Stefan Carrel

Subsets of human Ia-like molecules defined by monoclonal antibodies

Abstract Two human Ia molecule subsets were denned by their activity with hybridoma antibodies raised against Daudi cell membranes. The first subset (NG-1) was recognized by two distinct hybridoma products D1-11 and D1-12. The second subset (NG-2) was recognized by the other hybridoma, D4-22. The two subsets are present in all Ia preparations tested irrespective of their HLA-DR phenotype. In addition, each particular HLA-DR phenotype can be expressed on both subsets. The relative contribution of these subsets shows considerable variations. Since both D1-12 and D4-22 recognize small Ia subunit, the two subsets appear to carry alternative forms of this subunit

Melanocyte tumor progression is associated with changes in angiogenesis and expression of the 67‐kilodalton laminin receptor

Background. A number of experimental studies have substantiated changes in angiogenesis and in laminin/ laminin‐receptor interactions during tumorigenesis and tumor progression. However, these observations have never been verified objectively in tissues from a well‐defined model of tumor progression.

Characterization of an established human malignant glioma cell line: LN-18

SummaryA human malignant glioma cell line, LN-18, has been established in monolayer culture and subcultured for more than 115 passages. LN-18 cells grow in vitro as bipolar or stellate cells with pleomorphic nuclei, have a doubling time of about 72 h and a plating efficiency of 3%. The glial nature of these cells has been assessed by ultrastructural examination. The synthesis of glial fibrillary acidic and S-100 proteins could not be demonstrated, although the initial biopsy tissue and the early cultures were positive for the former. The presence of Ia-like antigens on the surface of these cells was demonstrated using allo and xeno antisera. LN-18 cells were also shown to synthesize large quantities of fibronectin. The injection of LN-18 cells into nude mice induced the formation of solid tumor masses that could be retransplanted every 3 weeks and showed a morphology comparable to that of the initial biopsy. Karyotype analysis revealed the presence of three marker chromosomes, constantly present before and after hetero-transplantation.

Cultured human fetal astrocytes can be induced by interferon-γ to express HLA-DR

Interferon-gamma (IFN-gamma) modulates the expression of Class II major histocompatibility antigens (MHC), thus providing a potential regulatory mechanism for local immune reactivity in the context of MHC-restricted antigen presentation. Within the central nervous system (CNS), the expression of MHC Class II antigens has been demonstrated on human reactive astrocytes and glioma cells. In order to investigate the modulation of HLA-DR on normal astrocytes, two cell lines were grown from a 20-week-old fetal brain. In situ none of the fetal brain cells expressed HLA-DR as determined by immunohistology on frozen tissue sections. The two cell lines, FB I and FB II, expressed GFAP indicating their astrocytic origin. FB I was HLA-DR negative at the first tissue culture passages, but could be induced to express HLA-DR when treated with 500 U/ml IFN-gamma. FB II was spontaneously HLA-DR positive in the early passages, lost the expression of this antigen after 11 passages and could also be induced to express HLA-DR by IFN-gamma. The induction of HLA-DR expression was demonstrated both by a binding RIA and by immunoprecipitation using a monoclonal antibody (MAB) directed against a monomorphic determinant of HLA-DR. The HLA-DR alloantigens were determined on FB II cells after IFN-gamma treatment, by immunofluorescence and by cytotoxicity assays, and were shown to be DR4, DR6, Drw52, DRw53 and DQwl. These results show that human fetal astrocytes can be induced to express HLA-DR by IFN-gamma in vitro and support the concept that astrocytes may function as antigen-presenting cells.

Comparative localization of glioma-reactive monoclonal antibodies in vivo in an athymic mouse human glioma xenograft model

