Rosette plaque assay: fine specificity differences among individual hapten primed lymph node cells

A combination rosette plaque assay was used to demonstrate simultaneously hapten binding and antibody secretion by hapten primed rabbit lymph node cells. A family of cross reacting haptens was employed to investigate fine specificity differences of hapten binding among early antibody forming cells. The compounds used included 3 nitro 4 hydroxy 5 iodophenyl acetic acid (NIP), 3,5 dinitro 4 hydroxyphenyl acetic acid (NNP), 3,5 diiodo 4 hydroxyphenyl acetic acid (DIP), and 3 nitro 4 hydroxyphenyl acetic acid (NP). The hapten primed lymph node cells were first rosetted with two different rosette target cells simultaneously; the priming (homologous) hapten coupled to a sheep red blood cell stabilized with pyruvic aldehyde (PASRBC). One of the cross reacting haptens was coupled to the other target PASRBC. The two rosette target PASRBC were distinguished by labeling one with a fluorescent marker. After rosette formation via centrifugation at 4°C, the cells were plaqued on fresh SRBC coupled with one of the several haptens. The simultaneous expression of the specificity of hapten binding and antibody secretion was manifested as a rosette in the center of a plaque. NIP primed lymph node cells produced three different types of rosettes within plaques. The majority bound only the homologous hapten coupled rosette target PASRBC (NIP PASRBC). A smaller number of cells bound only the cross reacting hapten coupled target cells, i.e., NNP PASRBC, DIP PASRBC, or NP PASRBC. The last group of cells bound both the homologous and cross reactive target PASRBC simultaneously. In animals immunized to either NNP or DIP, these being relatively non cross reactive, very little to no cross reactive binding was observed when the rosetted cells were plaqued on the homologous hapten conjugated plaque target SRBC. For example, NNP primed lymph node cells rosetted by employing NNP PASRBC plus DIP PASRBC, and then plaqued on NNP SRBC, were observed to bind the homologous NNP PASRBC exclusively. However, when the same hapten primed cells were rosetted as before, but not plaqued on the relatively non cross reactive plaque target cells (DIP SRBC), a significant increase was noticed in the cross reactive binding. It would appear that the use of the heterologous plaque target SRBC prejudiced the expression of heterologous binding which was not manifested when plaqued on the homologous plaque target SRBC. Moreover, the data considered collectively suggest a heterogeneity in the cross reactivity of hapten binding by plaque forming cells.