Georges E. Roelants

Methods for derivation and detection of anti-parasite monoclonal antibodies

We describe detailed methods for derivation and cloning of myeloma hybrids which secrete antibodies specific for antigens of protozoan parasites. The methods were designed to enable the derivation of large numbers of specific monoclonal antibodies and to give high cloning efficiencies of desired hybrids. Although special attention is paid to derivation and detection of anti-parasite antibodies, the methods can be applied to many different antibody-antigen systems. Using the described methods we have isolated more than 90 myeloma hybrids which secrete antibodies specific for antigens of African trypanosomes and Theileria parasites, thus illustrating their effectiveness.

Immunobiology of African Trypanosomiasis

African trypanosomes are flagellated Protozoa that include the causative agents of important human and animal diseases (Table I) and are fascinating biologically.

Antigen Recognition by B and T Lymphocytes

Selection theories of antibody formation (Jerne, 1955; Talmage, 1957; Burnet, 1959) postulate that lymphocytes are precommitted in their specificity. Thus a given lymphocyte would first “recognize” a single antigenic determinant and subsequently make identical antibodies able to combine with that determinant. The widespread acceptance of the “clonal selection of acquired immunity” has engendered a lot of efforts aimed to understand how lymphocytes recognize a determinant and how they are triggered to proliferate, differentiate and secrete antibody.

The bovine lymphoid system: II. Derivation and partial characterization of monoclonal antibodies against bovine peripheral blood lymphocytes.

We have raised monoclonal antibodies to produce reagents specific for bovine lymphocyte subpopulations. Spleen cells from mice immunized with bovine peripheral blood lymphocytes were fused with X63-Ag8 myeloma cells and eleven myeloma-hybrids which secreted antibody specific for bovine lymphocytes were doubly cloned. Five of the hybrids secreted antibodies which bound to the majority of bovine lymphocytes. Two of these antibodies were specific for polymorphic antigens. One antibody bound to B lymphocytes and serum IgM molecules. The remaining five bound to subpopulations of lymphocytes. Four monoclonal antibodies bound only to bovine cells while six also bound to lymphocytes from other bovidae. None bound to human lymphocytes. We discuss the difficulty of correlating the specificities of monoclonal antibodies to functional lymphocyte subpopulations in outbred animals where few other defined markers are available.

The regulation of the T-lymphocytes precursor pool by a humoral factor released by the thymus

Abstract Studies in congenitally athymic nude mice grafted with syngeneic thymus enclosed or not in a cell-impermeable Millipore chamber show that the thymus secretes one or several humoral factors controlling the size of the pool of early T-lineage cells produced by the bone marrow.

SUPPRESSOR CELLS IN TRYPANOSOMA CONGOLENSE-INFECTED MICE

Spleen cells from mice infected with T. congolense strongly suppressed lymphocyte stimulation induced in normal spleen cells by incubation with mitogens or allogeneic cells. Cell dilution studies showed that suppressor activity was extremely strong. Suppressor cell activity was markedly reduced by treatment of spleen cell populations with mitomycin-C and was unaffected by treatment with anti-Thy.1 sera and complement. Removal of cells which bound carbonyl iron or which bound to nylon columns, decreased but did not abolish suppressor activity.

Ontogeny of T Lymphocytes Studied in Athymic and Foetal Mice

Earlier studies on T lineage lymphocytes in congenitally athymic nude mice (1, 2) have been extended to surgically T deprived mice and early stages of embryogenesis. They permit the further characterisation of a new type of lymphocyte and its implication in T cell ontogeny.

RECEPTORS FOR ANTIGEN ON “B, T, B-T AND NUL” LYMPHOCYTES IN NORMAL AND “NUDE” MICE

ABSTRACT A rabbit anti-mouse θ reagent was made specific by absorbtion in nude mice in vivo . When used in double fluorescence with anti-Ig reagent it allows the recognition of 4 lymphocyte types: Ig + θ - , Ig - θ + , Ig - θ - , Ig + θ + . In nude mice spleen more than 20% of the lymphocytes are of T lineage. Antigen binding receptors are found among all cell types. By using combined autoradiography for 125 I-antigen and fluorescence for surface Ig and θ it is found that prior anti-Ig treatment induces the polar redistribution of receptors for antigen on both B and T cells followed by their removal from the cell surface. When put in culture for a few hours, both B and T cells are able to bind antigen again and the receptors are again cleared by anti-Ig. These results show that both B and T cells synthesize their Ig receptors for antigen.

Immune Depression in Trypanosoma Congolense-Infected Mice

The capacity of spleen cells from Trypanosoma congolense-infected mice to respond to the mitogens concanavalin A and bacterial lipopolysaccharide and to allogeneic lymphocytes is severely depressed or abolished. Moreover these cells cannot serve as stimulators of DNA synthesis in mixed lymphocyte reactions. The lack of responsiveness or of stimulation cannot be attributed to the dilution of appropriate B or T lymphocytes by the large number of “null” cells found in the spleen of infected mice. These “null” cells bear approximately ten times more H-2 antigen than normal lymphocytes but are devoid of Ia antigen.

Recognition of Antigen by B and T Lymphocytes

At the end of the session on “Lymphoid cell receptors and antigen recognition”, I would like to make a few brief remarks about a subject which paradoxically has barely been touched: that of the recognition structure of B and T lymphocytes.