Leukocyte-specific protein1 (LSP1) is an important regulator of actin cytoskeleton remodeling, modulatingleukocytes motility, due to its F-actin binding domain. We have previouslyshown that Lsp1- /- mice have an impaired CTL response after antigenexposure. Moreover, Lsp1-/- dendritic cells (DCs) fail to induce a strong CTL responsein vivo, migrate to lymphoid tissues, present antigens and produce IL-12 whentransferred into WT mice. In order to deepen the mechanisms operating thisdiminished CTL generation in Lsp1-/- mice, we first analyzed the ability of their CD8+ T cellsto proliferate and become activated. After in vitro stimulation with αCD3/CD28,Lsp1-/- CD8+ T cells proliferated and up-regulated CD25 as stronglyas WT CD8+ T cells. Granzyme B production and IFNg release wereslightly lower (p<0.05) in Lsp1-/-vs WT CD8+ T cells,with no difference in IFNg production. Then, we evaluated in vitro activationof CD8+ T cells purified from OT I (Lsp1+/+) miceafter culture with BMDCs (differentiated from bone marrow precursors withFlt3-L), previously pulsed with latex beads coated with ovalbumin, plusCpG-ODN. Cell proliferation, CD25 up-regulation and IFNg secretion wassignificantly lower in T cells cultured with Lsp1-/- BMDCsthan with WT BMDCs (p<0.01). Finally, we compared the ability of Flt3L-BMDCsfrom Lsp1-/- and Lsp1+/+ mice to translocate antigen from endosomes to cytosolby performing the βLactamase export assay. Lsp1-/- BMDCsshowed a slower escape of βLactamase to the cytosol, indicating a slower Ag translocationthan in WT BMDCs. In conclusion, theseresults reveal that the diminished generation of CTLs we observed in Lsp1-/- mice is due to adeficiency in the ability of DCs to cross-present Ag rather than a failure intheir CD8+ Tcells. This deficiency in Lsp1-/-DCs is related to an alteration in the escape of theantigen from endosomes to the cytosol, which led to fewer antigens associated to MHC-I to be presented toCD8+ T cells.