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In vitro treatment of goat red blood cells (GRBCs) with the sulphydryl compound 2-aminoethylisothiouronium bromide (AET) increases their specific reactivity with human T lymphocytes without affecting the specificity of the reaction. AET-GRBCs bind to only part of T lymphocytes rosetting with AET-sheep red blood cells (SRBCs): the receptors for both types of RBCs are very simular if not identical, but display higher affinity for AET-SRBCs than for AET-GRBCs. Rosetting of T lymphocytes with AET-GRBCs may be useful to enumerate T lymphocyte subsets in patients with abnormality of the immune system and to fractionate T lymphocyte subpopulations.

T lymphocytes from patients with solid tumors and from aged donors are abnormal in their expression of Ia antigens following in vitro stimulation with phytohemagglutinin (PHA). Ia antigens were not detected on PHA-activated T lymphocytes from 15 of 27 patients with solid tumors. The abnormality in T lymphocytes from 25 donors older than 60 years was evidenced by a reduction in the percentage of T cells acquiring Ia antigens following stimulation with suboptimal amounts of PHA and by a delayed appearance of those antigens. In both groups of donors the defect in Ia antigen expression by PHA-activated T cells did not correlate with the reduced [3H]thymidine uptake. PHA-activated T cells from aged donors and from patients with solid tumors were poorly stimulatory in autologous and allogenic mixed lymphocyte reactions. Furthermore, T lymphocytes from these two groups of donors displayed a reduced proliferative response to autologous non-T cells, but a normal proliferative response to allogeneic PHA-activated T cells and to non-T cells from control subjects.

The specificity of DR xenoantisera was assessed by testing them with a panel of HLA typed B-lymphocytes in a cytotoxic test with rabbit or human complement. The pattern of reactions of some xenoantisera correlated with DR allospecificities. These results confirm that injection of DR antigens across species barrier can elicit antibodies to DR allospecificities.

Soluble extracts of melanoma specimens were prepared by 3 M KCl extraction. Delayed cutaneous hypersensitivity reactions to these antigens were noted in 25 of 39 melanoma patients and 7 of 30 patients with other neoplasms. Only 4 of 34 melanoma patients reacted to an extract of autologous muscle. Maximum reactivity to these antigens occurred at 24 hr and was demonstrated histologically by skin biopsy. Chromatography on Sephadex G-150 resulted in two fractions that elicited delayed cutaneous hypersensitivity reactions in melanoma patients. These fractions were subjected to polyacrylamide gel electrophoresis. The first region of the Sephadex 1 gel reacted in 8 of 13 melanoma patients and only 1 of 7 patients with other neoplasms. Some activity was found in other regions of the gel. Skin test activity was confined to the second polyacrylamide gel electrophoresis region of the Sephadex II gel, to which 7 of 9 melanoma patients reacted compared with 1 of 7 patients with other tumors. Recovery of antigenic activity in excess of the total present in the original extract after partial purification indicated that inhibitors of delayed cutaneous hypersensitivity reactions present in the crude KCl extract were removed. A 20-fold increase in antigenic activity per unit protein was achieved.

Rabbits were immunized with hybrids constructed with human and murine cells. Serological and immunochemical studies showed that xenoantiserum 1595 is operationally specific for HLA-A2, xenoantisera 0806 and 0746 for HLA-A3 and xenoantiserum 0745 for HLA-B27. The Fab2 blocking assay suggests a spatial relationship between allotypic determinants recognized by the xenoantisera and those reacting with the HLA-A, B-specific monoclonal antibodies (MoAb) Q1/28, 6/31, Q6/64, and CR-1 and the anti-beta 2-microglobulin (beta 2-mu) MoAb NAMB-1.

Peripheral blood lymphocytes and skin fibroblasts from 12 cancer patients were infected with Epstein-Barr virus (EBV) or SV40 virus. The EBV-transformed lymphoblasts and SV40-transformed fibroblasts were grown as continuous cell lines and expressed the same histocompatibility antigens as tumor cell lines established from the same cancer patients. Sera from 350 melanoma and 195 sarcoma patients were tested for antibody reactive with membrane antigens on three of these tumor cell lines (two melanomas and one sarcoma) by immune adherence (IA) and indirect immunofluorescence (IMI) assays. Antibodies to HLA and other non-tumor-related antigens were completely removed from the most reactive sera by quantitative absorption with 4 x 10(7) lymphoblasts or 10(7) transformed fibroblasts autologous to the tumor target cells. These paired cell lines were used to monitor humoral immune responses in melanoma and sarcoma patients receiving allogeneic tumor cell vaccines.

