Mode of Action of a Supernatant Activity from T-Cell Cultures that Nonspecifically Stimulates the Humoral Immune Response

Abstract

The mode of action of "allogeneic supernatant" (the culture supernatant of a 24-hr mixedlymphocyte reaction), has been studied. This factor stimulates the response of spleen cell cultures depleted in thymus-derived lymphoid cells (T-cells) to antigens that elicit a thymus-dependent response. We used a limiting dilution analysis, in which the frequency and size of response of individual bone-marrow-derived lymphoid cells (B-cells) could be measured. In confirmation of other reports, the occurrence of B-cells responding to antigen under different conditions was shown to follow a Poisson distribution in mouse spleen cell suspensions. Allogeneic supernatant increased responses to thymusdependent antigens, both by increasing the frequency of B-cells whose response is initiated and by increasing the numbers of antibody-forming cells obtained from each responding B-cell. Two fractions were obtained by dialysis of the supernatant. The nondialyzable fraction contained factors able to increase both the frequency of B-cells responding to sheep erythrocytes, and the size of the responding unit. The dialysate contained factors that were only able to increase the numbers of antibody-forming cells obtained per responding B-cell from B-cells whose response had already been initiated by antigen-specific T-cells.Since the nondialyzable factors were active in the absence of detectable functional T-cells, it was concluded that these factors, produced by T-cells, might represent one mechanism whereby T-cells cooperate with B-cells in the initiation or development of a humoral immune response.The humoral response to a haptenic determinant coupled to a carrier structure (such as a heterologous erythrocyte) involves some cooperative activity between a hapten-specific bone-marrow-derived lymphoid cell (B-cell) whose progeny will eventually synthesize antibody and a carrier-specific thymus-derived lymphoid cell (T-cell) that performs some "helper" function but does not itself make antibody.Abbreviations: ATXBM, adult thymectomized irradiated bonemarrow-reconstituted mouse; B-cell, bone-marrow-derived lymphoid cell; T-cell, thymus-derived lymphoid cell; LPS, lipopolysaccharide W, from Escherichia coli 055:B5; RBC, red blood cells; SRBC, sheep red blood cells; HRBC, horse red blood cells; Tnp, trinitrophenyl; thymus-dependent (-independent) antigen, an antigen, the response to which is severely depleted in (unaffected by) the absence of thymus-derived lymphoid cells; thymusdeprived mice, mice deprived of thymus and thymus-derived cells.

Histocompatibility antigen-activated cytotoxic T lymphocytes. II. Estimates of the frequency and specificity of precursors.

“…The use of conditioned medium increased both the number of positive wells and the amount of '~lCr release in these wells; it may have worked by increasing the activation efficiency and may be in addition to the clone size. We do not know whether this effect was brought about by nutrients, processed antigen, allogeneic effect factor (27), T-derived medium factor (28), or by yet other mechanisms.…”

T cell regulation of immunoglobulin class expression in the antibody response to trinitrophenyl-ficoll. Evidence for T cell enhancement of the immunoglobulin class switch.

“…In the splenic focus assay we were concerned with the positivity or negativity of a given fragment for the production of a given isotype ofanti-TNP Ab. The cutoffin counts per minute anti-isotype bound which was used to categorize supernatants as positive or negative in our RIA was determined in two ways: (a) by calculating the X ± 3 SD of the counts per minute bound in all splenic fragments from mice given B cells only (no antigen) or B and T cells (no antigen), and (b) by constructing a histogram of the percentage of fragments (from mice which received cells and antigen) that fall into ascending counts per minute-bound categories and determining the point at which a trough in the bimodal distribution of positive and negative fragments appeared (24). In general, the cutoff point for positivity was the same with both of these approaches.…”

Separate Signals for the Initiation of Proliferation and Differentiation in the B Cell Response to Antigen

“…It was shown that the response of T depleted cell populations could be restored by irradiated allogeneic T cells (Hirst & Dutton 1970) or by the culture supematants of allogeneic spleen cultures (Dutton et al 1971). If the activity of such culture supematant is assayed in a limiting dilution assay it can be shown that both the apparent frequency of responding B cells and the number of PFC per responding B cell is increased by the presence of the T cell factor (Hunter & Kettman 1974). This was, at first, interpreted as indicating that both signals, antigen and T cell factor were necessary for the initiation of the response.…”