The localization of antigen in relation to specific antibody-producing cells. I. Use of a synthetic polypeptide [(T,G)-A--L] labelled with iodine-125

Abstract

A synthetic multichain polypeptide, (T,G,)-A--L 509, was trace labelled with ¹²⁵I in the tyrosine end groups, composing the main antigenic determinants, and used to study the distribution of antigen in the draining lymph nodes after injection into the footpads of mice. The polypeptide was administered: (a) in saline solution into unprimed mice—in which form it is not demonstrably immunogenic; (b) in Freund's adjuvant into unprimed mice—in which form it is immunogenic; and (c) in saline solution to primed mice, so as to give a booster response. Experiments comparable to (c) were also done with [¹²⁵I]haemocyanin.

At various time intervals from 12 hours to 21 days later sections of the draining nodes were examined by radioautography and methylgreen—pyronine staining, or by a combination of immunofluorescent staining for antibody containing cells with radioautography. In unprimed mice, irrespective of whether they made antibody, labelling was found predominantly in the phagocytic cells of the medulla and the cortical sinus, but definite weaker labelling was also seen in the germinal centres. The label persisted throughout the period of observation, but tended to become more prominent with time in the germinal centres of mice making antibody in response to antigen in Freund's adjuvant. In primed mice the label was rapidly and predominantly concentrated in germinal centres, where it persisted throughout the period of observation while its intensity gradually diminished in other sites. The distribution of label in germinal centres was in a lace-like or dendritic pattern, similar to that described by Nossal and co-workers, and did not correspond to the lymphocytes or pyroninophilic cells in the centres.

Grain counts were made over specific antibody containing cells in both primary and booster responses to (T,G)-A--L. Such cells sometimes lay close to and sometimes many cell diameters distant from phagocytic cells containing concentrations of the antigen, but the cells themselves did not have grain counts significantly different from the background count. If the antigen remained undegraded, under the conditions of the experiment it would have been possible to detect the presence of fifteen molecules or less in a cell. Reasons are given for supposing that ¹²⁵I constituted a valid label of the main antigenic determinant groups, of which there were a maximum of 100 per molecule.