The occasional and sometimes dramatic clinical responses in recent cancer vaccine trials suggest that vaccination against tumor-expressed antigens can induce clinically meaningful anti-tumor immunity, however currently these responses are only sporadic. One reason for the disappointing results of vaccine trials may be failure to induce adequate numbers of tumor-specific CD8+ T cells. While clinical vaccination trials demonstrate circulating antigen-specific CD8+ T cell responses on the order of 0.05-5% of total circulating CD8+ T cells (1
), levels achieved during viral infection can reach up to 20% or more of circulating CD8+ T cells (40
) which may better suggest the magnitude of response necessary for efficacy against established tumors. Vaccine-elicited T cells may also become functionally impaired in the tumor-bearing host due to lack of innate immune activating signals such as TLR ligands, inhibition by regulatory T cells (41
), or active immunosuppression by the tumor itself. Finally, tumor-specific CD8+ T cells may expand initially but fail to persist long enough to mediate total tumor destruction if they do not receive continued survival signals (such as common gamma-chain cytokines like IL-2, IL-7, and IL-15) (42
) or are programmed improperly during priming and initial expansion (43
Since the synthetic peptides that are widely used as vaccines are poorly immunogenic by themselves, adjuvants are required to induce significant T cell responses. Type I IFNs provide a critical link between the innate and acquired immune response during viral infection, suggesting that they may also be efficacious as vaccine adjuvants. Long-term systemic treatment with IFN-α is currently approved for the management of chronic viral infections such as with Hepatitis B and C viruses and several cancers, including melanoma. For our studies, we tested the vaccine adjuvant activity of IFN-α by inducing systemic IFN-α levels with the hydrodynamic gene transfer method. This method uses a brief hydrostatic pulse to transfect a microgram-amount of injected plasmid into normal hepatocytes, leading to long-term expression of the plasmid-encoded gene of choice. This method overcomes the significant obstacle of long-term daily injections of very costly recombinant IFN-α protein that has all but made impossible effective studies of the effects of prolonged, systemic in vivo treatment with IFN-α. Since even this small amount of plasmid DNA could conceivably contain sufficient CpG signals to trigger TLR9 on dendritic cells to enhance the vaccine adjuvant effect of IFN-α, we repeated the experiment with recombinant IFN-α and observed similar effects, effectively proving that the observed adjuvant activity does not depend on TLR9 triggering.
We found that IFN-α was capable of significantly boosting the antigen-specific (pmel-1) CD8+ T cell response to gp100 peptide vaccination. In order to achieve this effect, both interferon and vaccination were required; IFN-α alone did not produce a measurable rise in the pmel-1 CD8+ T cell response. IFN-α also boosted the response of the endogenous CD8+ T cell repertoire in response to ovalbumin peptide vaccination, demonstrating that Type I IFN can act as an adjuvant for vaccination of either endogenous or adoptively transferred T cells against both non-self and self antigens. However IFN-α adjuvant activity was limited to antigen-specific CD8+ T cells; the absolute number of non-specific bystander CD8+ T cells, and the absolute numbers of other leukocytes (CD4+ T cells, NK cells) were not significantly increased in the peripheral blood, spleen, or tumor of IFN-α-treated mice. The boost in pmel-1 T cell numbers required the expression of the IFNAR on the pmel-1 T cells, demonstrating that in addition to exerting indirect effects on T cells through antigen presenting cells, exogenous IFN-α also acts directly on activated antigen-specific CD8+ T cells.
When we measured T cell apoptosis and proliferation to determine their relative contribution to the IFN-induced accumulation of pmel-1 T cells, we found that exogenous IFN-α enhanced both proliferation and survival of vaccine-induced pmel-1 T cells. In the presence of IFN-α, antigen-specific pmel-1 T cells were protected from apoptosis in peripheral blood, spleen, and tumor, in a manner similar to the IFN-α-mediated protection of CD8+ T cells from apoptosis during viral infection. However, in contrast to the effect of endogenous IFN-α on CD8+ T cells during viral infection (11
), which seems limited to protecting them from apoptosis, we found a slight but consistent increase in the rate of antigen-specific CD8+ T cell proliferation after vaccination in the presence of exogenous IFN-α. This may be due to the continuous stimulation of the IFNAR on CD8+ T cells with high levels of IFN-α in our system compared to the more short-lived (1-5 days) systemic increase in IFN-α during acute viral infection in these reports(36
). Since these effects were seen over a range of non-toxic levels of IFN-α expression, our data suggest that adjuvant interferon at doses lower than required for anti-melanoma IFN-α monotherapy may increase both tumor-specific CD8+ T cell survival and proliferation in patients undergoing peptide vaccination.