Radioiodinated murine monoclonal antibodies (Mabs) 81C6, Me 1-14, C12, D12, and E9, made against or reactive with human gliomas but not normal brain, and Mab UJ13A, a pan-neuroectodermal Mab reactive with normal human glial and neural cells, were evaluated in paired label studies in the D-54 MG subcutaneous human glioma xenograft model system in nude mice. Following intravenous injection in the tail vein of mice bearing 200-400 mm3 tumors, specific localization of Mabs to tumor over time (6 h-9 days) was evaluated by tissue counting; each Mab demonstrated a unique localization profile. The comparison of localization indices (LI), determined as a ratio of tissue level of Mab to control immunoglobulin with simultaneous correction for blood levels of each, showed Mabs 81C6 and Me 1-14 to steadily accumulate in glioma xenografts, maintaining LI from 5-20 at 7-9 days after Mab injection. Mab UJ13A peaked at day 1, maintaining this level through day 2, and declining thereafter. Mabs D12 and C12 peaked at days 3 and 4, respectively, and E9 maintained an LI of greater than 3 from days 3-9. Percent injected dose localized/g of tumor varied from a peak high of 16% (81C6) to a low of 5% (Me 1-14 and UJ13A). Immunoperoxidase histochemistry, performed with each Mab on a battery of primary human brain neoplasms, revealed that Mabs 81C6 and E9, which demonstrated the highest levels of percent injected dose localized/g of tumor over time, reacted with antigens expressed in the extracellular matrix. This finding suggests that extracellular matrix localization of antigen represents a biologically significant factor affecting localization and/or binding in the xenograft model used. The demonstration of significant localization, varied kinetics and patterns of localization of this localizing Mab panel warrants their continued investigation as potential imaging and therapeutic agents for human trials.

Cyclic amp induces differentiation in vitro of human melanoma cells

Treating human melanoma lines with dibutyryl adenosine 3′:5′‐cyclic monophosphate (dbc AMP) resulted in morphologic changes associated with the altered expression of cell surface antigens. After treatment, cells developed long cellular projections characteristic of mature melanocytes and showed the presence of an increased number of Stage II premelanosomes. In addition, induction of melanin synthesis, detected as brown perinuclear pigmentation, was observed. The AMP further drastically reduced the growth rate of the five melanoma cell lines that were tested. The influence of dbc AMP was completely reversible 3 days after the agent was removed from the culture medium. The antigenic phenotype of the melanoma lines was compared before and after dbc AMP treatment. This was done with four monoclonal antibodies directed against major histocompatibility complex (MHC) Class I and II antigens and II monoclonal antibodies defining eight different melanoma‐associated antigenic systems. Treatment with dbc AMP reduced the expression of human leukocyte antigen (HLA)‐ABC antigens and beta‐2‐microglobulin in five of five melanoma lines. In the two HLA‐DR‐positive cell lines dbc AMP reduced the expression of this antigen in one line and enhanced it in the other. No induction of HLA‐DR or HLA‐DC antigens was observed in the Class II negative cell lines. Furthermore, dbc‐AMP modulated the expression of the majority of the melanoma antigenic systems tested. The expression of a 90‐kilodalton (KD) antigen, which has been found to be upregulated by interferon‐gamma, was markedly decreased in all the five cell lines. A similar decrease in the expression of the high molecular weight proteoglycan‐associated antigen (220‐240 KD) was observed. The reduced expression of Class I and II MHC antigens as well as the altered expression of the melanoma‐associated antigens studied were shown to be reversible after dbc AMP was removed. Our results collectively show that the monoclonal antibody‐defined melanoma‐associated molecules are linked to differentiation. They could provide useful tools for monitoring the maturation of melanomas in vivo induced by chemical agents or natural components favoring differentiation.

Characterization of four human malignant glioma cell lines

SummaryIn this paper, the characterization of four human malignant glioma cell lines is described. The four lines are positive for glial fibrillary acidic protein (GFAP) in variable amounts. One of them, LN 992, is positive for S-100 protein. Myelin basic protein could not be detected in any of the four lines. The four lines had high levels of CNPase activity. The karyotype shows polyploidy for all lines, with modal numbers ranging from 80 to 120 and various numbers of marker chromosomes. Particular attention has been paid to the surface phenotype and a panel of three antiglioma monoclonal antibodies (Mabs), five antimelanoma Mabs, one anti-CALLA Mab, and two anti-HLA-DR Mabs has been used in an antibody-binding radioimmunoassay for the four cell lines. Lines LN 215 and LN 235 are positive with two antiglioma Mabs, LN 992 is negative. The four lines are positive with all five antimelanoma Mabs, except for LN 992 which ist negative with Mab D5. LN 992 and LN 215 are positive with the anti-CALLA Mab N2A12. LN 308 and LN 992 are positive with anti-HLA-DR Mab D4-22. There was no correlation between the in vitro morphology of the lines and the expression of the various biochemical or surface markers. These results stress the heterogeneity of the phenotype of human malignant glioma lines. These lines will be useful tools for further immunologic studies.

Selective tumor localization of radiolabeled anti-human melanoma monoclonal antibody fragment demonstrated in the nude mouse model.

