Differential effect of interferon on the expression of tumor-associated antigens and histocompatibility antigens on human melanoma cells: relationship to susceptibility to immune lysis mediated by monoclonal antibodies

Incubation of cultured human melanoma cells with human leukocyte interferon did not change the expression of melanoma-associated antigens (MAA) recognized by monoclonal antibodies and of Ia-like antigens but significantly increased the expression of HLA-A,B antigens and of beta 2-microglobulin (beta 2-mu). The effect is dependent on the dose of interferon and on the incubation time. Interferon-treated melanoma cells showed an increased susceptibility to lysis mediated by monoclonal antibodies to HLA-A,B antigens and to human beta 2-mu; on the other hand, interferon-treated melanoma cells did not change in their susceptibility to murine natural killer (NK) cell lysis and to immune lysis mediated by monoclonal antibodies to MAA and to Ia-like antigens, and they displayed a reduced susceptibility to human NK cell lysis. Therefore, the increased susceptibility of interferon-treated melanoma cells to lysis mediated by anti HLA-A,B and anti beta 2-mu monoclonal antibodies is likely to reflect the increase in cell surface expression of the corresponding antigens.     

Serologic and immunochemical characterization of the specificity of four monoclonal antibodies to distinct antigenic determinants expressed on subpopulations of human Ia-like antigens

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.     

Distribution of antigenic determinants recognized by three monoclonal antibodies (Q2/70, Q5/6 and Q5/13) on human Ia-like alloantigens and on their subunits

The distribution of antigenic determinants recognized by the anti-Ia-like antigen monoclonal antibodies (MoAb) Q2/70, Q5/6 and Q5/13 on molecules coded for by the DR locus and by non-DR loci was investigated using a binding assay with 125I-labeled Ia-like antigens isolated from four B lymphoid cell lines. The determinants reacting with the MoAb Q2/70 and Q5/13 are expressed on all DR alloantigens tested and on BR4X7 specificities, while those reacting with the MoAb Q5/6 are not detectable on DRw7 and BR4X7 molecules. None of the monoclonal antibodies reacted with DC1 molecules. The MoAb Q5/6 and Q5/13 reacted with the isolated beta subunit of the Ia-like antigenic complex, while the MoAb Q2/70 did not react with the isolated chains.     

Immunochemical and functional analysis of Ia-like antigens on human monocyte-macrophages with monoclonal antibodies

The distribution, structural profile and functional properties of Ia-like antigens synthesized by human monocyte-macrophages have been analyzed using monoclonal antibodies to common determinants of these antigens. Up to 45 and 70%- of monocyte-macrophages isolated from the fluid of blisters induced with cantharidin and from peripheral blood, respectively, react with monoclonal antibodies to human Ia-like antigens. The level of Ia-like antigens on monocytes-macrophages appears to be similar to that on cultured B lymphoid cells. Monoclonal antibodies to common determinants of Ia-like antigens specifically block antigen presentation by monocyte-macrophages to T lymphocytes as well as proliferative response of T lymphocytes to autologous and allogeneic monocytes-macrophages. These results indicate that common determinants of Ia-like antigens play a role in the interaction of monocytes-macrophages with T lymphocytes.     

HLA-A,B antigens Ia-like antigens, and tumor-associated antigens on prostate carcinoma cell lines: serologic and immunochemical analysis with monoclonal antibodies

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.     

Purification of immunologically functional subsets of human Ia-like antigens on a monoclonal antibody (Q5/13) immunoadsorbent

Serologic and immunochemical asays have shown that the monoclonal antibody Q5/13 recognizes an antigenic determinant expressed on a subset of human Ia-like antigens. Testing with a panel of HLA typed B lymphoid cells has shown that this determinant is different from those defining the serologic polymorphism of HLA-DR antigens. The monoclonal antibody Q5/13 has been used to purify subsets of human Ia-like antigens, which are immunologically functional. These reagents should facilitate the characterization of structural and functional properties of human Ia-like antigens.     

