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Preconditioning a recipient host with lymphodepletion can markedly augment adoptive T cell therapy. However, the precise mechanisms involved are poorly understood. In a recent study, we observed a significant increase in the circulating levels of dendritic cells (DCs; CD11c(+)CD11b(+)) during the recovery from cyclophosphamide (CTX)-induced lymphodepletion. Herein, we demonstrate that the CTX-induced DC expansion was not altered by adjuvant chemotherapy or tumor burden but was augmented by coadministration of granulocyte-colony stimulating factor. Although the increase in the number of DCs was preceded by a systemic expansion of a population expressing the phenotype of myeloid-derived suppressor cells (Gr-1(+)CD11b(+)), depletion of these Gr-1(+) cells had no effect on the noted expansion. Moreover, when Gr-1(high)CD11b(high) cells were sorted from CTX-treated mice and adoptively transferred into control or CTX-treated recipients, they did not differentiate into DCs. Post-CTX expansion of DCs was associated with proliferation of DCs in bone marrow (BM) during the lymphopenic phase and in the blood and spleen during the recovery phase. Furthermore, adoptive transfer of BM cells from CTX-treated mice produced equal numbers of DCs in the blood of either CTX-treated or untreated recipients. CTX induced a dynamic surge in the expression of growth factors and chemokines in BM, where CCR2 and Flt3 signaling pathways were critical for DC expansion. In sum, our data suggest that CTX induces proliferation of DCs in BM prior to their expansion in the periphery. Targeting DCs at these phases would significantly improve their contribution to the clinical application of lymphodepletion to adoptive immunotherapy.

Recent preclinical studies suggest that vaccination following adoptive transfer of CD8(+) T cells into a lymphopenic host can augment the therapeutic antitumor responses of the transferred cells. However, the mechanism by which the lymphopenic microenvironment benefits Ag-specific CD8(+) T cell responses remains elusive. We show herein that induction of lymphodepletion by a single 4 mg cyclophosphamide (CTX) treatment induces a marked expansion of immature dendritic cells (DCs) in the peripheral blood on days 8-16 post-CTX (termed restoration phase). In vitro, these DCs were functional, because they showed normal phagocytosis and effective Ag presentation capability upon activation. In vivo, administration of the TLR3 agonist poly(I:C) at the peak of DC expansion (day 12 postlymphopenia) induced inflammatory cytokine production and increases in the number of activated DCs in lymph nodes. Importantly, boosting with gp100(25-33) melanoma peptide combined with poly(I:C) 12 days after an initial priming with the same regimen significantly increased the expansion and the antitumor efficacy of adoptively transferred pmel-1 CD8(+) T cells. These responses were abrogated after depletion of activated DCs during Ag boosting. In conclusion, our data show that CTX treatment induces, during the restoration phase, expansion of immature DCs, which are functional and can be exploited in vivo to foster more effective antitumor adoptive immunotherapy strategies.