Four lymphoid cell lines, presumably recent derivatives from non-neoplastic human tonsils, were identified in actuality as established Burkitt lymphoma and lymphoblastoid cell lines (LCL) when tested by chromosome banding and typing for HLA antigens. Additionally, the morphology, growth characteristics, tumorigenicity, expression of Epstein-Barr virus antigens and surface membrane immunoglobulins (SmIg) of the four lymphoid cell lines confirmed the correct identifications. In determining the possibility of cross contamination, the most reliable criteria for the identification of lymphoid cell lines were found to be HLA antigenic profile, karyotype, cellular morphology, and growth characteristics.

Serologic and immunochemical analysis of the antigenic profile of the 2 human prostate carcinoma cell lines DU-145 and H494 with a battery of monoclonal antibodies has shown that both cell lines express HLA-A,B alloantigens and the 94,000 m.w. tumor-associated glycoprotein recognized by the monoclonal antibody 376.96S. In addition, the cell line H494 unexpectedly expresses Ia-like antigens, which are similar in their antigenic profile and structure to B lymphoid cell derived Ia-like antigens. Both Ia-like antigens and tumor-associated antigens can function as targets of cell-dependent lysis mediated by the corresponding monoclonal antibodies.

Studies of human peripheral lymphocytes (HPL) from healthy subjects and of human lymphoblastoid cell lines (HLC) revealed spontaneous formation of rosettes with Macaca speciosa monkey red blood cells (MRBC). The percentage of HPL forming rosettes with MRBC paralleled the percentage of HPL exhibiting markers ascribed to human B cells. The MRBC-rosetting cells were positive for membrane-bound immunoglobulin (MBIg). Furthermore, by employing a mixed rosette method, lymphocytes carrying both EAC3dmo and FITC-MRBC were encountered. In contrast, no lymphocytes carrying both SRBC and FITC-MRBC were observed. Ninety-six per cent of purified B cells obtained at the interface after interaction of HPL with SRBC and Ficoll-Isopaque centrifugation formed rosettes with MRBC. In contrast, only 8% of an enriched population of T cells obtained at the interface after interaction with EAC3dmo and Ficoll-Isopaque centrifugation formed MRBC-rosettes. Incubation of HPL with MRBC and subsequent centrifugation on Ficoll-Isopaque resulted in a highly enriched T cell population at the interface. Several lymphoblastoid cell lines with B cell characteristics, but not two cell lines with T cell characteristics, formed spontaneous rosettes with MRBC. Inhibition experiments showed that the structure on the lymphoid cell surfaces mediating the adherence of MRBC was independent of the MBIg as well as the recptors for IgGFc, C3b, and C3d. It appears that rosetting with MRBC will provide a new detection marker for human cells classified as B type and a technique for obtaining an enriched T cell population.

Murine L cell fibroblasts, and derivatives were found to rosette with sheep red blood cells (SRBC). Primary fibroblast explants from the parent murine strain, C3H, did not possess this potential. No rosettes were observed with primary fibroblast explants from C57BL and B10Br mice, with a human fetal lung fibroblast, with baby hamster fibroblasts or their polyoma transormed derivative, or with a cell line, 1T-22, derived from BALB/c mice. Hybridization of 1T-22 and L cells, by Sendai virus-mediated cell fusion, suppressed the rosette potential of the L cell parent. The receptor for SRBC on L cells appears to result from the expression of a recessive characteristic.

The anti-H-2 alloantiserum D-32 [(B10.A(2R) x C3H.SW) anti-C3H] is cytolytic to human lymphocytes. Fab2 blocking assays, indirect immunoprecipitation and sequential immunoprecipitation experiments showed that the anti-H-2 alloantiserum D-32 recognizes antigenic determinants which are expressed on the heavy chain of subpopulations of HLA-A, B antigens. These determinants are different from those defining the serological polymorphism of the HLA-A, B, C system, are the same as or spatially close to those recognized by the anti-HLA-A, B monoclonal antibody Q6/64 and are expressed on rabbit, rat or guinea pig lymphocytes.

Biological and molecular properties of receptors specific for human T- and B-lymphoid cells are discussed. Sensitive simple and reproducible methods to enumerate B- and T-lymphocytes are described: their possible application in clinical immunology is indicated. A new method to obtain enriched populations of human T- and B-lymphocytes from peripheral blood is described. This method appears to be suitable to investigate the properties of antigens specific for B- and T-lymphoid cells.

