Magali Schreyer

Monoclonal antibody against carcinoembryonic antigen (CEA) identifies two new forms of crossreacting antigens of molecular weight 90,000 and 160,000 in normal granulocytes

Two new forms of non-specific crossreacting antigens (NCAs) were identified in the Nonidet P40 (NP-40) extracts of normal granulocytes by precipitation with the monoclonal antibody (MAb) 192 directed against carcinoembryonic antigen (CEA) and already known to crossreact with the perchloric acid soluble NCA-55. The NP-40 soluble NCAs recognized by MAb 192 have apparent mol. wts of 90,000 and 160,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Both NCAs appear to consist of a single monomeric polypeptide chain, since they have the same electrophoretic mobility in SDS-PAGE under reduced and non-reduced conditions. When granulocytes were extracted with perchloric acid instead of NP-40, only the 55,000 mol. wt antigen, corresponding to the previously described NCA-55, was precipitated by MAb 192. Furthermore, it was shown that NCA-55 is not a degradation product of NCA-90 or NCA-160 due to the perchloric acid treatment because exposure to perchloric acid of NCA preparations purified from NP-40 extracts did not change their apparent mol. wts in SDS-PAGE. It was also shown that NCA-160 is not a granulocytic form of CEA because it was not precipitated by the MAb 35 reacting exclusively with CEA. Immunocytochemical studies of granulocytes and macrophages showed that MAb 192 stained both types of cells whereas MAb 47 stained only the granulocytes and MAb 35 none of these cells. In granulocytes both MAbs reacted with antigens associated with granules and also present at the periphery of the nucleus as well as in the Golgi apparatus. The NCA-90 identified by MAb 192 was found by sequential immunodepletion to be antigenically distinct from the NCA-95 precipitated by MAb 47. The epitope recognized by MAb 192 on CEA and NCA molecules appears to be on the peptidic moiety because the antigens deglycosylated by the enzyme Endo F were still precipitated by this MAb. Taken together, the results indicate that MAb 192 identifies two novel forms of NCA (NCA-90 and NCA-160) in NP-40 extracts of granulocytes, which are distinct from CEA and the previously described NCA-55 and NCA-95 identified by MAbs 192 and 47, respectively, in perchloric acid extracts of granulocytes.

Production and characterization of a rat monoclonal antibody against the murine CD3 molecular complex

We describe the production of a rat monoclonal antibody, 17A2, that detects the T cell receptor-associated CD3 molecular complex. 17A2 cross-competes with a CD3 epsilon-specific reagent and similarly stimulates IL-2 production by T cells. Immunohistological and cell separation applications are shown.

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.

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.

Sparse distribution of γ/δ T lymphocytes around human epithelial tumors predominantly infiltrated by primed/memory T cells

SummaryEvidence from the mouse system has suggested that T lymphocytes accumulating in non-lymphoid tissue, in particular epithelia, may preferentially express the T cell receptor (TCR) γδ. In this study, we characterize the T cell receptor αβ or γδ phenotype of lymphocytes infiltrating human tumors of epithelial origin using monoclonal antibodies (mAb) for immunohistology and flow cytometry on cells extracted by enzyme digestion. This report shows that the majority of CD3+ tumor-infiltrating lymphocytes are TCR αβ+ but a small percentage of TCR γδ can be clearly defined scattered throughout the tumor tissue with apparently no microanatomical selection. So far there has been little evidence for an accumulation of activated T cells in human tumor tissues as defined by mAb against molecules appearing transiently during the acute phase of activation. Now mAb are available that can identify primed or memory T cells such as mAb UCHL-1 recognizing the CD45RO antigen. Here we show that CD3+ tumor-infiltrating lymphocytes have a statistically significant accumulation of primed T cells, as compared to the autologous peripheral blood lymphocytes, suggesting their immune stimulation by tumor cells.

Mouse mammary tumor virus superantigens and murine autoimmune gastritis.

BACKGROUND/AIMS Neonatal thymectomy induces autoimmune gastritis in BALB/c (minor lymphocyte-stimulating antigen [Mls]-1b) mice, whereas DBA/2 (Mls-1a) mice are resistant. Resistance has been linked to the Mls-1a locus, which encodes a retroviral superantigen, and to superantigen reactive T cells that express V beta 6+ T-cell receptors. V beta 6+ T cells are known to be deleted in mice expressing Mls-1a superantigens. METHODS Neonatal thymectomized BALB/c and Mls-1a congenic BALB.D2.Mls-1a mice were analyzed to examine directly the role of Mls-1a self-superantigens and V beta 6+ T cells in autoimmune gastritis. RESULTS Autoimmune gastritis was detected in thymectomized BALB.D2.Mls-1a mice with high incidence. Autoantibodies to the gastric H+,K(+)-adenosine triphosphatase were present independent of the Mls phenotype in sera of gastritic mice. Severe gastritis had already appeared 1 month after thymectomy in BALB.D2.Mls-1a mice. V beta 6+ T cells were deleted in the stomach lymph nodes of 1-month-old gastritic BALB.D2.Mls-1a mice but could be detected by immunocytochemistry in the stomach lesions. CONCLUSIONS Endogenous Mls-1a self-superantigens and Mls-1a reactive V beta 6+ T cells are not involved in resistance to autoimmune gastritis in BALB.D2 mice.

Use of Monoclonal Antibodies for the Histological Localization of Melanoma Associated Antigens on Fresh Tumor Material

Abstract For the staining of fresh tumor tissue with monoclonal antibodies, a three-stage biotin-avidin-peroxidase system was used. All tumor specimens were frozen within 15 min after excision and stored at −80° C until use. A series of primary and metastatic melanoma have been analyzed using 7 monoclonal anti-melanoma antibodies and 3 anti-glioma antibodies crossreacting with melanomas. Several other monoclonal antibodies, generated in our laboratory, were included in each experiment such as an anti-HLA-DR, an anti-T, an anti-CALLA and an anti-CEA. The results obtained showed a distinct staining pattern of the tumor masses with each monoclonal anti-melanoma tested. Almost no staining of the surrounding normal tissue was observed. The monoclonal anti-CALLA antibody displayed a faint staining of some of tumor nodules. With the monoclonal anti-HLA-DR antibody a positive staining of both the tumor cells and the infiltrating inflammatory cells was observed. The anti-T antibody stained the lymphoid cells infiltrating some of the tumors.