No abstract

Antibody-mediated suppression of the in vitro immune response to polymerized flagellin of Salmonella adelaide and to sheep erythrocytes was studied at the cellular level. Normal mouse spleen cells, preincubated in vitro with mixtures of antigen and antibody for short periods of time before being washed, did not respond to an optimal antigenic challenge in vitro, whereas similar cells treated with antibody alone gave a normal response. The degree of immune suppression was found to depend on the time of preincubation. Significant immune suppression could be induced in as short a time as 15 min, whereas profound suppression (90%) required the incubation of cells with mixtures of antigen and antibody for 4–6 hr. Mouse spleen cells treated similarly were also unable to respond subsequently to the antigen upon transfer to lethally irradiated hosts, as measured at both the level of the antigen-reactive cell and that of serum antibody production. These results were taken as evidence that in vitro an effect of antibody-mediated suppression occurred at the level of the immunocompetent cell. Similarities between immune tolerance and antibody-mediated suppression in vitro were described, and the significance of the findings discussed in the light of current concepts of the mechanism of antibody-mediated suppression.

Tissue culture techniques were combined with cell separation procedures to investigate the cellular requirements for a response to antigen, leading to the production of antibody-forming cells. Mouse spleen was dissociated, and the cells were separated into various groups on the basis of density, size, and active adherence. The ability of fractions to initiate a response in vivo, on transfer to an irradiated recipient, was compared to the response in vitro; and this ability was correlated with the presence or absence of phagocytic cells. Two different antigens were studied, sheep erythrocytes (SRC) and polymerized bacterial flagellin (POL). Density distribution analysis of spleen showed a wide density range of cells responding to both antigens in vivo. The same fractions responded to POL in vitro as in vivo. By contrast, only the light density regions responded in vitro to SRC. Response occurred in regions of overlap between lymphocytes and phagocytic macrophages. Separation by active adherence on columns of large glass beads gave a preparation containing large, medium, and small lymphocytes but no detectable phagocytic macrophages and very low levels of phagocytic polymorphs. This lymphocyte preparation responded to both antigens in vivo. In vitro it gave a full response to POL, but no response to SRC. Addition of a small quantity of the adherent fraction, enriched for phagocytic cells, restored response to SRC. The use of strain-specific antisera in a mixed culture containing a C57 phagocytic fraction and CBA lymphocytes showed that the lymphocyte fraction contributed the precursors of the final antibody-forming cells. The accessory cells from C57 spleen banded in the light regions of the density gradient where phagocytic macrophages were found. Irradiated spleen cells also activated the lymphocyte preparation, suggesting that the irradiated host provided the accessory cells for the in vivo response to SRC. Small lymphocytes were purified from spleen by the small glass bead size filtration technique. This sample of small lymphocytes responded less well to POL than the total lymphocyte population, but it responded as well in vitro as in vivo. The small lymphocyte preparation responded in vivo to SRC but not in vitro. Addition of a small quantity of the phagocyte-rich fraction from adherence columns restored the in vitro response to SRC. The results indicated that phagocytic cells are not required in the initiation of an immune response to POL. By contrast some accessory cell, possibly a phagocytic macrophage, is required for a response to SRC. The basis for this marked difference is discussed.

Smmnaiy. The tuiitara, Sphcnodon punctatum, has been shown to produce :mtil)odit!s t(» Salmonella adelaide fliiKt'llin. Tlie serum of immunized itnimals contained immnuoylobulins which pusst'ssed sedinientiUion coefficients of approx. 18S and 7S. The 18S iminiiiK)^luhiilin.s roscmliled tho yM immiinogloltiilins of otlier vertehrates in size and polypeptide chain stnictnrf. Thr 7S immunolobiilins were antijienically related to the lSS proteins and possessed li^ht chains resembling those of the latter on gel electrophore.sis in urea. Tbe heavy chains of the 7S protein dilfered from those of the macroglobnlin. thereby indicating the presence of distinct imniunoglubidin cla-Sfies. Although the immunization period lasted for over eight months, antibody activity was found only in the 18S imuiimoglobulin.The lymphoid system of the tuatara was found to be primitive. Tbe only clearly recognizable lymphoid organ in the adult animal was tbe spleen which showed a definite demarciihon into wbite pnlp and red pulp, Injected carbon particles were taken up only by tlie bver. The forHgii particles in this organ were associated with phagocytie cells whith resembled the Kuplfer-cells of the mammalian liver.The fact that this ancient reptile possessed multiple classes of immnnoglobulins is consistent with previous findings that distinct inunimoglobulin classes, defined by the presence of different heavy chaim. were present by the phylogenetic level of higher amphibians.