Melanoma-associated antigens (MAA) were isolated and their functional immunologic properties were evaluated. Spent fetal calf serum-free culture media and 3-m KCI extracts of cultured human melanoma cells grown in this medium were used as antigen sources. Ultracentrifugal flotation on KBr was used to separate MAA and HLA antigens present in the extracts or spent culture media; thus interference by histocompatibility antigens was prevented in subsequent tests of tumor antigenic activity. MAA purified in this manner retained their immunologic functions as evidenced by their ability to produce delayed cutaneous hypersensitivity reactions in patients with melanoma, specifically combine with antimelanoma xenoantibody, and elicit production of functionally specific xenoantibody. Possible structural differences between HLA antigens and MAA were considered in evaluation of the data.

Human T lymphocytes activated with PHA express Ia-like antigens and acquire the ability to stimulate autologous T lymphocytes in mixed lymphocyte reaction. This reaction is immunological in nature since it has specificity and memory. Ia-like antigens play a role in the stimulation of T lymphocytes by autologous PHA-T lymphocytes since monoclonal antibodies to Ia-like antigens can significantly, although not completely, inhibit the stimulation.

The hybridoma 653.40S, constructed with splenocytes from an inbred BALB/c mouse immunized with cultured human melanoma cells, secreted an antibody that had been shown to recognize an antigenic determinant restricted to human melanoma cell lines. The monoclonal antibody (MoAb) 653.40S showed immunoprecipitation of two glycopolypeptides synthesized by melanoma cells, one with the apparent molecular weight of 280,000 and the other one with a molecular weight larger than 500.000. These two glycopolypeptides were not bridged by disulfide bonds and were peripheral rather than integral to the plasma cell membrane. Comparison of the reactivity of cells of the melanocyte lineage with the MoAb 653.40S and with the MoAb Q5/13 to human Ia-like antigens showed that the former reacted with proliferating melanocytes and melanoma cells, whereas the latter reacted only with melanoma cells. The MoAb 653.40S did not react with a large variety of surgically removed normal and tumor tissues except for some instances of basal cell and squamous cell carcinomas. These results suggested that double staining of pigmented skin lesions with the MoAb 653.40S and with an MoAb to Ia-like antigens may help to solve controversial diagnosis of melanoma. Furthermore, the MoAb 653.40S may be useful for radioimaging and immunotherapy of melanoma.

Murine 6C3HED lymphoma cells were found to rosette with sheep red blood cells (SRBC). Normal C3H lymphocytes did not exhibit this property. This rosetting capacity of 6C3HED cells was found to be an accurate and reproducible means for discriminating between normal and tumor cells. The SRBC receptor on these lymphoma cells appeared to be serologically distinct from that expressed by normal human T lymphocytes since reciprocal blocking experiments demonstrated that inhibitory antisera were not cross-reactive. The expression of the SRBC receptor by the 6C3HED cells appeared to correlate with the expression of a tumor-associated antigen and was spatially related to an antigen expressed by 6C3HED and normal neonatal but not adult mouse cells.

The human beta2-microglobulin antigen-antibody system was used as a model to illustrate the versatility of a microradioimmunoassay technique using protein A-bearing Staphylococcus aureus Cowan I strain (SACI) bacteria as a non-specific immunoadsorbent in place of a second antibody. Experimental conditions are described for a sensitive microassay which makes it possible to process large numbers of samples more rapidly and with minimum handling. Furthermore, SACI coated with specific antibodies by mixing with unfractionated antisera are a versatile reagent not only for radioimmunoassays but also for use as molecular probes to characterize cell surface antigens. Antibody-coated SACI could be lyophilized and proved extremely stable in storage thus providing a unique advantage for use in binding inhibition assays and as versatile reagent for clinical and investigative immunology.

In patients with different types of solid tumors who had no detectable metastasis and had received no radio- and chemotherapy the levels of Ia-like antigen bearing lymphocytes was determined by indirect immunofluorescence staining of peripheral lymphocytes with xenoantisera to framework determinants of Ia-like antigens. The level of Ia-like antigen bearing lymphocytes was increased in patients with urinary bladder carcinoma, was decreased in patients with carcinoma of the gastrointestinal tract and was within normal ranges in patients with melanoma, carcinomas of the breast, lung and genital tract. In patients with carcinomas of the gastrointestinal tract as well as in patients with carcinoma of lung and genital tract the levels of T lymphocytes measured by rosetting with sheep red blood cells, was reduced while it was not different from normal in other groups of patients. Determination of the level of lymphocyte subpopulations may provide information about the immunocompetence status of patients with solid tumors.

