The production of a mouse monoclonal antibody, Ki-67, is described. The Ki-67 antibody recognized a nuclear antigen present in proliferating cells, but absent in resting cells. Immunostainings with Ki-67 revealed nuclear reactivity in cells of germinal centres of cortical follicles, cortical thymocytes, neck cells of gastrointestinal mucosa, undifferentiated spermatogonia and cells of a number of human cell lines. The Ki-67 antibody did not react with cells known to be in a resting stage, such as lymphocytes, monocytes, parietal cells and Paneth's cells of gastrointestinal mucosa, hepatocytes, renal cells, mature sperm cells, brain cells, etc. Expression of the antigen recognized by Ki-67 could be induced in peripheral blood lymphocytes after stimulation with phytohaemagglutinin, whereas it disappeared from HL-60 cells stimulated with phorbol esters to differentiate into mature macrophages in a resting stage. These findings suggest that Ki-67 is directed against a nuclear antigen associated with cell proliferation. A first series of immunostainings of tumour biopsies indicated that Ki-67 may be a potent tool for easy and quick evaluation of the proportion of proliferating cells in a tumour.

Production of a mouse monoclonal antibody reactive with a human nuclear antigen associated with cell proliferation

The crystal structures of major histocompatibility complex (MHC) molecules contain a groove occupied by heterogeneous material thought to represent peptides central to immune recognition, although until now relatively little characterization of the peptides has been possible. Exact information about the contents of MHC grooves is now provided. Moreover, each MHC class I allele has its individual rules to which peptides presented in the groove adhere.

Allele-specific motifs revealed by sequencing of self-peptides eluted from MHC molecules.

https://www.cell.com/cell/pdf/S0092-8674(94)90462-6.pdf

Inhibitors of the proteasome block the degradation of most cell proteins and the generation of peptides presented on MHC class I molecules

Production of monoclonal antibodies to group A erythrocytes, HLA and other human cell surface antigens-new tools for genetic analysis.

Monoclonal antibodies (O1 to O4) to oligodendrocyte cell surfaces: An immunocytological study in the central nervous system

Fine specificity analysis with monoclonal antibodies of antigens controlled by the major histocompatibility complex and by the Qa/TL region in mice.

The cell hybridization technique was used for the production of 12 monoclonal antibodies against H-2Kk, H-2Db, I-Ak and I-Ek antigens. The strain distribution pattern indicated that three antibodies reacted with new H-2 and Ia determinants, respectively, while the majority of determinants defined by the monoclonal antibodies showed good correlation with H-2 and Ia determinants described by conventional alloantisera. Monoclonal Ia antibodies showed strong reactivity with about 90% of surface IgM positive B cells, but not with T cells. In double fluorescence studies, both I-A and I-E determinants were always found to be coexpressed on the same B cells. When the high sensitivity of the fluorescence activated cell sorter was utilized, about 30 to 40% of purified lymph node T cells were found to carry both I-A and I-E antigens, although in a much lower density than B cells. In conclusion, monoclonal Ia antibodies appear to display the same serological and cellular reactivity pattern as do conventional antisera.

Isolation of twelve monoclonal antibodies against ia and h-2 antigens. Serological characterization and reactivity with b and t lymphocytes.

HYBRIDISATION of a myeloma cell line with hyperimmunised spleen cells can provide hybrid cell lines secreting monoclonal antibodies of predefined specificity1,2. Lines have thus been established which secrete homogeneous antibodies against antigens such as sheep red blood cells1, the hapten 2,4,6-trinitrophenyl2, influenza virus3, horse red blood cells, the haptens (4-hydroxy-3-nitrophenyl)acetyl and (4-hydroxy-5-iodo-3-nitrophenyl)acetyl, group A streptococcal carbohydrate, hen egg lysozyme, chicken γ globulin, the synthetic polypeptide poly-L-(Tyr, Glu)-poly-D,L-Ala-poly-L-Lys4 and antigens encoded by the major histocompatibility complex (MHC) of the rat5. We report here the derivation of three cloned hybrid cell lines which synthesise antibodies detecting different public antigenic specificities of the mouse H–2 complex. These hybrid cell lines can be grown in the mouse as ascites tumours, and the ascites fluids obtained contain large quantities of homogeneous anti-H–2 antibodies.

