Proliferative Responses by Immune Murine Popliteal Lymph Node Cells to High-Molecular-Weight Basic Allergen of Ryegrass Pollen and Its Cyanogen Bromide Fragments

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Abstract
High-molecular-weight basic allergen (HMBA) of ryegrass pollen and its fragmented form obtained by cleavage with cyanogen bromide (CNBr) (HMBA-frag) were evaluated for their immunogenicity in terms of their capacity to stimulate the in vitro proliferative responses of the in vivo antigen-primed popliteal lymph node (PLN) cells. For this purpose, mice (C3H/HeJ) were immunized in the hind foot pads with solutions containing 1–5 μg of HMBA or HMBA-frag emulsified in Freund’s complete adjuvant (FCA). Eleven days later, single-cell suspensions of the PLN were cultured in vitro for 4 days in the presence of either HMBA or HMBA-frag at concentrations ranging from 0.625 to 20 μg/ml. 3H-thymidine was added to the cultures 6 h prior to harvesting the cells. The degree of proliferation was assessed from the extent of intracellular incorporation of the 3H-thymidine by the PLN cells. Comparable degrees of lymphocyte proliferation were consistently obtained on stimulation with either HMBA or HMBA-frag of PLN cells from mice immunized with the allergen HMBA. A similar finding was made using PLN cells from mice immunized with the HMBA-frag. This study demonstrated that the CNBr-derived fragments of HMBA retained the immunogenicity of the parent molecule HMBA in terms of being able to (i) induce the in vivo priming of PLN cells and (ii) elicit the in vitro proliferative response of the antigen-primed PLN cells.