The specificity of A9 antiserum produced in rabbits immunized with high density lipoprotein-associated HLA-A9 from serum was investigated by using serologic and immunochemical approaches. After suitable absorption, the xenoantiserum No. 2958 is monospecific for A9 antigens in direct cytotoxic test. Data from lysostrip experiments and from blocking of cytotoxicity with Fab2 fragments indicate that the A9 xenoantiserum No. 2958 react with the same or closely associated antigenic structures recognized by A9 alloantisera. Immunoprecipitates obtained with the specific xenoantiserum No. 2958 and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis contained only proteins of the size of detergent-solubilized HLA and beta2-microglobulin (45,000 and 12,000 m.w.). Immunodepletion experiments indicated that the precipitation was mediated by antibodies directed to the heavy chain, and not the light chain of the complex. These results indicate that monospecific HLA-A9 xenoantisera have the same discriminatory capacity of alloantisera and can be useful for the serologic and immunochemical characterization of HLA antigens.

Four out of 8 thrombocytopenic patients without detectable cytotoxic antibodies to human peripheral lymphocytes contained cytotoxic antibodies to cultured human lymphoid cells. The presence of such antibodies was associated with reduced survival of infused allogeneic platelets in these patients. The antibodies reacted in a distinct fashion with a panel of cultured human lymphoid cells and are directed to B cell antigens. Cytotoxic antibodies to cultured human lymphoid cells did not react in vitro with platelets suggesting that the antibodies play no significant role in the accelerated destruction of infused allogeneic platelets, although their presence predicts it. Therefore, screening of sera with cultured human lymphoid cells appears to be a useful test in addition to those now used to select patients for transfusion of allogeneic platelets. Sera from polytransfused patients without cytotoxic antibodies to peripheral lymphocytes may be a useful source of antibodies to B cell antigens.

Murine strains vary greatly in their content of natural antibodies to human lymphocytes; sera from NZB, B10.BR/TSn, DBA/J, and B10.S mice reacted with more than 90% of the panel of human lymphocytes, yet those from other strains did not react. These levels of natural antibodies to human lymphocytes did not correlate with the H-2 type of mice, but appeared to be a dominant character with incomplete penetrance. Since sera from germfree mice also contained natural antibodies to human lymphocytes, their formation was not influenced substantially by bacteria.

H-2 cross-reactive antibodies present in HLA xenoantisera were purified by absorption on and elution from murine cells. Antibodies isolated from one of these antisera, 78-E48, were found to mediate complement-dependent lysis of both human and murine lymphoid cells but not of Daudi cells or of human lymphoid cells coated with Fab2 fragments from a cow anti-human beta 2-microglobulin (beta 2 m) antiserum. In indirect immunoprecipitation analyses 78-E48 reacted only with proteins of approximately 45,000 and 12,000 MW present in NP-40 extracts of both human and murine lymphoid cells. Sequential precipitation experiments with rabbit anti-human beta 2 m and allo-anti-H-2Kk, Iak sera established that these proteins were in fact H-2 and HLA-A,B antigens. It was also found that 78-E48 reacted only with the heavy chain of HLA-A,b and H-2 antigens, since this eluate was unreactive with beta 2 m in a radioimmunoassay, and its capacity to immunoprecipitate the 45,000 and 12,000 MW proteins from human cell extracts was unaffected by prior reaction with purified human beta 2 m. These data show for the first time that H-2 and HLA-A,B antigens share properties that probably depend upon their teritary structure.

Attempts were made to superinfect two epithelial-Burkitt hybrid cell lines, designated D98/HR-1 and D98/Raji, with Epstein-Barr virus (EBV) and to investigate the expression of some cell surfacr markers including histocompatibility antigens, and the presence of B-cell markers, such as receptors for the third complement component and for monkey red blood cells. Successful superinfection of D98/HR-1 cells with EBV was made evident by the expression of early antigen and, to a lesser extent, virus capsid antigen. Only a rare D98/Raji cell was found to be positive for early antigen. The histocompatibility antigens of the parental cell lines D98, HR-1, and Raji were expressed on the surfaces of the hybrid cells. Receptors for third complement components b and d were not detected on the hybrid cells or on the D98P OR HR-1 cell lines; they were found, however, on the Raji cells, indicating that EBV receptors and complement receptors can be separated. The significance of the infection of the hybrid cells with EBV and the expression of cell surface markers is described.

