We compared the adjuvant efficacy of two chemokines, MCP-3 and IP-10, and the cytokine Flt3-Ligand (Flt3L) to the established adjuvant cytokine GM-CSF (Supplemental Table 1
). Retroviral vectors were constructed with a marker gene directly linked to the adjuvant gene ensuring that levels of the two genes were proportional (Supplemental Figure 1A
). Cell lines were normalized by cell sorting to equivalent high levels of marker gene expression (Supplemental Figure 1B,C
) and then expression of the adjuvant gene was confirmed by intracellular cytokine staining (Supplemental Figure 1D
). This system allowed rapid generation of vaccine cell lines expressing levels of all adjuvant genes within or exceeding the known ED50 ranges by ELISA (Supplemental Table 1
Flt3-Ligand and IP-10 are effective adjuvants for B16 prophylactic vaccination
Mice were vaccinated twice with irradiated tumor cells prior to being challenged with B16-BL6 melanoma cells on the opposite flank. Compared to B16-YFP, B16-GMCSF (Gvax), B16-Flt3L (FL3vax), and B16-IP10 all showed statistically significant activity in preventing tumor growth (). Although MCP3 protected more mice than B16-YFP alone, its effect was not significant.
Fl3vax, like Gvax, synergizes with CTLA-4 blockade to treat 3-day pre-implanted B16 melanomas
The YFP marker created high variability in the B16-YFP control(), and was difficult to normalize in expression between lines (Supplemental Figure 1B
), so for all subsequent experiments a non-immunogenic truncated Lyt-2 marker was used instead. These Lyt2 vectors yielded similar prophylactic data to the YFP vectors (Supplemental Figure 2
) but were easier to normalize (Supplemental Figure 1C
) and lacked the potentially confounding immunogenicity of YFP. Compared to B16 alone, B16-Lyt2 grew at the same rate in untreated mice (Data not shown).
B16-Flt3L is as effective as B16-GMCSF in treating 3-day implanted B16 melanomas when combined with antibody blockade of CTLA-4
To compare our adjuvant genes to GM-CSF, we tested their ability to synergize with CTLA-4 antibody blockade to treat 1×104 B16 melanoma cells implanted 3-days earlier. Individually neither any of the cellular vaccines nor CTLA-4 blockade alone slows tumor growth () or is curative. When coupled with CTLA-4 blockade, treatment with Gvax or Fl3vax resulted in 63% and 60% tumor-free mice(). By comparison, IP-10 and MCP3 showed much less capacity to prolong survival of B16-bearing mice. Gvax and Fl3vax were also most effective in slowing tumor growth (), while B16-MCP3 had no effect on tumor growth and was not studied further.
FL3vax and B16-IP10 increase in effectiveness when administered at the tumor site, while Gvax fails to prevent tumor outgrowth in any mice
As melanoma lesions can sometimes be directly accessed for treatment, we tested the efficacy of Gvax, Fl3vax, and B16-IP10 both locally (at the tumor site) and distally (on the opposite flank) in combination with CTLA-4 blockade.
Surprisingly, Gvax showed vastly diminished efficacy in treating mice which had been challenged with 2×104 B16 cells 3-days prior when administered locally versus distally both in terms of tumor free survival () and tumor size at first measurement (). No tumor growth curve is provided because granulomas evoked by the vaccine at the tumor site cannot be distinguished from tumor for 3–4 weeks. Unlike Gvax, Fl3vax was significantly more effective when administered at the tumor site versus the opposite flank resulting in 75% tumor-free mice. B16-IP10 which showed little effect distally, proved to be effective when given at the tumor site.
Fl3vax with CTLA-4 blockade prevents tumor outgrowth in nearly all mice when given at the tumor site where Gvax proves ineffective, while B16-IP10 is effective locally but not distally
Gvax can induce myeloid suppressor cells under certain conditions suggesting that the failure of Gvax proximal to tumor may be due to the interaction of the myeloid cells evoked by the vaccine and the tumor microenvironment(32
). Our Gvax line produces 360ng/1×106
cells/24hrs of GM-CSF which is within the optimal immunogenicity range, and well below the levels which have been described as inherently suppressive(32
). These data indicated there were likely to be significant differences in the cellular subsets evoked by each vaccine, therefore sought to determine if Gvax and Fl3vax would cooperate in eradicating B16 melanomas.
Combination treatment with Fl3vax and Gvax is no more effective than using either vaccine alone
In order to test the potential for Fl3vax and Gvax to synergize in rejecting B16 melanomas, we waited until 5 days following tumor challenge to begin treatment. Gvax and FL3vax demonstrated similar efficacy; however, combination treatment with both vaccines prevented tumor outgrowth in equal or fewer mice compared to either alone(). In addition, tumors in mice receiving the dual cytokine vaccine grew at similar or faster rates compared to mice receiving either vaccine alone ().
