In the present study we observed substantial increases in both the relative and absolute numbers of DCs with an immature phenotype during the restoration phase after CTX-induced lymphodepletion. This observation is consistent with recent studies that reported increases in the number of DCs during the restoration phase in the peripheral blood of cancer patients receiving combinatorial treatment with CTX and the growth factors G-CSF and GM-CSF (42
). Although it was not clear in these studies whether the increase in the frequency of DCs was due solely to the effects induced by CTX or by the growth factors, our results demonstrate that CTX is capable of inducing DC expansion in the peripheral blood in a murine model. The potential clinical significance of our observation is evidenced by the substantial increases in the antitumor efficacy of adoptively transferred CD8+
T cells when these cells were boosted with peptide vaccination and poly(I:C) at the peak of post-CTX DC expansion. These Ag recall responses were markedly abrogated when DCs were depleted during Ag boosting; indicating to the essential contribution of post-CTX expanded DCs.
Despite their immature phenotype, CTX-expanded DCs showed phagocytic () and Ag-presenting () capabilities, indicating that they are biologically functional. As expected, this post-CTX surge in DCs bearing immature phenotype did not enhance the responses after vaccination with gp10025–33
alone (). Provision of poly(I:C) at this surge of DCs, however, created an inflammatory microenvironment (TNF-α, MCP-1, IFN-γ, and IL-6) (), resulting in the appearance of activated CCR7+
, and CD80+
DCs in LNs (), which could be contributing to the augmented Ag-specific responses of CD8+
T cells to gp10025–33
peptide when delivered along with poly(I:C) at the peak of DC expansion (). The appearance of DCs in LNs after poly(I:C) was associated with a significant decrease in the numbers of DCs in the peripheral blood. Given that CCR7 is essential for migration of activated DCs to LNs (40
), its up-regulation would suggest that the appearance of DCs in LNs after poly(I:C) treatment is, at least in part, due to their homing from PBL into LNs after maturation. Because resident DCs in LNs express CD8α while migratory DCs do not (41
), and because DCs expanded in the blood were CD8α−
and those increased in LNs were CD8α+
, we do not exclude the possibility of local expansion and activation of the resident DCs in LNs of CTX-treated hosts in response to poly(I:C). Alternatively, migratory blood-born DCs may acquire the phenotype of lymphoid (CD8α) DCs upon their arrival to LNs as previously reported (45
), and thus become indistinguishable from resident DCs. Regardless of the mechanisms mediating the increase in the number of DCs in LNs, depletion of these cells before boosting of both PBS- and CTX-treated hosts with peptide/poly(I:C) significantly abrogated the expansion of effector pmel-1 cells (), indicating to the importance of DCs to the Ag recall responses of CD8+
T cells to active vaccination in general, and to CTX preconditioning in particular. Similar requirement of DCs to Ag recall responses has been reported in the viral setting (41
). Indeed, recent studies by Wang et al. have pointed to the importance of hematopoietic-derived APCs during the lymphopenic phase to the Ag priming of CD8+
T (pmel-1) cells (47
). Together, DCs appear to be required during Ag priming and boosting for generation of memory CD8+
The expansion of DCs during the restoration phase post-CTX induced lymphodepletion was preceded by the activation of these cells during the lymphopenic phase (). Indeed, previous reports, including ours, have demonstrated a rapid activation of DCs at the lymphopenic phase after chemotherapy and TBI, associating with significant increases in the Ag-specific responses of adoptively transferred T cells (3
). Paulos et al. demonstrated that this rapid DC activation is attributed to the significant damage to the integrity of mucosal barriers and translocation of bacterial products (LPS; a TLR4 ligand) (3
). These events would lead to induction of inflammatory cytokines (8
) and activation of DCs. Therefore, the transient activation of DCs during the lymphopenic phase (up to day 4) after CTX treatment may be because of the rapid clearance of LPS from circulation. There is further evidence that exogenous LPS can substitute for the endogenous TBI-induced LPS for augmentation of the antitumor responses of CD8+
T cells to active vaccination when they were adoptively transferred to immune cell (NK cells and CD4 T cells)-ablated recipient mice (3
). Similarly, we have reported that addition of poly(I:C) to OVAp vaccination during the lymphopenic phase after CTX preconditioning markedly augmented CD8+
T cell expansion, which was associated with significant activation of DCs in spleen and liver (14
). In the present study, we were able to extend this observation by showing the capability of poly(I:C) to induce activation of DCs expanded during the restoration phase, resulting in robust increases in the CD8+
T cell responses to active vaccination (). Taken together, we would suggest that the host microenvironment created after induction of lymphodepletion can be successfully exploited both at the lymphopenic and restoration phases by TLRL (e.g., TLR3L and TLR4L) to benefit adoptive T cell therapy combined with active vaccination.
