Augmenting major histocompatibility complex class I expression by murine tumors in vivo enhances antitumor immunity induced by an active immunotherapy strategy

Exposure of B16.F10 cells to rmIFNγ in vitro increases H-2Kb MHC class I expression. B16.F10 cells were exposed to 500 U/mL of rmIFNγ for 48 hours, followed by staining with FITC-labeled mouse anti-mouse H-2Kb MAb or an isotype-matched control antibody, and subjected to flow cytometry; 0.42% of untreated B16.F10 cells bound the fluorescent label, compared with 95% of B16.F10 cells that had been exposed to rmIFNγ. The experiment shown represents 1 of multiple performed.

Intraperitoneal administration of rmIFNγ enhances the expression of H-2Kb MHC class I by established B16.F10 flank tumors. B16.F10 tumors were established in C57BL/6 mice and rmIFNγ (5000 U) was injected as a single IP dose. The tumors were resected 24 or 48 hours after rmIFNγ administration and evaluated using immunohistochemistry. The dark brown surface membrane staining represents MHC class I expression. A, Twenty-four hours after rmIFNγ treatment. B, Forty-eight hours after rmIFNγ treatment. C, Tumor from an untreated mouse.

Up-regulation of H-2Kb MHC class I expression enhances the binding of CD8+ CTLs to B16.F10 tumor cells in a conjugate-formation assay. Naive B16.F10 cells or rmIFNγ-exposed B16.F10 cells were stained with hydroethidine (red), and tumor-specific CD8+ CTLs were stained with sulfofluoresceindiacetate (green). The 2 cell populations were then cocultured and subjected to 2-color flow cytometry. The binding of CD8+ CTLs and B16.F10 cells is defined as the double positive population in the right upper quadrant. A to C, Cytograms from a representative experiment of 3 independent experiments. A, Naive B16.F10 cells cocultured with CD8+ CTLs. B, rmIFNγ-exposed B16.F10 cells cocultured with CD8+ CTLs. C, rmIFNγ-exposed B16.F10 cells cocultured with CTLs and anti-MHC class I MAb. D, Summary data from 3 separate experiments performed. The % binding is defined as: Binding (%) = (no. of green-red double positive cells)/(no. of green positive cells) × 100. Each bar represents the mean ± SEM.

Pretreatment of B16.F10 cells with rmIFNγ results in more efficient stimulation of tumor-specific CTLs in vitro. CTLs were harvested from mice and evaluated for IFNγ release (activation) in the ELISPOT assay as described in Materials and Methods. Each bar represents the mean number of spots ± SEM from a total of 3 wells from each group. A, Splenocytes from AdCD40L-treated, tumor-bearing mice. B, Splenocytes from tumor-bearing mice receiving no treatment. C, Splenocytes from AdNull-treated, tumor-bearing mice. D, Splenocytes from untreated, tumor-free mice. *No spots detected.

In vivo administration of a single dose of rmIFNγ to tumor-bearing mice enhances tumor-specific CTL generation induced by AdCD40L. CTLs were harvested from tumor-bearing mice and evaluated for IFNγ release (activation) using the ELISPOT assay as described in Materials and Methods. Each bar represents the mean number of spots ± SEM from a total of 3 wells for each group. Target cells for this experiment consisted of only untreated B16.F10 tumor cells.

A single dose of rmIFNγ enhances the antitumor effect and cure rate obtained with intratumoral injection of AdCD40L. Twelve days after B16.F10 flank tumor initiation, C57BL/6 mice were randomized to 6 groups: AdCD40L plus IP rmIFNγ (n = 6); AdCD40L alone (n = 6); AdNull plus IP rmIFNγ (n = 6); AdNull alone (n = 5); IP rmIFNγ alone (n = 5); or PBS (n = 5). All vectors were administered intratumorally (5 × 10¹⁰ particle units/100 μL of PBS), and mIFNγ (5000 U) was administered concomitantly with the adenovirus vectors. The tumor area was assessed at 2- to 3-day intervals with microcalipers. The data points represent the mean tumor area + SEM. The mice were killed when the largest tumor diameter reached 15 mm. A, Tumor area. B, Survival. The arrows indicate the day of treatment.

Intratumoral MAb blockade of MHC class I attenuates the synergistic effect of rmIFNγ on antitumor immunity induced by AdCD40L. Eight days after B16.F10 flank tumor initiation, mice were randomized to 3 groups: intratumoral AdCD40L plus IP rmIFNγ, intratumoral AdCD40L plus IP rmIFNγ plus 3 successive intratumoral injections of an anti-MHC class I MAb (20 μg), or no treatment. Mice were followed over time for measurement of tumor area. In addition, mice from each group were killed 7 days after treatment for splenocyte harvest and evaluation of CTL generation using the ELISPOT assay as previously described. A, Tumor area. Each arrow represents 1 intratumoral injection of anti-MHC class I MAb. B, CTL generation using the ELISPOT assay. Each bar represents the mean number of spots ± SEM of 3 wells performed for each group.