The relationship between HL-A antigens and rosetting of sheep red blood cells (SRBC) with peripheral human lymphocytes has been investigated by incubating them with HL-A antibodies. Although sensitizing the lymphocytes with HL-A alloantisera had no effect on their ability to form rosettes with SRBC, further sensitization with C6 deficient rabbit serum as a source of early complement components inhibited the formation of rosettes with SRBC. The involvement of HL-A alloantibodies in the inhibition of rosette formation was shown first by correlating the HL-A phenotype of the lymphocytes and the HL-A specificity of the alloantisera and, second, by specifically absorbing the HL-A alloantibodies from the alloantisera. Complement was needed to inhibit rosette formation since this effect was lost when rabbit serum was treated to inactivate complement. The participation of complement's classical pathway in rosette inhibition was shown by chelating the Ca2+ ions by EGTA treatment of the C6 deficient rabbit serum. Perhaps, binding of HL-A antibodies and early complement components to the lymphocyte surface disturbs the distribution of the receptors or affects the charge of the cell membrane, thus inhibiting the rosette formation with SRBC.

Human urine was shown to be a good source for the isolation of immunologically functional HLA-A9 antigens. The use of complex solubilization procedures can be avoided since the antigens are present in soluble form and are not complexes with membrane fragements. Purification in excess of 400-fold could be achieved by the application of cellulose ion exchange chromatography, isoelectric focusing, and acrylamide gel electrophoresis. The purified HLA-A9 antigen is composed of a glycoprotein of m.w. 38,000 and beta2-microglobulin, a peptide of m.w. 12,000. HLA-A9 antigens isolated from urine proved to be immunologically functional since they not only reacted specifically with anti-HLA-A9 alloantibody but also elicited anti-HLA-A9 xenoantibodies. These antibodies when covalently attached to Sepharose 4B specifically bound HLA-A9 antigens isolated from both serum and urine.

Serologic and immunochemical assays showed that the monoclonal antibodies Q2/70, Q2/80, Q5/6, and Q5/13 react with human Ia-like antigens. Each monoclonal antibody recognizes distinct antigenic determinants that are different from those defining the serologic polymorphism of Ia-like antigens defined by conventional alloantisera and are expressed on subpopulations of Ia-like antigens. The determinants recognized by the MoAb Q2/70 and Q5/13 are expressed on all HLA-DR allospecificities tested, whereas those reacting with the MoAb Q2/80 and Q5/6 are not detectable on HLA-DR5 and HLA-DR7 allospecificities, respectively.

Melanoma-associated antigens were isolated from human melanoma cells in long-term tissue culture and from the spent culture fluid of these cells propagated in chemically defined, serum-free media. The 3 M KCl extracts from such cells and their concentrated spent culture media elicited specific delayed cutaneous hypersensitivity reactions in patients with malignant melanoma but not in patients with other neoplasms. HLA antigens present in these extracts could be specifically removed by ultracentrifugation in KBr at a density of 1.23 g/ml. Purification of melanoma-associated antigens was achieved by this step, followed by ion-exchange chromatography and preparative isoelectric focusing on Pevikon C870. Another approach is described for the isolation of carcionembryonic antigens from metastatic lesions with an approximately 70% yield utilizing the least denaturing procedures, which avoid lyophilization and involve essentially 0.9% NaCl solution extraction, specific adsorption, elution from concanavalin A Sepharose, and subsequent gel-exclusion chromatography on Ultrogel AcA 22. For effective isolation of carcinoembryonic antigens freely shed from cultured cells derived from a primary colon tumor, a system was devised based on the use of Amicon hollow fiber culture units, in which cultured tumor cells were introduced in the extracapiliary spaces of such a unit. The extracapillary fluid, containing carcinoembryonic antigens but no fetal calf serum components, is removed and further purified by affinity chromatography.

A new method was developed which utilizes fractional salting out, gel filtration, ion exchange chromatography and polyacrylamide gel electrophoresis for the isolation and purification of soluble HL-A antigens from serum. The method produced purified antigens possessing HL-A 5,9 specificities with yield up to 60% of that detected in the original serum. Up to 150-fold increase in specific HL-A antigenic activity above that in the starting material could be achieved as measured by the ability of the purified antigens to specifically inhibit the cytotoxic activity of HL-A alloantisera against selected target cells. Electrophoretically purified HL-A antigens with different specificities appear to have a similar molecular size, i.e. 33 000, but differ in their electric charge properties, beta2-microglobulin (beta2m) seems to be noncovalently associated with HL-A antigens present in serum, but during purification beta2m in progressively lost until at the final purification by polyacrylamide gel electrophoresis, the material is completely devoid of beta2m.