A. Pellegrino

A simple microabsorption technique for HL-A typing.

Summary A simple, reproducible microabsorption method for HL-A typing of peripheral leukocytes and of human cultured lymphoid cells is described. The test is economical as it requires only few cells and small amounts of diluted typing HL-A allo-antisera. The method can be used to quantitate different HL-A determinants on the cell surface.

A rapid microtechnique for in vitro stimulation of human lymphocytes by phytohemagglutinin

Abstract A simple, rapid micromethod to determine in vitro stimulation of lymphocytes with phytohemagglutinin is described. The test utilizes whole blood instead of purified lymphocytes and a γ-emitter, 125 I deoxyuridine in place of 3 H thymidine to pulse the cultures, thus greatly facilitating the procedure. This test is suitable in aiding the clinical evaluation of immune-deficiency diseases.

Solubilization of fetal HL-A antigens. A preliminary report.

SUMMARY Water-soluble substances bearing HL-A antigenic determinants were extracted from the spleen, lung, liver, and kidney of 3-, 4-, and 5½ -month fetuses. These substances inhibited alloantisera in a pattern consistent with the phenotype of the antigen donor. There were significant differentials in the HL-A antigens which were solubilized from various tissues. Fetal kidney was the best source for the solubilization of HL-A antigens by sonic energy.

A quantitative study of cross reactivity in the HL-A system with human cultured lymphoid cells and soluble HL-A antigens.

SUMMARY Cross reactivity in the HL-A system has been evaluated quantitatively by utilizing human cultured lymphoid cells and soluble HL-A antigens in cross absorption and cross inhibition assays. Most of the cross reactions among different HL-A specificities determined with platelets or peripheral lymphocytes were confirmed in this study. Some cultured lymphoid cells or soluble HL-A antigens possessing a cross reacting specificity cross absorbed a certain anti-HL-A antiserum, whereas others with the same specificity did not. The number of cells required to absorb cytotoxic HL-A alloantisera directed against cross reacting specificities is significantly greater than that necessary to absorb alloantisera directed against HL-A determinants present on the cell surface. This observation suggests that relatively small numbers of available antigenic determinants react with the cross reacting antibodies. The differential absorbing capacity of different cultured cell lines for the same cross reacting HL-A alloantibodies suggests a variability in the cell surface expression of cross reacting HL-A determinants.

An antiglobulin microcytotoxicity assay to analyze non-complement fixing monoclonal antibodies to human histocompatibility antigens.

An antiglobulin microcytotoxicity assay has been used to analyze non-complement fixing monoclonal antibodies to human histocompatibility antigens. The assay utilizes methodology similar to that of the widely used microcytotoxicity assay for HLA typing, requires low numbers of target cells and is suitable to test large numbers of samples. The sensitivity of the assay is influenced by the anti-mouse Ig antiserum used, by the sequence of addition of the various reagents and by the incubation time. The assay is suitable to screen supernatants of clones derived from hybridization experiments and to characterize the serological specificity of anti-HLA monoclonal antibodies.

The expression of HL-A antigens during the growth cycle of cultured human lymphoid cells☆

Abstract An extensive study of the relationship between the expression of HL-A antigens and the growth cycle of human cultured lymphoid cells revealed that the density of these genetically determined cell surface markers did not change during the cell cycle. However, the yield and the immunologic potency of extractable soluble HL-A alloantigens was found to be greater with cultures in the late log or resting phase as opposed to those in early log phase. These results indicate that the variation in the yield of soluble HL-A antigens extracted from cultured lymphoid cells depends on factors other than the number of detectable HL-A antigenic determinants present on the cell membrane. Our data suggest that the turnover of cell membrane components or the presence of pools of histocompatibility antigens during the cell growth cycle may account for the observed differences in both potency and overall yield of extractable, soluble HL-A antigens.

Linkage between the B-cell specific receptor for monkey red blood cells and HL-A antigens in man-mouse hybrids

The established human lymphoid cell lines 6410 and WI-L2 exhibit the recently discovered receptor for monkey red blood cells (MRBC). This receptor is specific for B cells. These lymphoid lines were fused with the BUdR-resistant murine fibroblast line IT22 by Sendai virus-mediated cell fusion. In 107 subclones of the primary fusion events, it was shown that the parental MRBC receptor and HL-A antigens segregated concordantly. Twenty-two clones expressed both, while 85 expressed neither marker. No discordant clones were observed. This syntenic relationship may be exploited in mapping the MHC region and in rapid demonstration of that chromosmal region in somatic cell hybrids. Hybrid clones expressed the HL-A specificities characteristic of the human parent line and no new nonparental specificities were detected.

Inhibitory effect of normal mammalian sera on the rosette formation of human lymphocytes with sheep red blood cells.

Abstract Sensitization of human peripheral lymphocytes with sera from some animal species could prevent the rosette formation with normal sheep red blood cells. Further, sera which did not block rosette formation by themselves did so after the first complement components were added. The inhibitor present in one normal rabbit serum was characterized as IgG by immunochemical means. In contrast, the inhibitor which required the first complement components had IgM characteristics. The rabbit IgG reacted specifically with human T cell structures, since it was absorbed by T cell but not by B cells or human γ-globulins. The titer of these blocking antibodies was increased by immunizing the rabbit with autologous blood in CFA.

Inhibition of T Lymphocyte Rosetting by HL-A Alloantisera and Complement

Summary The relationship between HL-A antigens and rosetting of sheep red blood cells (SRBC) with peripheral human lymphocytes has been investigated by incubating them with HL-A antibodies. Although sensitizing the lymphocytes with HL-A alloantisera had no effect on their ability to form rosettes with SRBC, further sensitization with C6 deficient rabbit serum as a source of early complement components inhibited the formation of rosettes with SRBC. The involvement of HL-A alloanti-bodies in the inhibition of rosette formation was shown first by correlating the HL-A phenotype of the lymphocytes and the HL-A specificity of the alloantisera and, second, by specifically absorbing the HL-A alloanti-bodies from the alloantisera. Complement was needed to inhibit rosette formation since this effect was lost when rabbit serum was treated to inactivate complement. The participation of complements classical pathway in rosette inhibition was shown by chelating the Ca2+ ions by EGTA treatment of the C6 deficient rabbit serum. Perhaps, binding of HL-A antibodies and early complement components to the lymphocyte surface disturbs the distribution of the receptors or affects the charge of the cell membrane, thus inhibiting the rosette formation with SRBC.