Immune Responses in Vitro

Abstract

The suppressive effects of monospecific goat anti-mouse globulins on primary immunoglobulin class-specific plaque-forming cell responses in mouse spleen cell cultures were investigated. Anti-µ suppressed responses in all immunoglobulin classes, whereas anti-γ1 and anti-γ2 suppressed the γ1 and γ2 responses but not γM or γA responses, and anti-γA suppressed only γA responses. The mechanism of action of the anti-µ was studied in detail because of its suppression of responses in all immunoglobulin classes. The anti-µ was specific for µ-chain determinants; its activity was dose dependent, but was not mediated by killing cells with surface µ-chain determinants. Free γM but not γG myeloma proteins in solution effectively competed with µ-bearing cells for the anti-µ. An excess of anti-µ was necessary in the cultures for 48 hr to insure complete suppression of 5-day responses. However, after removal of excess anti-µ at 48 hr, responses could be stimulated by newly added antigen in cultures where incubation was prolonged to 7 days. Anti-µ was most effective when added at the initiation of cultures and had no suppressive effect when added at 48 hr. Excess antigen did not effectively compete with anti-µ for antigen receptors. Precursors of antibody-forming cells were shown to be the cell population where the suppressive activity of anti-µ was mediated. The experiments suggest that anti-µ combines with µ-chain determinants in antigen-specific receptors on the surfaces of antibody-forming cell precursors, prevents effective stimulation by antigen and subsequent antibody production. To explain suppression of responses in all Ig classes by anti-µ, several models were proposed. It is not possible to determine from the data whether stimulation of precursor cells with γG or γA receptors requires concommitant stimulation of separate cells with only γM receptors, or whether cells bearing γM receptors are precommitted to or differentiate into cells capable of synthesis of other Ig classes, or whether receptors of γM and another Ig class are present on some virgin precursors or the second Ig receptor appears after antigenic stimulation.

Immune Responses in Vitro

“…However, the selective suppression of lgG responses by antisera against alloantigens on lymphoid cells indicates that interactions involved in the generation of IgG responses are affected more than interactions involved with IgM responses. Two mechanisms can be dismissed: (a) the alloantiserum is not interfering with antigen recognition by B cells, since this function is mediated by immunoglobulin receptors (16,21), and normal IgM responses do develop; and (b) the alloantiserum is probably not activating a population of nonspecific suppressor T cells, since such cells do not interfere with initiation of the PFC response (22) which the alloantiserum does. Furthermore, we have been unable to detect suppressor T-cell activity after interaction of spleen cells with these alloantisera.…”

“…phenomenological point of view, is not unlike that of anti-u-chain antisera (16), and antibody to the stimulating antigen (10), both of which mediate their effect early in a specific, noncytotoxic fashion and interfere with the successful initiation of the immune response. The possibility that alloantisera suppress PFC responses in a nonspecific fashion by combining with any membrane alloantigens on responding spleen cells and producing membrane perturbations which preclude development of immune responses can be ruled out since antisera against H-2D region alloantigens failed to suppress responses.…”

Functional Maturation of Thymic Lymphocyte Populations in Vitro

“…Lymphoid cells were labeled with alCr by incubation with 0.1 mCi Na2~lCrO4 (The Radiochemical Centre, Amersham, England)/50 X 106 cells in L-15 medium (Microbiological Associates, Inc.) supplemented with 10% bovine serum for 1 hr at 37°C (27). The cells were washed twice with 50 ml L-15, then mixed with appropriate dilutions of anti-0 serum and guinea pig complement and incubated at 37°C for 30 min.…”

“…The amount of ~lCr released into the supernate was assessed using a Packard gamma scintillation counter (Packard Instrument Co., Inc., Downers Grove, Ilk). The percentage 51Cr released was calculated using a freeze-thaw control as 100% release and a complement-alone control as 0% (27). Supporting Cell Populations.--Three cell populations were tested for their ability to promote thymic lymphocyte maturation.…”

Synthesis of two classes of antibody, gammaM and gammaG or gammaM and gammaA, by identical cells. Amplification of the antibody response to pneumococcal polysaccharide type III.

“…(c) Blockage of receptors with anti-yM before stimulation in vitro resulted in the inhibition of yM as well as yG and yA PFC responses to sheep erythrocytes in the unprimed spleen cell population, whereas after priming there was a gradual loss of suppression ofyG1 (yl) and yG2 (y2) PFC by anti-yM, yA PFC were always inhibited with anti-# (13,14).…”