Functional defect of heat-inactivated human lymphocytes in mixed-lymphocyte culture

Possible causes were examined for the inability of heat-inactivated lymphocytes to induce proliferative responses in mixed-lymphocyte cultures (MLC). Cells heated at 45 degrees C for 60 min lost greater than 90% of their capacity to stimulate in primary (1 degree) or secondary (2 degrees) MLC. This was not due to accelerated or delayed proliferation, nor to a simple quantitative loss of antigen since a 10-fold increase in stimulators or sequential addition of heated stimulators at 4-hr intervals was ineffective. Heated B lymphocytes had approximately 80% expression of HLA-DR and DQ antigens compared to unheated B cells when measured by flow cytometry using monoclonal antibodies detecting both monomorphic and polymorphic antigens. Contrary to some reports, there was no evidence of direct suppression or induction of suppression by heated stimulators or their supernatants. Reconstitution of 1 degree and 2 degrees MLC with crude MLC supernatants or more purified interleukin 1 (Il-1) or interleukin 2 (Il-2) was unsuccessful. The results indicate the heat-induced defect occurs immediately and is not due to direct or indirect suppression, insufficient amounts of Il-1 or Il-2, nor loss of polymorphic Class II HLA determinants. Heat inactivation of stimulator function may result from failure to present an "immunogenic grid" or loss of accessory molecules required in lymphocyte interactions.