Both B16-Flt3L and B16-GMCSF vaccination are known to elicit Tregs which dampen anti-tumor responses(35
). To determine if these two cytokines would synergize in the absence of Tregs, we repeated the above experiments in mice which had been pre-depleted of CD25+ cells. Once again, the combination of Gvax and Fl3vax was clearly no more effective than either vaccine alone (), although the potency of all vaccines was enhanced by Treg pre-depletion. Tumors in mice vaccinated with both B16-Flt3L and B16-GMCSF, although small, grew at similar or faster rates compared to mice receiving either vaccine alone (). To further understand the mechanisms of action of each these vaccines, we examined the cells infiltrating both the vaccine and tumor sites for each.
Fl3vax treatment relies on CD8+ T cells, NK1.1+, and CD4+ cells and is opposed by the action of CD4+CD25+ Treg cells
Published analysis for Gvax indicated that CD8+ T-cells and NK1.1+ cells were the critical populations for rejecting B16 tumors(26
). We found in mice depleted of CD8+ cells or NK1.1+ cells, Fl3vax + α-CTLA-4 vaccination lost virtually all capacity to promote rejection of tumors implanted 3 days earlier (Supplemental Figure 3A
), and tumors grew at rates similar to untreated mice (Supplemental Figure 3B
). Also, a much higher percentage of CD4 depleted mice developed tumors and those tumors grew faster than in mice pre-depleted of Tregs (which are also depleted by α-CD4). Thus, we found that CD8+ T-cells, CD4+ T-cells, and NK1.1+cells all play important roles in rejecting tumors following Fl3vax vaccination with CTLA-4 blockade. We hoped that a more detailed study of the vaccine and tumor infiltrating lymphocyte populations evoked by each vaccine would further clarify the adjuvant mechanics of each, and perhaps suggest reasons for their lack of synergy.
Relative to Gvax, Fl3vax vaccination sites contain a higher percentage of CD8+ T-cells, NK cells, plasmacytoid DCs, and CD11b−/lo DCs
Mice were given 2 vaccinations with Gvax, FL3vax, or a combination of both with α-CTLA-4 antibody and 24hrs later the lymphocytes infiltrating the vaccine site were isolated and typed (Supplemental Table 2
). For both Gvax and Fl3vax, the vaccination site infiltrate was dominated by granulocytes and macrophages (). Despite this similarity, the B16-Flt3L site contained a 2–3 fold higher fraction of B cells, NK cells, and CD11b−/lo
DCs relative to the B16-GMCSF site. Most strikingly, plasmacytoid DCs (pDCs) and CD8+ T-cells comprised 5.5-fold and 8.5-fold higher fractions of the Fl3vax site compared to that of Gvax. Tregs, granulocytes, and monocytes/CD11bmed/hi
DCs were more abundant in the B16-GMCSF site. The CD11b−/lo
DC population favored by Flt3L consists primarily of “sentinel” and pDCs, whereas the CD11c+CD11bmed/hi
population elicited by GM-CSF is a mixture of monocytes and “inflammatory” DCs(37
). The most striking difference between the Gvax/Fl3vax combination and either vaccine alone was the relative lack of NK cells and T-cells, especially CD8+ T-cells.
Fl3vax elicits strong infiltration of the vaccine and tumor sites by CD8+ T-cells, “sentinel” DCs, and plasmacytoid DCs
We next decided to investigate how the differences observed in the composition of the vaccine sites would impact the profile of the lymphocytes infiltrating the tumor.
Fl3vax induces higher percentages of CD8+ T-cells, Tregs, “sentinel” and plasmacytoid DCs and fewer CD11b+GR1+ cells in tumors relative to those of mice receiving Gvax
To dissect the differential effects of Gvax, Fl3vax, and combination vaccination in treating B16, we undertook a more comprehensive analysis of the tumor infiltrating lymphocyte (TIL) population. Following tumor implantation and 3 vaccinations, TIL were isolated and characterized (). We found that overall TIL frequency (i.e. the extent of infiltration) in mice receiving the Gvax, Fl3vax, and Gvax/Fl3vax vaccines with CTLA-4 blockade was similar and substantially higher than in untreated mice (Supplemental Figure 4
). Similar to the vaccine site infiltrate, TIL from Fl3vax treated mice contained higher proportions of “sentinel” DCs, pDCs, NKDCs, and CD8+ T-cells and lower levels of “inflammatory” DCs relative to TIL from mice receiving Gvax. Unlike the vaccine site, FL3vax inoculated mice also had higher levels of Tregs in their tumors. Despite having large numbers of granulocytes, macrophages, and DCs, TIL from the combination vaccinated mice contained the lowest percentage of CD8+ T-cells.
