Dendritic Cells Crosspresent Antigens from Live B16 Cells More Efficiently than from Apoptotic Cells and Protect from Melanoma in a Therapeutic Model

Dendritic cells (DC) are able to elicit anti-tumoral CD8+ T cell responses by cross-presenting exogenous antigens in association with major histocompatibility complex (MHC) class I molecules. Therefore they are crucial actors in cell-based cancer immunotherapy. Although apoptotic cells are usually considered to be the best source of antigens, live cells are also able to provide antigens for cross-presentation by DC. We have recently shown that prophylactic immunotherapy by DC after capture of antigens from live B16 melanoma cells induced strong CD8+ T-cell responses and protection against a lethal tumor challenge in vivo in C57Bl/6 mice. Here, we showed that DC cross-presenting antigens from live B16 cells can also inhibit melanoma lung dissemination in a therapeutic protocol in mice. DC were first incubated with live tumor cells for antigen uptake and processing, then purified and irradiated for safety prior to injection. This treatment induced stronger tumor-specific CD8+ T-cell responses than treatment by DC cross-presenting antigens from apoptotic cells. Apoptotic B16 cells induced more IL-10 secretion by DC than live B16 cells. They underwent strong native antigen degradation and led to the expression of fewer MHC class I/epitope complexes on the surface of DC than live cells. Therefore, the possibility to use live cells as sources of tumor antigens must be taken into account to improve the efficiency of cancer immunotherapy

Regulatory T cells increase the avidity of primary CD8+ T cell responses and promote memory.

Although regulatory T cells (T(regs)) are known to suppress self-reactive autoimmune responses, their role during T cell responses to nonself antigens is not well understood. We show that T(regs) play a critical role during the priming of immune responses in mice. T(reg) depletion induced the activation and expansion of a population of low-avidity CD8(+) T cells because of overproduction of CCL-3/4/5 chemokines, which stabilized the interactions between antigen-presenting dendritic cells and low-avidity T cells. In the absence of T(regs), the avidity of the primary immune response was impaired, which resulted in reduced memory to Listeria monocytogenes. These results suggest that T(regs) are important regulators of the homeostasis of CD8(+) T cell priming and play a critical role in the induction of high-avidity primary responses and effective memory

DC cytokine profile after culture with live or apoptotic B16 cells.

<p>DC were cultured alone (DC) or with live (zVAD treated) or apoptotic (γ-irradiated) B16 cells for 24 h and stimulated or not with LPS and IFNγ. As controls, B16 zVAD and B16γ cells were also cultured alone. DC were then assessed for their ability to produce IL-12p70 (A) or IL-10 (B) by ELISA. In the upper panels, data from one out of three independent experiments are shown. In the bottom panels, for LPS and IFNγ stimulation, relative IL-12p70 and IL-10 productions are expressed as a percentage of the cytokine production obtained for DC alone. Mean percentage values±SEM are shown. The significance of differences between series of results was assessed using a paired t test (n = 3, 3 independent experiments).</p

More native antigen in live than in apoptotic donor cells: improved antigen crosspresentation by DC.

<p>A, B, 30.10⁶ live (zVAD treated) or apoptotic (γ-irradiated) B16 cells (A) or L-OVA cells (B) were lysed for protein extraction. Lysates from B16 cells were analysed using anti-gp100, anti-TRP2 and anti-actin antibodies (A). Lysates from L-OVA cells were analysed using anti-OVA and anti-actin antibodies antibodies (B). Anti-actin antibodies were used as controls. C, DC were cultured with different numbers of live (filled triangle) or apoptotic (open triangle) L-OVA cells or live L cells (filled square). DC were then purified by magnetic sorting using anti-CD11c microbeads and cultured with B3Z-T cells hybridoma cells for 18 h. Activation after recognition of the SIINFEKL-K^b complex was detected by optical density measurement at 560 nm after addition of the CPRG substrate. The significance of differences between series of results was assessed using a two-tailed unpaired t test. **p<0.01, *p<0,05, n.s. not significant. Mean ± SEM, representative of two independent experiments.</p

DC maturation after culture with live or apoptotic B16 cells.

<p>DC were cultured with live (zVAD treated) or apoptotic (γ-irradiated) B16 cells for 24 h in the presence of culture medium or LPS and IFNγ. The expression of maturation molecules was then tested by flow cytometry. One representative experiment out of three independent experiments is shown.</p