A quantitative assay for GvH in the irradiated mouse by uptake of 3H thymidine in vitro

A rapid in vitro assay of graft versus host (GvH) reactivity in the adult inbred mouse has been developed, which quantitatively measures the response of lymphocytes to allogenic host antigens. C³H/HeJ mice were lethally irradiated with 1000R using a bilateral cobalt source. One day later, animals received graded doses of syngeneic or allogeneic (BALB/c) spleen cells intravenously. Animals were sacrificed daily up to 7 days, spleens weighed and individual cell suspensions made. These cells (6 x 10⁵/well) were incubated in microculture plates for 4 hrs in the presence of 1 microcurie of [³H]thymidine ([³H]Tdr) prior to harvesting on a multiple automated sample harvester. There was a 3-5 fold increase in incorporation of [³H]Tdr when animals received 12-50 x 10⁶ allogeneic spleen cells compared to syngeneic recipients 4 days post transplant. Comparing allogeneic transplants with radiation exposed animals receiving no cells results in a 20-30 fold increase in incorporation of isotope. An H2 identical M locus different strain (CBA) produced intermediate incorporation values. Cells from recipient spleens had cytotoxic activity against tumor cells of host H2 type and H2 typing of recipient spleens revealed donor type cells only 4 and 6 days post transplant.