Monoclonal antibodies (MAb) directed against distinct epitopes of the human 240 kD melanoma‐associated antigen have been evaluated for their capacity to localize in human melanoma grafted into nude mice. A favorable tumor to normal tissue ratio of 13 was obtained with intact 131I‐labeled MAb Mel‐14. This ratio was further increased to 43 and 23 by the use of F(ab′)2 and Fab fragments, respectively. The specificity of tumor localization was demonstrated by the simultaneous injection of F(ab′)2 fragments from MAb Mel‐14 and anti‐CEA MAb 35, each labeled with a different iodine isotope, into nude mice grafted with a melanoma and colon carcinoma. The fragments from both MAb localized with perfect selectivity in their relevant tumor as shown by differential whole body scanning and by direct measurement of the two isotopes in tumors and normal tissues. These in vivo experimental results suggest that the F(ab′)2 fragment from MAb Me1–14 is suitable for melanoma detection by immunoscintigraphy in patients. Cancer 58:655‐662, 1986.

Monoclonal antibodies against recombinant-MAGE-1 protein identify a cross-reacting 72-kDa antigen which is co-expressed with MAGE-1 protein in melanoma cells

The MAGE‐1 gene codes for tumor‐associated peptides recognized by cytolytic T lymphocytes in association with MHC‐class‐I molecules such as HLA‐AI and HLA‐Cw16. In the course of a study aiming at the immunohistochemical detection of the MAGE‐1 gene product in tumor samples, 2 mouse monoclonal antibodies (MAbs) directed against a full‐length recombinant MAGE‐1 fusion protein were found to react strongly not only with the 46‐kDa MAGE‐1 protein, but also with a 72‐kDa product in immunoblots of lysates obtained from several MAGE‐1‐mRNA‐positive melanoma cell lines. Pre‐incubation of the antibodies with the recombinant MAGE‐1 fusion protein abolished their reactivity both with MAGE‐1 protein and with the 72‐kDa product, thus confirming the occurrence of antigenic determinant(s) shared by the 2 proteins. The 72‐kDa protein is not an alternative product of MAGE‐1, since it was still detected in lysates of a MAGE‐1 loss variant derived from a MAGE‐1‐positive melanoma cell line. Moreover, the 72‐kDa protein does not appear to be a product of the other members of the MAGE gene family known to be expressed in tumors (such as MAGE‐2, ‐3, ‐4 and ‐12). Interestingly, expression of the 72‐kDa protein was found to be correlated with that of MAGE‐I protein. Thus, in 30 tumor cell lines analyzed by immunoblotting and RT‐PCR, the 72‐kDa protein was never detected in MAGE‐1‐mRNA‐negative cell lines, while it was co‐expressed with MAGE‐1 protein in 12 out of 15 cell lines expressing MAGE‐1. Furthermore, the 72‐kDa protein was detected in lysates of human testis, the only normal tissue known to express MAGE‐1. Finally, treatment of MAGE‐1‐mRNA‐negative cell lines with 5‐Aza‐2′‐deoxycytidine, a hypomethylating agent known to induce MAGE‐1 expression, resulted in the expression of the 72‐kDa protein. Taken collectively, these findings suggest that expression of the gene encoding the 72‐kDa protein identified in this study through antigenic determinant(s) shared with MAGE‐1 protein is regulated in a way similar to that of MAGE‐1.

Phenotyping of 60 cultured human gliomas and 34 other neuroectodermal tumors by means of monoclonal antibodies against glioma, melanoma and HLA-DR antigens

The reactivity spectrum of three monoclonal antibodies (Mabs) to human malignant glioma, five Mabs to melanomas and one Mab anti-HLA-DR was investigated by an indirect antibody binding radioimmunoassay on a panel of cells derived from 60 glioma lines, including 47 malignant astrocytomas, 11 low-grade astrocytomas and two malignant ependymomas as well on cells from 12 melanoma, three neuroblastoma, three medulloblastoma, two schwannoma, two retinoblastoma, two choroïd plexus papilloma, ten meningioma and 12 unrelated tumor lines. The anti-glioma Mabs BF7 and GE2 reacted preferentially with gliomas, while the anti-glioma Mab CG12 reacted with gliomas, melanomas, neuroblastomas and medulloblastomas. The five anti-melanoma Mabs reacted with gliomas, neuroblastomas and medulloblastomas. The anti-HLA-DR Mab D1-12 reacted with gliomas, melanomas and some meningiomas. On the basis of the data presented, we describe three different antigenic systems; the first one is glioma-associated, the second one is related to differentiation antigens expressed on cells derived from the neuroectoderm and the third is represented by HLA-DR antigens which are expressed not only on B-lymphoblastoid cells but also on melanomas and gliomas.