Ontogeny of human Ia antigens

Indirect immunofluorescence (IIP) staining of tissues from human fetuses (ages ranging from 8 to 32 weeks of intrauterine life) with monoclonal antibodies (MoAb) to monomorphic determinants of Ia antigens and HLA-A,B,C antigens has shown that both types of antigens are already detectable in tissues of 8-week-old fetuses. Ia antigens and HLA-A,B,C antigens reach their almost-complete tissue distribution after 32 and 24 weeks of intrauterine life, respectively. The structure of Ia antigens synthesized by fetal thymus cells is similar to that of B-lymphoid cell-derived Ia antigens. Ia antigen-bearing thymic fetal cells can stimulate allogeneic lymphocytes in mixed lymphocyte reactions (MLRs). These reactions are blocked by monoclonal antibodies to monomorphic determinants of human Ia antigens and of HLA-A,B, antigens.     

Expression of HLA-DR, MB, MT and SB antigens on human mononuclear cells: identification of two phenotypically distinct monocyte populations

The expression of HLA-DR, SB, MB, and MT antigens in different populations of human mononuclear cells was investigated with the use of monoclonal antibodies that recognize distinct human Ia-like antigens. Our results indicate that in man, as previously reported in other species, two phenotypically distinct populations of monocytes or macrophages can be identified on the basis of expression of Class II MHC antigens. Virtually all circulating monocytes displayed determinants associated with HLA-DR, SB, and MT. In addition, a subpopulation of human monocytes expressed MB/DS-associated antigens, as detected with monoclonal antibodies specific for MB1, MB3, and DS-framework determinants. Most B lymphocytes expressed antigens associated with HLA-DR, and the specificities SB2, SB3, MB1, MB3, MT2, and MT3 were also present. Resting T lymphocytes were unreactive with antibodies that recognize all of the Class II MHC antigens tested. T lymphocytes activated by soluble antigen or alloantigens, and expanded in culture, expressed DR, SB, MB, and MT. The majority of the MB/DS+ cells present in the adherent population were monocytes, because they were phagocytic and had the monocyte-specific marker 63D3. The rest of the cells were not identified. They are likely to include mostly B lymphocytes. The presence of other cells, such as dendritic cells, in this subset needs to be determined.     

Discordant expression of human Ia-like antigens on hematopoietic progenitor cells

The expression of HLA-DR, SB, MT2, and DC antigens on human hematopoietic progenitor cells has been determined by using monoclonal antibodies with complement (C)-mediated cell lysis and immune separation techniques. HLA-DR was detected on greater than 85% of CFU-G/M, myeloid clones (MyCl), BFU-E, and CFU-E. CFU-E were less susceptible to C-mediated lysis at suboptimal C concentrations. The polymorphic MT2 and SB antigens were also present on all categories of progenitor cells, although a lesser proportion of cells were positive. Because in most individuals the antigen density of MT2 and SB, as determined by monoclonal antibody staining, was also lower on B cells and monocytes when compared to HLA-DR expression, the lower number of positive progenitor cells probably reflects lower antigen density rather than distinct positive and negative progenitor cell populations. The DC antigen is expressed weakly on monocytes and B cells, although there is considerable individual variation. In some individuals, distinct DC-positive and -negative monocyte populations are detectable. The DC antigen was not detected on myeloid progenitor cells, even in those individuals with moderate DC expression on their monocytes and B cells. This discordant expression of DC and other Ia-like antigens on hematopoietic progenitor cells may be of physiologic significance and may assist in the purification of progenitor cells from blood and marrow.     

HLA antigen expression at the single cell level on a K562 X B cell hybrid: an analysis with monoclonal antibodies using bacterial binding assays

The influence of different genetic environments on the expression of HLA complex-controlled antigens has been investigated using cell lines with various defects in the synthesis of these molecules and a somatic cell hybrid derived from them. A very sensitive bacterial binding assay allowing simultaneous evaluation of the morphology of a given cell and the quantity of a surface molecule has been developed for these studies. The fetal erythroid cell line K562, the Burkitt's lymphoma-derived cell line DAUDI, and their hybrid DUTKO1 have been employed. K562 and the hybrid, but not DAUDI, expressed HLA-A,B,C heavy chains as detected by the monoclonal antibody W6/32.HL, while two monoclonal antibodies (TU48 and 2BC4) against the supertypic specificities HLA-Bw4 and Bw6 showed no reactivity. The presence of human Ia-like antigens on the cell surfaces was investigated with a panel of eight monoclonal antibodies. K562 cells were completely unreactive, and DAUDI cells gave the expected positive reaction, but about 1% or less of the cells in the DUTKO1 population appeared to express these antigens as well. We discuss possible reasons for the failure to detect HLA-B antigens with monoclonal antibodies and the lack of complete "dominance" of the K562 genome in the hybrid cell line.     

