Tumor-Specific CD4⁺ T Lymphocytes from Cancer Patients Are Required for Optimal Induction of Cytotoxic T Cells Against the Autologous Tumor

MLTC-activated CD4⁺ T cells proliferate in response to the stimulatory autologous tumor cells in the presence of autologous DCs as APC. CD4⁺ T cells were collected from MLTC and tested for proliferative responses in microculture plates in the presence of whole autologous tumor cells (AuTu) and autologous DCs (A) and autologous DCs pulsed with AWE from the autologous tumor cells (AuTu-AWE-DC) (B). All mAbs were added at 10 μg/ml final concentration throughout the culture period. Bars represent mean values ± SD from 19 experiments (all 19 patients were tested). Control anti-TNP mAb, isotype-matched to anti-MHC class II mAb, was included in four experiments.

Specificity of CD4⁺ T cells activated during the MLTC. CD4⁺ T cells from patients 1, 9, 5, and 6 were tested in a proliferation assay against autologous DCs pulsed with AWE from autologous or allogeneic tumor cells. Columns indicate mean cpm ± SD from triplicate cultures. Pairs of columns indicate results from the same donor obtained with CD4⁺ T cells in parallel cultures set up in the absence (A) or presence (B) of anti-MHC-class II mAb.

Specificity of CD8⁺ T cells activated during the MLTC. CD8⁺ T cells from patients 1, 9, 5, and 6 were tested in cytotoxicity assays for killing of the autologous DCs pulsed with AWE from autologous or allogeneic tumor cells. Columns indicate mean percent cytotoxicity from triplicates ± SD. Pairs of bars indicate results obtained with CD8⁺ T cells from the same patient in parallel cultures set up without (A) or with (B) anti-MHC-class I mAb.

Cytotoxicity against autologous tumor targets mediated by CD8⁺ T cells collected from MLTC cultures set up using as responders whole MEAMNC (▪); CD4⁺ T cell-depleted MEAMNC (▴); CD14⁺ cell-depleted MEAMNC (•) (A); or CD8⁺CD4⁺ T cells and AuTu-AWE-DC (▪); CD8⁺ T cells and AuTu-AWE-DC (▴); CD8⁺ and CD4⁺ T cells pulsed with AWE (•) (B) (see text for details). Killing of K562 (▵) was marginal in both A and B. □, Inhibition with anti-MHC class I mAb. Inhibition with isotype-matched to anti-MHC class I mAb at the same E:T ratio is also shown (▿). Results are given as mean percent cytotoxicity ± SD from four experiments conducted with an equal number of patients.

Activated, AuTu-AWE-DC are capable of stimulating CD8⁺ T cells to lyse autologous tumor targets in the absence of CD4⁺ T cells. AuTu-AWE-DC were treated for 48 h as indicated in “Preincubation system.” Thereafter, CD4⁺ T cells and/or excess mAbs were removed (see also Materials and Methods) and the treated AuTu-AWE-DC were cocultured with autologous CD8⁺ T cells (stimulation phase). In one group (control group; ♦), CD4⁺ T cells from the preincubation system were added back to the culture during the stimulation phase. After 10 days of incubation, 3 rounds of restimulation were performed (restimulation phase) with MEAMNC depleted of CD4⁺ T cells and pulsed with AWE (MEAMNC (−CD4⁺) + AWE) except in group ♦, in which CD4⁺ T cells were included in AWE-pulsed MEAMNC. CD8⁺ T cells recovered from each group were then tested against autologous tumor targets and the results are expressed as mean percent cytotoxicity ± SD from five experiments.