Arthur Kimura

A rapid method for the separation of functional lymphoid cell populations of human and animal origin on PVP-silica (Percoll) density gradients.

Subclasses of lymphocytes can be separated on gradients of non‐toxic polyvinylpyrrolidone‐coated colloidal silica (Percoll) by virtue of differential densities. Such gradients can yield functionally active lymphocyte populations after brief centrifugation. Gradients can he generated in a discontinuous step fashion and centrifuged in standard table‐top laboratory centrifuges or as self‐generating gradients duriny ultracentrifugation. The density medium has low viscosity and can be made isotonic for virtually any use. Gradients have proved useful in both human and experimental animal studies, and high percentage yields allow for separations from small cell numbers. Methods are described for separation of whole blood and lymphotd subpopulations. including blasts stimulated with mitogens or in mixed lymphocyte reactions. The cytotoxic capability of various density fractions was evaluated for mixed lymphocyte culture‐induced allogeneic killing and spontaneous, so‐called ‘natural’ killer cell activity. The lower density associated with blast transformation allows for significant enrichments of stimulated cells from in vitro cultures. Higher thymidine incorporation, restimulation in mixed lymphocyte reactions, and greater cytotoxic capacity are associated with these‘blast’fractions.

The specificity of the combining site of the lectin from Vicia villosa seeds which reacts with cytotoxic T-lymphoblasts☆

Abstract The combining site of purified Vicia villosa lectin was studied by quantitative precipitin and precipitin inhibition assays. The lectin, which specifically hemagglutinated blood group A erythrocytes nevertheless precipitated to different extents with blood group A1, A2, H, B and precursor I substances from saliva and ovarian cysts, and with different blood group substances of the same specificity indicating an interaction of the lectin with a non-blood group determinant site. The agglutinating and precipitating activities of the lectin were not strongly affected by EDTA or bivalent cations. Precipitates of Vicia villosa lectin with certain blood group glycoproteins showed unusually high solubilities as compared to other lectin glycoprotein and antigen-antibody precipitates. Inhibition assays with various monosaccharides, glycosides and oligosaccharides indicate that the Vicia villosa lectin is specific for terminal, non-reducing, α-linked d GalNAc. Of the monosaceharides tested, methyl α d GalNAc, p-NO2-phenyl α d GalNAc and d GalNAc were best. They were about 100 times better inhibitors than the corresponding d Gal compounds. The most potent inhibitor was methyl α d GalNAc which was 10 times more active than d GalNAc and 18 times more potent than the β-anomer. Among disaccharides tested, d GalNAcαl → 3 d Gal was most active, about as potent as methyl α d GalNAc, twice as active as d GalNAcαl → 6 d Gal and 8 times more potent than d GalNAc αl → 6 d GalNAc , indicating the importance of a subterminal α l → 3- linked d Gal in the binding. d GalNAc α l → 3 d Gal β l → 3 d GlcNAc was 7 times less active and the blood group A determinant was less than 1 40 as active as d GalNAc α l → 3 d Gal . These findings indicate that the combining site of the Vicia villosa lectin is at least as large as the disaccharide d GalNAc α l → 3 d Gal and that the αl → 3 linkage is important in the binding. The unique nature of this site is consistent with its high specificity for a glycoprotein on cytotoxic T-lymphocytes.

Cytotoxic T lymphocyte membrane components: an analysis of structures related to function.

Linus Pauling once stated that “life is the harmony between molecules and not the property of any one.” As immunologists concerned with the “disharmony between molecules,” we seek to unravel and understand the complexities of cellular recognition of disturbing structures, cellular activation, and finally the relief of the disharmony by the various effector mechanisms of the immune system.

Cell surface characteristics of human histiocytic lymphoma cell lines. II. Expression of Helix pomatia a hemagglutinin binding surface glycoproteins, HLA-DR and common acute lymphocytic leukemia (cALL) antigen

Abstract A panel of human cell lines, derived from diffuse histiocytic lymphoma (HL), have been analyzed for the expression of Helix pomatia A hemagglutinin (HP) binding surface glycoproteins, HLA-DR and common acute lymphoblastic leukemia (cALL) antigen. The expression of HP receptors and HLA-DR was compared to that of other normal and malignant lymphoid and non-lymphoid cell lines. It is concluded that tumors of the histopathological entity HL, according to Rappaports [31] classification, are heterogenous in their expression of HP receptors, HLA-DR and cALL. Taken together with other features the present findings suggest that at least three types of HL exist, origin from (a) monocytic cells (extremely rare) (b) B-lymphocytes (most common) (c) non-T, non-B lymphocytes (relatively rare).

Cytotoxicity to allogeneic cells in the chicken: II. Specific cytotoxic T cells and macrophages in the spleens of agammaglobulinemic and normal alloimmune chickens☆

Abstract Repeated intravenous injections with allogeneic RBC or spleen cells caused a rapid but brief appearance of specific cellular cytotoxic activity in both NL and Aγ chickens. The activity was much higher in Aγ than in NL chickens, of longer duration, and contained a significant cytotoxic T-cell component which could not be demonstrated in NL chickens. A large fraction of the cytotoxic T cells adhered to Vicia villosa and showed relatively low density on Percoll gradients. Specificity of the cytotoxic cells in Aγ alloimmune chickens appeared to be directed at antigens of the major histocompatibility locus. Plastic-adherent cells from Aγ animals showed some specific and nonspecific killer activity. The specific component was removed by treatment with rabbit anti-T-cell serum and C. Specific cytotoxic activity in Aγ spleen cells was approximately equally distributed over Fc+ and Fc− cells. No evidence for specific cytotoxicity of nonT cells was obtained with cells from Aγ alloimmune chickens.

Receptor specificity, functional characteristics, and cell-surface phenotype of a highly selected anti-I-Ab-specific, long-term T-cell line.

A highly selected alloreactive T-cell line was developed by repeated restimulation of B10.D2/n lymph-node cells with irradiated C57BL/10Sn (BIO) spleen cells in long-term MLC for up to 2 1/2 years. Continuous growth of the line requires restimulation every 2 to 4 weeks with fresh H-2b stimulator cells. The line proliferates strongly against H-2b but not againstH-2d,H-2f,H-2q,H-2r, orH-2s stimulators. Analysis of recombinant mouse strains showed that the proliferative response is directed against I-Ab but not Kb or Db determinants. During the growth period of the line, strong cross-reactivity with H-2p (B10.P) and weak cross-reactivity with H-2k strains (e.g., CBA/J and B10.BR) was observed. A clone with exquisite specificity for I-Ab, but with no cross-reactivity with H-2p or H-2k was isolated from the line; thus clonal heterogeneity of the line still exists despite the highly selective growth conditions. — The majority of T cells from the line or clone were shown to bind I-Ab but not Kb or Db determinants either spontaneously during restimulation with fresh B10 stimulator cells or via membrane vesicles expressing I-Ab determinants. No killing activity by the line in either specific or nonspecific cytolytic T-cell assays was observed nor was the T 145 glycoprotein, characteristic of killer T cells, detected.