Transformation of LMTK- cells with purified class I genes. V. Antibody-induced structural modification of HLA class I molecules results in potentiation of the fixation of a second monoclonal antibody

A potentiation phenomenon was observed with HLA-A3 and CW3 transformed murine L cells between anti-HLA class I B10.6 (potentiated) and B10.8 (potentiating) monoclonal antibodies (m.Ab.). Further studies of this phenomenon with these transformed L cells indicated that: 1) no significant specific binding of B10.6 m.Ab. to HLA-A3 and CW3 transformed L cells could be demonstrated by conventional radioimmunoassay or cytofluorometric study in the absence of B10.8 m.Ab.; 2) potentiation of the fixation of B10.6 m.Ab. was induced by other anti-HLA class I m.Ab., which all reacted with the same cluster of antigenic determinants; 3) potentiation reflects an increased specific fixation of B10.6 m.Ab. to HLA class I molecules implicating its combining site; 4) potentiation was mediated by B10.8 Fab fragments. These results indicate that potentiation of the fixation of B10.6 m.Ab. to the HLA-A3 and CW3 molecules expressed by the transformed L cells reflects conformational changes of these molecules after interaction with B10.8 m.Ab.     

Antigenic phenotype of TdT-positive cells in human peripheral blood

Double immunofluorescence studies for terminal deoxynucleotidyl transferase (TdT) and leucocyte surface membrane antigens have been used to characterize the small subpopulation of TdT-positive cells in human peripheral blood. The predominant antigens demonstrated were those coded for by the major histocompatibility complex, namely HLA-A,B and Ia-like antigens. A small proportion of TdT+ cells expressed antigens restricted to B lymphocytes and their precursors (BA-1+ CALLA+). In contrast, antigens associated with T-lymphocyte differentiation were not detected using a panel of T-cell-specific monoclonal antibodies. These results preclude the possibility that circulating TdT+ cells are immature cortical thymocytes that have "leaked" into the bloodstream. Although bone marrow-derived prothymocytes, which have not yet acquired T-cell lineage markers, may be included amongst this subset, the expression of B-cell related antigens by some TdT+ cells indicates the likely existence of lineage heterogeneity amongst this population of lymphoid cells. The relevance of these findings to the monitoring of human acute lymphoblastic leukaemia is discussed.     

Hybridomas synthesizing monoclonal antibodies to surface antigens of human lymphocytes

Three types of hybridomas were obtained by fusion of murine myeloma cells (NSI-1-Ag4-1) with splenocytes from mice immunized with human lymphoblastoid cells (RPMI-6410t line, acute myeloblastic leukemia). Hybridomas of the first type synthesize monoclonal antibodies Ma-1, which interact with 6410t-cells, but are not bound to the cells of human Burkitt lymphoma-Raji. Raji cells contain HLA-DRw5 and -DRw6 antigens on cell surface but there are no HLA-A2, -B7 and -B12 antigens (specific for 6410t). Thus, Ma-1 are probably derected against some of HLA antigens of loci A or B. Hybridomas of the second type synthesize Ma-2 antibodies which react with 6410t and Raji cells, but are not bound to peripheral blood lymphocytes (PBL). We suppose that Ma-2 antibodies to tumor specific antigens which have common antigen determinants both for Raji and RPMI-6410t cells. The third type of hybridomas synthesizes monoclonal antibodies Ma-3 reacting with all the three types of target cells: 6410t, Raji, and PBL. Ma-3 seems to be directed against human species-specific lymphocyte antigens which remained in 6410t and Raji cells.     