The activation of DCs during the lymphopenic phase and their expansion during the restoration phase may suggest a harmonized hierarchy of multiple mechanisms involved in the beneficial effect of CTX on adoptive T cell therapy. This hierarchy warrants the reconsideration of the timing between adoptive T cell transfer into a CTX-lymphodepleted host and any subsequent vaccinations. We tested this hypothesis by applying prime-boost vaccination with gp10025–33/poly(I:C) at the lymphopenic and restoration phases, so that pmel-1 cells could benefit from the activated DCs at the lymphopenic phase as well as from the activated large pool of DCs at the restoration phase. This regimen induced a substantial expansion of pmel-1 cells and prevention of B16 tumor growth (), which absolutely required CTX treatment because the same prime-boost regimen in PBS-treated mice could not prevent B16 tumor growth. Our results point to the role of post-CTX expanded DCs in this antitumor effects of pmel-1 cells because boosting of the CTX-treated mice with gp10025–33/poly(I:C) at the peak of DC expansion resulted in recruitment of activated DCs in LNs and spleen (), increases in the numbers (quantity) of the Ag-specific pmel-1 cells in LNs and spleen (), and tumor regression and better survival (). These results suggest that tumor regression was mediated by pmel-1 cells expansion in the presence of a large pool of activated DCs. Because DCs were required for pmel-1 cell expansion (), our results suggest that DCs are important for the antitumor effects of pmel-1 cells by augmenting the quantity of the latter. DCs might also play a role in the tumor regression by augmenting the quality of the Ag-specific pmel-1 cells because adoptive transfer of donor cells harvested from mice vaccinated with gp10025–33/poly(I:C) on days 2 and 12 induced higher antitumor responses than those harvested from mice vaccinated with gp10025–33/poly(I:C) only on day 2. Thus, we suggest that the presence of a large pool of activated DCs augment both the quantity and quality of the tumor-specific responses of pmel-1 cells.
Recent studies have also reported the capability of naive and effector gp10025–33
-specific pmel-1 cells to induce regression of established B16 tumor. The antitumor effects in these studies, however, required aggressive treatment protocols consisting of TBI-induced lymphodepletion or myelodepletion followed by hematopoietic stem cell transplant, adoptive transfer of in vitro cytokine-conditioned Ag-stimulated pmel-1 cell, vaccination with 2 × 107
plaque forming units of a recombinant fowlpox virus encoding gp10025–33
or with repeated ex vivo vaccination with peptide-pulsed DCs, and exogenous administration of high doses of IL-2 (4
). In addition to these established therapeutically effective antitumor regimens, our data presents another therapeutically effective antitumor regimen based on targeting a large pool of DCs by TLRLs during peptide vaccination, obviating the need for more complicated and potentially toxic treatment regimens such as IL-2 therapy.
We believe that our in vivo DC-based prime-boost vaccination with peptide/TLR3L at defined phases post-lymphodepletion is an effective treatment approach because it induces in vivo maturation of DCs by TLRLs, which, in contrast to ex vivo DC-based vaccination, also stimulate other critical factors in the host microenvironment. In line with this notion, several recent studies reported substantial improvement in T cell responses to active vaccination and generation of efficacious antitumor responses upon in vivo activation of Flt3L-mobilized DCs with TLRLs, in particular the TLR9L CpG (51
). In conclusion, our data showed that CTX therapy induces a biphasic effect on DCs. During the lymphopenic phase, DCs are activated, while during the restoration phase DCs are expanded but express immature phenotype, which can be exploited in vivo to favor generation of robust antitumor immunity against self-tumor Ag. Our data provide a useful foundation for a rationale design of anti-cancer immunotherapy regimens by combining lymphodepletion, adoptive T cell therapy, and TLRL-based tumor vaccines.