The corresponding antigenic determinants were tentatively designated Ek-1 to Ek-11. The pattern of these antigens among the cell lines is given in a table and it seems apparent that the Ek-series considerably increases the cell identification potential of the typing results. So far, five of the sera determining the Ek-antigens have failed to be blocked by anti-beta-2-microglobulin, indicating that the antigens do not belong to the HLA-A, B or C series. Preparatory work for HLA-D typing is under way.     

Surface markers of primate B and T lymphoid cell lines identified by antibodies to human and simian lymphocyte antigens

Simian B and T lymphoid cell lines were shown to maintain surface markers found on mature lymphocytes in vivo. The T lymphoid cell lines expressed Ia-like antigens on their surfaces, further suggesting that they represent mature, activated T cells. These Ia antigens show a structural similarity to Ia on human cells although some diversity exists. The Ia antigen expressed on T lymphoid cell lines was shown to be very similar to those on B lymphoid cell lines. Owl monkey and marmoset T lymphoid cell lines were also shown to express a VH immunoglobulin-related determinant, a marker which is thought to be associated with T cell antigen receptor. Owl monkey and marmoset T cell lines express a surface antigen which identifies the sheep erythrocyte receptor on human T cells and some of these lines express an antigen found on human helper T cells. It is noteworthy that substantial conservation of surface components has occurred within primate evolution such that monoclonal antibodies to human Ia, OKT-11a and Leu 3a markers can be used to type lymphocytes of lower primates.     

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.     

Avoidance of Certain Systematic Pitfalls in the Detection of HLA–DR Antibodies Defining Fine Specificities

A selective screening program has been established to identify rapidly and effectively the fine specificity of HLA-DR antibodies in pregnancy anti-HLA sera. Following initial HLA-A, B, C screening sera with extra- or multispecific reactions were selected and specifically tested after platelet absorption on isolated B- and T-lymphocyte populations of the serum donor's husband. Identification of the HLA-DR type of the husband, as well as screening and typing on HLA, A, B and -C typed heterozygous panel B cells led to a more precise characterization of the major specificity of a detected anti-HLA-DR serum. Multispecific anti-DR sera were defined and rendered operationally monospecific by titration.
Some critical steps in a reliable assessment of HLA-DR typing reagents could be worked out. Weak HLA-A, B antibodies, B-cell auto- and Lewis antibodies may cause positive reactivity preferentially or even selectively on B lymphocytes. Of particular importance was the hidden presence of HLA-C specific antibodies, since they cannot be absorbed out by stored platelets. In addition they are not readily detectable through screening on typed panel cells. Because of the frequently very high linkage disequilibrium between HLA-B and HLA-C alleles it is difficult to select appropriately dissecting panel cells. The two points demonstrated above gain even more weight when isolated T and B cell populations are used for HLA-DR typing, because HLA-C antibodies preferentially kill B cells. In this fashion contaminating HLA-C antibodies are not only difficult to detect but can mimic the presence of HLA-DR antibodies.