Figure 2. Knock-down of Beclin 1 in antigen donor cells decreased cross-presentation of tumor antigens by DC.

(A) B16 F10 melanoma cells were transduced with lentiviral vectors encoding a mir-30 modified short hairpin RNA (shRNAmir) specific for mouse Beclin 1 or a nonspecific control shRNAmir. The specific knockdown of Beclin 1 was confirmed by western blot analysis with anti-beclin 1 antibody. Anti- actin antibody was included as the control.

(B) F10 melanoma cells expressing either control or Beclin-1 shRNAmirs were treated with 20 nM rapamycin for 18 hours. Lysates were prepared from both untreated and treated cells. Ten microgram of total proteins were loaded and subjected to SDS PAGE and western blot analysis with rabbit anti-LC3 antibody.

(C) B16 F10 melanoma cells that were transduced with beclin 1 or control shRNAmir were injected into both flanks of C57BL/6 WT or TAP1 knock-out mice (n=4) that received 5 x10⁶ Thy1.1⁺ pmel-1 transgenic T cells. Six days after tumor injection, the CFSE profile of transferred pmel-1 T cells from the draining lymph nodes was determined.

(D) The percentage of pmel-1 T cells found among the lymphocytes in the draining LNs of each mouse was determined by flow cytometry. All values indicate the mean with standard deviation (n=4). The difference between control and Beclin-1 knockdown was significant; however, the difference between beclin-1knockdown and no antigen was not significant.

The data represent the results from three independent experiments.