Hybrid cell lines secreting monoclonal antibody specific for major histocompatibility antigens of the mouse

CD30 is a costimulatory receptor on activated lymphocytes and a number of human lymphoma cells. Specific ligation of membrane-bound CD30 or cellular stimulation by PMA results in a metalloproteinase-mediated down-regulation of CD30 and release of its soluble ectodomain (sCD30). In this report, it is demonstrated that PMA-induced CD30 cleavage from Karpas 299 cells was mediated by a membrane-anchored metalloproteinase which was active on intact cells following 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate extraction of membrane preparations. Moreover, CD30 shedding was blocked by the synthetic hydroxamic acid-based metalloproteinase inhibitor BB-2116 (IC(50), 230 nM) and the natural tissue inhibitor of metalloproteinases (TIMP)-3 (IC(50), 30 nM), but not by the matrix metalloproteinase inhibitors TIMP-1 and TIMP-2. This inhibition profile is similar to that of the TNF-alpha- converting enzyme (TACE) and, indeed, mRNA transcripts of the membrane-bound metalloproteinase-disintegrin TACE could be detected in Karpas 299 cells. The ectodomain of TACE was expressed in bacteria as a GST fusion protein (GST-TACE) which cleaved CD30 from the surface of Karpas 299 cells and concomitantly increased the level of sCD30 in the cell supernatants. Hence, TACE does not only control the release of TNF-alpha, but also that of sCD30.

CD30 shedding from Karpas 299 lymphoma cells is mediated by TNF-alpha-converting enzyme.

The mechanism by which 2-ME acts as a macrophagesubstitute for the induction of a primary PFC response to SRC in vitro was studied in macrophage-depleted mouse spleen cell cultures. 2-ME could replace macrophages only in FCS-supplemented cultures. Evidence is presented that the function of 2-ME is independent of residual macrophages. Neither normal nor macrophage-depleted spleen cell cultures from congenitally athymic nude mice supplemented with 2-ME, with or without FCS, could give rise to a primary in vitro anti-SRC immune response. 2-ME, at an optimal concentration of 10 -5 M, induced DNA synthesis in normal and macrophage-depleted spleen cells in both FCS-containing and serum-free cultures. The peak response occurred on day 3. This stimulation was accompanied by a polyclonal B cell activation to antibody secretion which was much more pronounced in FCS-containing than in serum-free cultures. Spleen cells from nude mice showed a weaker DNA stimulation than did cells from normal mice in FCS-containing cultures, and nearly no response under serum-free conditions. T cells obtained by a nylon column adherence method from normal mouse spleen cells showed good DNA synthetic responses in FCS-containing, but no response in serum-free cultures. These results show that 2-ME has weak mitogenic activity for B cells, and in combination with FCS, strong mitogenic activity for T cells. Since the macrophage provides stimulation to the T cell in the primary anti-SRC PFC response in vitro, these results suggest that the direct mitogenic activity of 2-ME with FCS on T cells provides the functional substitution for macrophages.

Hilmar Lemke

Papers

Fine specificity analysis with monoclonal antibodies of antigens controlled by the major histocompatibility complex and by the Qa/TL region in mice.

Isolation of twelve monoclonal antibodies against ia and h-2 antigens. Serological characterization and reactivity with b and t lymphocytes.

Hybrid cell lines secreting monoclonal antibody specific for major histocompatibility antigens of the mouse

CD30 shedding from Karpas 299 lymphoma cells is mediated by TNF-alpha-converting enzyme.

Function of 2-Mercaptoethanol as a Macrophage Substitute in the Primary Immune Response in Vitro