Monomorphic anti-HLA-A,B,C monoclonal antibodies detecting molecular subunits and combinatorial determinants

Thirteen monoclonal antibodies that react with monomorphic determinants on the HLA-A,B,C-beta 2-microglobulin (beta 2m) molecule were characterized. Analysis of antibody activity included inhibition by papain-solubilized HLA antigens and free beta 2m, antibody binding to mouse-human somatic cell hybrids containing human chromosome 6 or 15, and antibody cross-reactivity with lymphocytes from nonhuman species. Two criteria for monomorphism were established: 1) equal inhibition or absorption of antibody activity by all papain-solubilized HLA antigens or cell lines of different HLA specificities tested; and 2) nonpolymorphic cross-reactivity within another species or subspecies. On the basis of soluble antigen inhibition and binding to somatic cell hybrids, 3 classes of antibodies were detected: anti-beta 2m, anti-heavy chain, and anti-complex (against a combinatorial determinant formed by heavy chain and beta 2m). Antibody cross-reaction patterns in nonhuman species were suggestive that these monomorphic antibodies detect a limited number of determinants, minimally one on each chain and 2 combinatorial determinants. Examination of the known primary sequences for HLA-A2, HLA-B7, H-2Kb, and mouse, rabbit and human beta 2m provides a molecular explanation for this limited mouse anti-HLA monomorphic antibody activity.     

Cytotoxic T lymphocyte recognition of HLA-A/B antigens introduced into EL4 cells by cell-liposome fusion

HLA-A2 and -B7 antigens were introduced into EL4 (H-2b) cells by cell-liposome fusion and were used as targets or stimulators for cytotoxic T lymphocytes (CTL) generated in C57B1/6 (H-2b) mice. It was found that such EL4-HLA cells were not recognized by CTL that had been raised against either a human cell line bearing these HLA antigens or the purified HLA-A2 and -B7 antigens reconstituted into liposomes. In addition, EL4-HLA cells were not capable of inducing CTL that could recognize a human cell line bearing HLA-A2 and -B7 antigens. Instead, EL4-HLA cells induced CTL that specifically lysed EL4-HLA cells and not human cells expressing HLA-A2 and -B7. CTL recognition required the presence of HLA antigens on the EL4 cell surface and was inhibited by antibodies against either H-2b or HLA-A/B. Monoclonal antibody binding studies showed that the expected polymorphic determinants of the HLA-A2 and -B7 antigens were still present on EL4-HLA cells. However, the specificity of CTL or their precursors that are capable of recognizing HLA-A2 or -B7 was altered after these antigens became associated with the EL4 surface. Possible explanations for these results are discussed.     

Cross-reactivity between human and murine lymphocyte antigens

The anti-H-2 alloantiserum D-32 [(BlO.A(2R) × 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.     

Effects of monoclonal antibodies against lymphocyte surface antigens on interleukin 2 excretion by Epstein-Barr virus-specific human T-cell hybridomas

Monoclonal antibodies were used as probes to study the role of cell surface antigens in the response of Epstein-Barr virus (EBV)-specific human T-T hybridomas to autologous EBV-infected B lymphoblasts. Somatic cell hybrids were generated by fusing EBV-primed peripheral blood T lymphocytes with a mutant clone of the JM human T-lymphoblastoid-cell line. When exposed to autologous EBV-infected B lymphoblasts, the resulting hybrid clones released Interleukin 2 into the culture medium. Incubation of the EBV-infected B cells with two monoclonal antibodies against human Ia-like molecules blocked their ability to trigger the hybridomas. Under the same conditions, monoclonal antibodies against beta 2-microglobulin, and a 45,000 MW surface antigen common to EBV-infected B lymphoblasts, did not alter the capacity of the B cells to stimulate the hybridomas. None of four monoclonal antibodies against surface antigens on the T-cell hybridomas impaired their responsiveness to EBV-infected B lymphoblasts. These results suggest the possibility that naturally occurring or exogenously administered antibodies against Ia molecules might interfere with T-cell regulation of EBV-induced B-cell activation.     

