CD4⁺ T cells enhance the production of IFN-γ by CD8⁺ T cells in response to subcutaneous tumor digest. (A) CD4⁺ or CD8⁺ T-cell subsets derived from TDLN CD62L^low cells activated for 85 days were mixed with enzymatically digested single cell suspensions derived from subcutaneous MCA205 or MCA207 tumors, a single cell suspension of in vitro–cultured MCA205 or immobilized anti-CD3 mAb. Brefeldin A was added at hour 5 and cells were harvested at hour 20, permeabilized and stained for IFN-γ and CD4 and CD8. First column shows CD8⁺ T_Es alone; second column, CD4⁺ T_E cultures stained for CD4⁺ cells; third column, CD4⁺ T_E cultures stained for CD8⁺ cells; and fourth column, CD8⁺ T_Es and CD4⁺ T_Es mixed at a 1:1 ratio and stained for CD8⁺ cells. The percentage of total cells is indicated in each compartment, whereas the percentage of IFN-γ–producing cells among the CD8⁺ T-cell subset is indicated next to the fourth column. (B) Subcutaneous MCA205 or MCA207 tumors were enzymatically digested to a single cell suspension, treated with FcR blocking antibody and stained with anti–I-A^b or anti-CD11b mAb then analyzed by FACS.