CD40-Licensing Of Human Dendritic Cells Further Matures Tnfa-Treated Dcs But Does Not Mediate Resistance Against Inhibition Of T-Cell Proliferation By TREG
Authors: Eyrich Matthias , Rachor Johannes , Blume Marissa , Becker Isabelle , Stumpf Anne , Wajant Harald , Wolfl Matthias , Schlegel Paul G. , Lutz Manfred 
Published: October 2019

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 (Tregs).
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 Tregs during maturation led to a reduction of costimulatory molecules, presumably by transendocytosis of Tregs. 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 Tregs inhibited CD4+ T-helper proliferation by approx. 40%, this suppression was not alleviated by CD40-licensing of DCs. Interestingly, Treg proliferation in combined MLR-assays was increased in all experimental settings. Treg-suppression of CD4+ T-helper proliferation as well as the increased Treg-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 Tregs 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 Treg-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.

Disclosures:
No relevant conflicts of interest to declare.