Serological expression after sequential double transfection with purified HLA-11 gene of mouse fibroblasts carrying human beta-2 microglobulin

A genomic cosmid library constructed from DNA from a genotyped individual (JF = HLA-A11, Cw–, B38/A26, Cw7, B51) was screened for clones containing class I histocompatibility genes. Among these clones, one was found to carry a 4.8 kb Hind III fragment which is highly correlated with HLA-A11. This clone was used to transfect LMTK⁺ cultured mouse fibroblast transformants expressing human beta-2 microglobulin. The human beta-2 microglobulin heavy chain-associated determinant was positively detected by the M18 monoclonal antibody. HLA-A11 expression on these doubly transformed cells was specifically demonstrated by complement-dependent cytotoxicity with HLA-A11 + A3-specific but not with HLA-A3-specific monoclonal antibodies. Absorption studies with human alloantisera confirmed the presence on these cells of HLA-A11 determinants and of cross-reacting determinants which absorbed anti-HLA-A1 and –A3 alloantisera. The JF5-J27 transfected cell expressed both heavy and light chains of human class I histocompatibility genes.Abbreviations used in this paper ₂m beta-2 microglobulin - CTL cytolytic T lymphocytes - FCS fetal calf serum - HAT hypoxanthine-azaguanine-thymidine - kb kilobase pair - MHC major histocompatibility complex - MoAb monoclonal antibodies - PBL peripheral blood lymphocytes - PEG polyethylene glycol - r correlation coefficient This study is dedicated to the memory of Jean-Jacques Metzger.     

Human dendritic reticulum cells of lymphoid follicles: their antigenic profile and their identification as multinucleated giant cells

The B-dependent areas of human lymphoid tissue contain non-lymphoid, non-phagocytic cells known as dendritic reticulum cells (DRC). These cells can be detected only very occasionally in routinely stained histologic sections. Recently we were able to overcome this limitation by preparing a monoclonal antibody, termed R 4/23, that reacts selectively with DRC. Thus by using an optimized immunoperoxidase method applied to frozen sections, it is possible to detect DRC in situ. To determine the antigenic profile of DRC, serial frozen sections of human tonsils were immunostained with R 4/23 and a large panel of other monoclonal antibodies or conventional antisera. In addition, touch imprints of tonsils and cytocentrifuge slides of cell suspensions with increased concentrations of DRC were immunostained with these reagents. DRC proved to be positive for mu, gamma, alpha, kappa and lambda chains, complement component C3b, C3b receptors, C3d receptors, HLA-A,B,C antigens, human Ia-like antigens, common ALL antigen (cALLa), and antigens that are characteristic of the monocyte/macrophage lineages. DRC did not express delta chains, T cell antigens, or antigens that are expressed on interdigitating reticulum cells (IDC) and Langerhans cells. DRC in touch imprints and suspensions prepared from hyperplastic tonsils were found to be giant cells often with 10 or more nuclei. In certain cases of follicular hyperplasia and of centroblastic-centrocytic lymphoma, DRC with several nuclei were also detectable in situ. These results show that (1) the phenotype of DRC differs from that of all other cell types in lymphoid tissue, (2) this phenotype most nearly resembles that of cells of the monocyte/macrophage series, thus suggesting that DRC are related to these cell lineages, and (3) DRC are multinucleated giant cells.     

The free and the β2-microglobulin-associated heavy chains of HLA-A,B alloantigens share the antigenic determinant recognized by the monoclonal antibody Q1/28

Serological and immunochemical assays have shown that the monoclonal antibody Q1/28 recognizes an antigenic determinant which is expressed on the heavy chain of subsets of HLA-A, B antigens and is distinct from those defining the serological polymorphism of this system. Association of the HLA-A, B heavy chain with ₂-microglobulin is not required for expression of the antigenic determinant recognized by the monoclonal antibody Q1/28, since this antibody can immunoprecipitate a 45 000 m. w. component from radiolabeled lymphoid-cell glycoproteins immunodepleted with either an anti-human ₂-microglobulin xenoantiserum or the MoAb W6/32 to framework determinants of HLA-A, B, C antigens. Furthermore, the MoAb Q1/28 can immunoprecipitate a 45 000 m. w. component from an NP40lysate of radiolabeled Daudi cells, which lack the genetic information for ₂-microglobulin. The determinant recognized by the MoAb Q1/28 is relatively resistant to denaturing treatments and does not appear to be carbohydrate in nature. The MoAb Q1/28 is the first example of an antibody which recognizes an antigenic determinant expressed on both the ₂-microglobulin-associated and free HLA-A, B heavy chains.