The fractionation of lymphoid cells

The work described in this thesis set out to elucidate the heterogeneity of lymphocytes from rats by investigating methods for the separation and analysis of lymphocyte sub-populations.
In order to separate lymphocytes on the basis of differences in their size, the technique of velocity sedimentation under the influence of gravity in a shallow albumin gradient was applied to rat thoracic duct lymphocytes. Tractions containing small lymphocytes, contaminated by fewer than 0.3% large, dividing cells could be obtained and were tested for their immunological function. In addition, large lymphocytes could be enriched from the 5% or so found in normal lymph to 50 to 90% in fractions from the second sedimentation. The separation was checked by sedimenting thoracic duct lymphocytes in which the large cells were specifically labelled by tritiated thymidine (CHAPTER III).
The tests to which the fractions were subjected included (a) their ability to restore a primary response to Salmonella adelaide flagella in irradiated rats (b) the adoptive transfer of the secondary response to tetanus toxoid (c) their graft-versus-host activity across a strong histocompatibility barrier. In each ease, small lymphocytes purified by velocity sedimentation performed equally as well as unfractionated cells, confirming the results of earlier authors using other purification techniques, while in assays (b) and (c) fractions containing large lymphocytes were less active; such activity as there was could have been explained by small lymphocyte contamination. No positive immunological function of large lymphocytes could be checked, however, so that it was possible that their poor performance resulted from handling the cells and the conditions of sedimentation. With this reservation, it was concluded that small and not large lymphocytes are the cells responsible for the induction of the immune responses investigated. (CHAPTER III).
Using the same technique, it was shown that in the spleen of rats depleted of lymphocytes through a thoracic duct fistula, the immunological memory that the rats still possessed was carried by small lymphocytes and probably not large, dividing cells. The performance of these non-recirculating small cells was, surprisingly, as good as that of recirculating small lymphocytes from the thoracic duet (CHAPTER III). Possible explanations are discussed in CHAPTER VI.
The ability of the technique of velocity sedimentation to discriminate another heterogeneity was then established. Thymus-dependent (T) and thymus-independent (B) lymphocytes were distinguished and partially separated according to the following criteria. Rapidly sedimenting (approximately 4.5 to 5.0 mm/hr) small lymphocytes from the thoracic duct took up considerably more uridine on incubation in culture than more slowly sedimenting smell lymphocytes: the peak of greatest incorporation coincided with the position to which T lymphocytes from radiation chimaeras sedimented. In addition, the rapidly sedimenting cells migrated in the manner characteristic of T lymphocytes when intravenously transfused to syngeneic recipients; they were to be found, after 24 hours, in the peri-arteriolar regions of spleen and in the deep cortex of lymph nodes. In the sedimented fractions they also coincided with the peak of graft-versus-host activity, which is probably initiated by T lymphocytes. These studies (CHAPTER IV) therefore showed the existence of T and B lymphocytes in normal rat lymph, and indicated a way in which they might be partially separated.
Columns of fine siliconed glass beads have been used in the past to filter out large lymphocytes and thus to collect small lymphocytes in the filtrate. In CHAPTER IV it was suggested that such columns could also discriminate T and B lymphocytes, B lymphocytes being preferentially retained: the argument in support of this depended on the enrichment in the filtrate of cells from thoracic duct lymph able to take up relatively great quantities of uridine, shown earlier to correspond to T lymphocytes. The depletion of B lymphocytes by the columns could explain the results of certain experiments where small lymphocytes purified by this technique were found to be defective in their ability to induce humoral antibody formation (CHAPTER III). These experiments also allowed the derivation of a minimum estimate of 30 to 35% for the proportion of B lymphocytes in the rat thoracic duet (CHAPTER IV).
The possibility of developing a rapid and simple plaque assay to enumerate the frequency of lymphocytes in a test population bearing a receptor for a particular antigenic determinant was explored in CHAPTER V. The assay involved the binding of dinitrophenylated bacteriophage to thoracic duct lymphocytes from normal and immune donors and counting the numbers of phage eluted from the cells by competing free hapten, after their immobilisation on an agar plate containing indicator bacteria sensitive to the phage. Various factors influencing the measured frequency of antigen-binding cells were investigated, including temperature of the incubation, alterations in the cell:phage ratio and methods of washing the cells, A model system using antibody-coated Sephadex beads was also studied, which revealed the complication that binding of phage to the surface was very firm (possibly due to polyvalent attachment) and was not readily reversed by free hapten. The chief factor preventing the useful application of the assay was the variability of the results, for which the causes were investigated: suggestions were made for improving the assay (CHAPTER V).
To accompany the studies with dinitrophenylated bacteriophage the responses of rats to immunisation with dinitrophenylated bovine gamma globulin, and the performance of their thoracic duct lymphocytes when transferred to irradiated recipients were studied (CHAPTER V). A passive haemagglutination assay was developed to measure the responses, which used rat erythrocytes coated with a dinitrophenylated, non-agglutinating fragment of rabbit immunoglobulin whose antibody activity was directed against rat erythrocytes (APPENDIX I). It turned out to be a sensitive assay, and substantial titres of anti-DNP antibody could be neaaured following primary or secondary immunisation of rats with hapten-protein conjugate.

Advisor: Gowans, J