Young (less than 4 months of age) gp70-deficient (n=14) and -sufficient (n=25) mice were injected with 5x10⁴ CT26 tumor cells and tumor growth was monitored for 60 days by palpation of the injection site. The two groups are significantly different (p<0.0001) as determined by the log rank test.

A. Young (less than 4-months of age) gp70-sufficient (+/+) and –deficient (-/-) mice were injected with live CT26 tumor cells and then 4 and 6 days later, were injected with AH1 peptide in adjuvant [32]. TIL were extracted and stained on day 14. The relative avidity of T cells was extrapolated from the mean fluorescence intensity (MFI) of tetramer on a Mo-Flo flow cytometer (DakoCytomation Inc.). The relative tetramer staining of the CD8+ cells is shown in the histogram on the right: MFI of CD8+ tet+ cells is 58 for the gp70+/+ mouse (grey filled), and 72 for the gp70-/- mouse (black line). The MFI of CD8 staining of the tet+ cells was 904 for TIL from the gp70-sufficient and 766 from the –deficient mouse. The data are representative of 4 experiments. B. Mice were treated as in A and data were collected on a Cyan flow cytometer (DakoCytomation Inc.). Live events gated on CD8+ cells are shown. The relative MFI of tetramer staining of the CD3+ cells is shown in the histogram on the right: MFI of CD8+ CD3+ tet+ cells is 27.6 for the gp70+/+ mouse and 41.3 for the gp70-/- mouse. The MFI of CD3 staining of the tet+ cells was 36.9 for TIL from the gp70-sufficient and 30 from the –deficient mouse. The data are representative of 3 experiments. C. TIL from mice treated as in A were gated on L^d-tet/AH1+ CD8+ (y-axis) and costained with a panel of TCR V-beta antibodies (x-axis) from two mice. The y-axis scale is the same on both graphs.

mRNA was extracted from CT26 tumor cells (A), T2-Ld cells (B), and from the colons of gp70-sufficient (C), and gp70-deficient mice (D) (>8 months old). cDNA was produced and used in semi-quantitative RT-PCR reactions. Amplification was terminated at 20, 30, 35, and 40 cycles, and the DNA products were resolved on 2% agarose gels. Gp70 and β-actin cDNAs were assayed. NT, no-template was assayed to monitor contaminants.

⁵¹Cr-labeled CT26 (A) and peptide-loaded MC57G-Ld (B) cells were combined with increasing numbers of the CT-T cell clone [dashed lines, [30]] or splenocytes from young (less than 4-months old) congenic mice backcrossed 3 generations, then intercrossed: gp70-deficient (diamonds), and gp70-sufficient mice (circles). B. Percent lysis of MC57G-Ld cells incubated with the AH1 peptide minus the MCMV peptide is shown. Percent lysis was calculated after 4 hours at 37°. The negative values are due to more chromium release from the MCMV-loaded targets relative to the AH1-loaded targets. A representative assay is shown; assays were performed in triplicate. C. Gp70-sufficient and -deficient mice that were less than 6-months old, were vaccinated twice, one week apart with irradiated CT26-GM cells. Two million CSFE-labeled splenocytes, half incubated with the AH1 peptide and half with the βgal peptide, were injected and detected by flow cytometric analyses of the host spleen 24 h later. The bar represents the mean of the % specific lysis. The negative values in the gp70+/+ group are from mice that had more CFSE-low (βgal) cells than CFSE-high (AH1) cells, indicating that killing of AH1-loaded targets in this experiment was not detected. The 2 groups are significantly different (p=0.0061) as determined by a two-tailed unpaired t test.

A. Young (Y, <7-weeks old) and middle-aged (MA, >8-months old) gp70-sufficient (+/+) and –deficient (-/-) mice were vaccinated with irradiated CT26 tumor cells engineered to express GM-CSF two times, one week apart. Splenocytes were stained with AH1 peptide-loaded tetramer and anti-CD8 antibody either ex vivo or after one week in culture. The percentage of AH1-specific T cells ex vivo in Y gp70-sufficient and –deficient mice is significantly different using an unpaired two-tailed t test (p=0.0453). The difference continues to be significant after one week in culture (p=0.0132), as is Y vs. MA gp70-sufficient (p=0.0199) and MA gp70-sufficient vs. gp70 deficient (p=0.0035). B. Splenocytes from mice vaccinated as in A, but with CT26-βgal-GM were stained ex vivo with the H-2L^d tetramer loaded with the βgal peptide. The percentage of βgal-specific T cells in the Y and MA gp70-sufficient mice is the same (p=0.4999). C. Tissue from colon (C), thymus (T), and liver (L) was dissected from the young and middle-aged mice used in A, and PCR reactions were performed as described in Fig 1 and Table 2 for 30, 35, and 40 cycles. D. Young (squares, n=11) and middle-aged (circles, n=9) gp70-sufficient mice were injected with irradiated CT26-GM as in A, then one week after the last injection, were challenged and monitored as in Fig 2. The 2 groups are significantly different (p=0.0151) as determined by the log rank test.