Tregs suppress cytotoxic T-cells and, in the case of B16, elevated ratios of CD8+ T-cells to FoxP3+ Tregs within the tumor correlate with successful treatment(35
). Both Gvax and Fl3vax increase the ratio of CD8+ T-cells to Tregs within the tumor, especially in conjunction with α-CTLA-4 antibody (). Of the mice receiving CTLA4 blockade, this ratio is lowest in the combination vaccine group but not to an extent which explains it’s reduced immunogenicity. Although Tregs do make up a larger percentage of the TIL elicited by Fl3vax relative to Gvax, the even higher level of CD8+ T-cells also elicited by Fl3vax yielded the highest CD8/Treg ratios in TIL.
As CD11b+,GR1+ candidate myeloid suppressor cells can be classified as either macrophages or granulocytes in our typing system depending on the other markers they express, we decided to analyze their relative enrichment in the tumors of vaccinated mice as a separate population. We find that vaccination with Gvax, with or without CTLA-4 blockade, results in significantly higher percentages of CD11b+,GR1+ cells in TIL compared to mice receiving FL3vax or to untreated mice (). This enhanced infiltration by myeloid suppressor cells may explain the failure of Gvax to cure tumors when given at the tumor site where much higher concentrations of GMCSF would be present around the tumor.
Having demonstrated the efficacy of Fl3vax for treating pre-implanted B16 melanomas in conjunction with CTLA-4 blockade, we sought to determine whether it would also have utility for the treatment of other poorly immunogenic tumors.
Flt3-Ligand expression synergizes with CTLA-4 blockade in rendering irradiated autologous TRAMP-C2 cells capable of preventing the outgrowth of 2-day implanted TRAMP-C2 prostate adenocarcinomas
We used retroviral vectors to create GMCSF and Flt3L expressing vaccines derived from the TRAMP-C2 prostate adenocarcinoma. The majority of TRAMP-C2 tumors can neither be cured by Gvax or CTLA-4 blockade alone, but are susceptible to combination therapy. We found that both TRAMP-GMCSF and TRAMP-Flt3L could protect 100% of mice from outgrowth of 1-day pre-implanted low dose (5×105 cells) TRAMP-C2 tumors when combined with CTLA-4 blockade (Data not shown). For this reason we sought to block the outgrowth of a higher TRAMP-C2 challenge (1×106 cells) with vaccination on days 2, 5, and 8.
Treatment with TRAMP-GMCSF, TRAMP-Flt3L, and a combination of both resulted in similar percentages of tumor-free mice when combined with CTLA-4 blockade (). The overall tumor growth rate was also not significantly different between mice receiving either vaccine (). As with B16, combination therapy with TRAMP-GMCSF and TRAMP-Flt3L failed to show any efficacy beyond that of either treatment alone. These data demonstrate that the adjuvant utility of Flt3-ligand extends beyond the B16 melanoma system.
Flt3L converts irradiated TRAMP-C2 prostatic adenocarcinoma cells into an effective vaccine which synergizes with CTLA-4 blockade to protect against outgrowth of pre-implanted TRAMP tumors
An improved FL3vax synergizes with CTLA-4 blockade to protect more mice from outgrowth of B16 melanoma implanted 5-days prior to treatment than does Gvax
Whereas GMCSF has been shown to plateau in adjuvant efficacy at a relatively modest level(34
), we wondered if the efficacy of Flt3L might increase with higher levels of expression. A preliminary study suggested that Gvax decreased in efficacy with increasing dose, while FL3vax maintained or slightly increased its effect at higher doses (Supplemental Figure 5
). This system suffered from many limitations, however, including veterinary complications due to the large vaccine dose (5×106
/injection), as well as the potential to saturate the amount of serum protease available to release Flt3-ligand from the cell membrane in its bioactive form. We decided instead to create two new vaccines which constitutively secrete Flt3L. One of these is truncated at the site where membrane cleavage occurs in the wild type form (secFL3vax), and the other is truncated at the same site and joined to a mouse IgG2a constant region for increased stability and to enable dimerization (sFL3vaxIg). Each of these lines was normalized to the same Lyt2 expression level as FL3vax and found to produce between 900 and 1000 ng/1×106
cells/24hr of Flt3L in vitro
To compare these new vaccines to the original FL3vax and Gvax, we tested their ability to synergize with CTLA-4 blockade to block outgrowth of B16 melanoma cells injected 5-days prior to treatment. In this setting, sFL3vaxIg cured a significantly higher percentage of mice than did Gvax or any of the other Flt3L-based vaccines (). The tumors of mice receiving the sFL3vaxIg vaccine with CTLA-4 blockade also grew at a significantly slower rate than those of any other treatment group in each of 3 individual experiments (). These experiments demonstrated that this enhanced Flt3L-expressing B16 vaccine was superior to Gvax for use in treating B16 melanoma in conjuction with CTLA-4 blockade.