CD40-Licensing Of Human Dendritic Cells Further Matures Tnfa-Treated Dcs But Does Not Mediate Resistance Against Inhibition Of T-Cell Proliferation By T_REG

Vaccination with cytokine-matured dendritic cells (DCs), loaded with autologous tumor lysate, represents a promising strategy in cancer immunotherapy, however, in clinical trials vaccination with this kind of DCs has shown only limited efficacy so far. In animal experiments, CD40-licensing of DCs conveys resistance to tumor-induced immunosuppression including immunomodulation of regulatory T cells (T_regs).

DCs were generated by differentiation of monocytes for 7 days with GM-CSF/IL-4 followed by 48h maturation with TFNα/IL-1ß ± CD40L. Then, DC were analyzed with respect to phenotype, cytokine production, and T-cell stimulatory capacity.

CD40-licensed DCs showed higher expression of CD86 (MFI 3074±630 vs. 2433±359 with vs. without CD40L, respectively, p<.05) and a trend towards higher CD80, CD83 expression. Coculture of DCs with T_regs during maturation led to a reduction of costimulatory molecules, presumably by transendocytosis of T_regs. This phenomenon could be partially abrogated by CD40-licensing. TNFα/IL1ß-matured DCs produced significant amounts of IL-6, IL-8, TNFα, and IL-1ß. CD40-licensing did further increase cytokine production, however, no IL-12 could be detected. In contrast to murine DCs, a second round of LPS-stimulation after TNFα/IL1ß could not trigger IL-12 production. Control DCs matured with LPS/IFNγ showed up to 16% IL-12⁺ DCs. Using Melan-A as a model tumor antigen, priming capacity of CD40-licensed DCs to induce Melan-A specific CD8⁺ CTLs was slightly but not significantly improved compared to nonlicensed DCs, as demonstrated by somewhat higher frequencies of Melan-A multimer⁺ and TNFα⁺IFNγ⁺ CTLs after 11 days of culture with CD40-licensed DCs and rechallenge, respectively. Again, T-cell priming was best with control DCs matured with LPS/IFNγ. In contrast to T-cell priming, CD40-licensed DCs did not show any improved capacity to stimulate CD4⁺ T-helper cell proliferation. Furthermore, in a classcial MLR-suppression assay T_regs inhibited CD4⁺ T-helper proliferation by approx. 40%, this suppression was not alleviated by CD40-licensing of DCs. Interestingly, T_reg proliferation in combined MLR-assays was increased in all experimental settings. T_reg-suppression of CD4⁺ T-helper proliferation as well as the increased T_reg-proliferation in the combined MLR-assays could not be prevented by the lysosomal inhibitor Bafilomycin A. This suggests, that other mechanisms than transendocytosis of costimulatory molcules by T_regs mediate these effects.

In summary, these data show that CD40-licensing is a feasible tool to improve maturity of cytokine-treated DCs. However, CD40-licensing cannot induce IL-12 production in human DCs without TLR-stimulation and was not able to confer resistance against T_reg-mediated T-cell proliferation inhibition. Thus, in order to strengthen DCs for cancer immunotherapy, CD40-licensing should be further investigated in combination with TLR-triggering DC-maturation cocktails.