Even though we used agonistic CD40 antibodies as an adjuvant, which licenses DCs and thus bypasses CD4+ T cell help for priming CD8+ T cells, 46 CD4+ T cell help is needed for optimal generation and maintenance of CD8+ T cells

Even though we used agonistic CD40 antibodies as an adjuvant, which licenses DCs and thus bypasses CD4+ T cell help for priming CD8+ T cells, 46 CD4+ T cell help is needed for optimal generation and maintenance of CD8+ T cells.47 In fact, memory CD8+ T cells may be formed in mice vaccinated with the DC-SIGN targeting formulations, as only in those mice long-term tumor protection was observed when the vaccination was combined with Treg depletion. portion of mice. This novel strategy resulted in optimal generation of antigen-specific activated CD8+ T cells which accumulated in regressing tumors. Notably, Treg depletion also allowed the local appearance of effector T cells specific for endogenous B16 antigens. This indicates that antitumor immune responses can be broadened by therapies aimed at controlling Tregs in tumor environments. Thus, transient inhibition of Treg-mediated immune suppression potentiates DC targeted antigen vaccination and tumor-specific immunity. rich tumor microenvironments.7-9 Here, nTreg actively expand and suppress other immune cells in a cell-contact dependent manner.3,8 Thus, it is clear that various subpopulations of Tregs endowed with various suppressive functions co-exist in cancer patients. Together, these events enable tumors to escape the immune system and result in uncontrolled growth and expansion of the tumor cells. The identification of the immunodominant epitopes of several tumor antigens facilitated the use of protein or peptide antigens as vaccines to boost tumor-immunity.10 However, these types of vaccines require high amounts of antigens to be effective as Triptorelin Acetate they will also be internalized and/or offered by other cells than DCs.11-15 Additionally, the efficacy of these vaccines is often limited in a therapeutic setting. To enhance cross-presentation of tumor antigens and to achieve a better priming of T cells, current vaccination TRAILR-1 strategies focus at the delivery of tumor-antigens as proteins or peptides specifically to DCs. Hereto, antigens can be tagged with antibodies or ligands specific for any DC-expressed receptor.16 A particularly promising target in this respect is the endocytic C-type Lectin Receptor (CLR) DC-SIGN, which is expressed on human immature DCs, providing the opportunity to specifically target DCs and additionally mediate fast and efficient uptake of antigens. Antigens taken up via DC-SIGN end up as epitopes in MHC class II and I molecules enhancing antigen-specific CD4+ and CD8+ T cell responses.17-19 As no functional homolog of DC-SIGN exists in mice,20 we generated humanized mice expressing human DC-SIGN (hSIGN) on conventional DCs.21 Importantly, delivery of antigens via anti-DC-SIGN monoclonal antibodies (aDC-SIGN) enhances T cell responses and < 0.05. Results shown are representative of three impartial experiments. BMDCs from hSIGN and WT mice were loaded with equimolar amounts of OVA-aDC-SIGN or OVA conjugated with isotype control Abs (OVA-isotype) and subsequently co-cultured with OVA-specific CD4+ or CD8+ T cells. Internalized OVA-aDC-SIGN is Triptorelin Acetate usually shuttled into the MHC class II presentation route as obvious from vigorous proliferation of OVA-specific CD4+ T cells (Fig. 1B). Moreover, the response induced by DC-SIGN mediated targeting was much more efficient than that induced by control OVA-isotype, as the same degree of CD4+ T cell proliferation could be induced with >80-fold less OVA. OVA-aDC-SIGN also efficiently joined a cross-presentation route resulting in presentation on MHC class I molecules and activation of OVA-specific CD8+ T cells (Fig. 1C). The enhanced presentation of OVA antigens in MHC-II and I was specifically induced upon DC-SIGN-mediated uptake, as neither OVA-isotype nor WT DCs evoked such strong OT-II and OT-I T cell proliferation. Similarly, and as reported Triptorelin Acetate earlier,28 glycan-modified OVA internalized by DC-SIGN is usually shuttled into both MHC class II and I presentation routes as revealed from increased proliferation of OVA-specific CD4+ and CD8+ T cells (Figs. 1D and E). Yet, while targeting DC-SIGN with OVA-LeB induces comparable activation of CD4+ T cells as OVA-aDC-SIGN, we found that cross-presentation of OVA is much more enhanced using OVA-aDC-SIGN than OVA-LeB. Moreover, we found that approximately 10- to 50-fold lower amounts of OVA were sufficient when conjugated to aDC-SIGN to evoke comparable CD8+ T cell responses as OVA-LeB (i.e., 3?nM vs. 183?nM, respectively). Thus, both DC-SIGN targeting formulations increased specific activation of CD4+ and CD8+ T cells by enhancing antigen presentation, albeit with some differences in cross-presentation. We next assessed whether these differences are also reflected in the generation of endogenous effector CD4+ and CD8+ T cells re-stimulation. Compared to native OVA/anti-CD40, immunization with OVA-LeB and OVA-aDC-SIGN induced higher percentages of IFN- and TNF-double-producing CD8+ T cells (Fig. 2A). Similarly, IFN single-producing CD8+ T cell Triptorelin Acetate responses were highest in mice immunized with DC-SIGN targeting formulations (Fig. 2B). By contrast, antigen-specific TNF single-producers.

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