Efficacy of the anti-tumor CD8+ T cell response depends not only on the induction of specific T cells but also on their effector function. We found increased numbers of vaccine-induced pmel-1 T cells producing IFN-γ in tumor, PBMC, and spleen, as well as higher mean IFN-γ release by pmel-1 T cells from IFN-α-treated mice. The observed increased production of TNF-α, MIP-1α, and IFN-γ by purified pmel-1 T cells from vaccinated, IFN-α-treated mice is consistent with enhanced development of an activated, effector CTL phenotype (44
). We and others have shown that the T cell-mediated regression of B16 melanoma is partly dependent on tumor-specific T cell-derived IFN-γ (45
). MIP-1α is a member of the group of CC chemokines, and plays a role in chemotaxis of T cells from the circulation into inflamed tissue (46
). TNF-α is also produced by activated CTL, and has diverse pro-inflammatory and some direct anti-tumor activities (47
). The coordinated upregulation of these cytokines in vaccinated mice treated with IFN-α is consistent with generation of a pro-inflammatory environment which supports the infiltration and anti-tumor activity of vaccine-induced CTL.
Prevention of tumor recurrence may depend on the induction of a stable and long-lasting population of specific CD8+ effector T cells. The increase in antigen-specific CD8+ T cell numbers and CTL activation correlated with inhibition of tumor growth in mice bearing established subcutaneous B16 melanoma, a fast-growing and poorly immunogenic tumor. While transient growth inhibition was seen in mice treated with IFN-α alone, uncontrolled tumor growth resumed within 2 weeks after treatment and rapidly killed these mice; however, tumor growth was suppressed for over 4 weeks in mice that received both vaccine and IFN-α. This correlates with the persistent increase in anti-tumor T cells in vaccinated mice treated with IFN-α. Even in vaccinated IFN-α-treated mice, tumor growth ultimately resumed, possibly due to the number of protective T cells dropping below a critical threshold, exhaustion of persistently-stimulated pmel-1 T cells, or outgrowth of resistant tumor clones. Further experiments will determine whether long-surviving tumor-specific cells are functional or have undergone exhaustion (48
), and whether they depend on chronic IFN-α stimulation, persistent antigen stimulation, or both.
The phenotypic profile of IFN-α-induced CD8+ T cells may provide clues to their function and efficacy in vivo
. IL-15 and IL-7 are constitutively produced common cytokine-receptor γ chain-family cytokines which play an important role in maintenance of antigen-specific CD8+ T cells, with IL-7 supporting the proliferation and survival of naïve cells, and IL-15 acting primarily on memory cells. IL-15 signals through a heterodimeric receptor composed of the IL-2/IL-15R β chain and the common γ chain which are expressed on CD8+ memory T cells. The IL-15Rα is expressed primarily on activated monocytes and dendritic cells where it acts to “trans-present” IL-15 to IL-15Rβ/γ-expressing target cells. IL-7Rα is expressed at high levels on naïve CD8+ T cells, and downregulated in response to signaling through the TCR. The phenotype of persistent IFN-α-induced pmel-1 cells (CD44hi
) is similar to that of effector memory cells, which would also be consistent with their residence in peripheral tissues (lung). One potential application for cancer vaccine therapy is in long-term control of minimal residual disease after debulking therapy. CD8+ T cells of effector memory phenotype may be suitable for this purpose, since they would allow prolonged maintenance of highly active tumor-specific T cells to allow for immune surveillance. Alternatively, these IL-7Ralo
CD8+ T cells are reminiscent of a “short-lived effector” memory CD8+ T cells that have been recently identified (49
). These cells have a longer survival than pure effector cells, but a shorter life span than memory T cells. Furthermore, these “short lived” memory CD8+ T cells have increased effector activities, and are induced by high levels of inflammation, yet still respond to a secondary stimulation.
The upregulation of IL-15Rα and requirement for host-derived IL-15 in supporting persistence of IFN-α-induced pmel-1 T cells suggests that at least some of the adjuvant activity of IFN-α is mediated by IL-15Rα-mediated transpresentation of IL-15. Our in vitro
data demonstrating that abolishing either IL-15 or IL-15Rα expression by mDCs completely abrogates the IFN-α-mediated enhancement of pmel-1 T cell persistence suggests that expression of IL-15 and IL-15Rα by DCs may also be important in vivo
. The upregulation of IL-15Rα we observed on IFN-α-stimulated mDCs in vitro
and in vivo
provides a straightforward mechanism by which Type I IFN may enhance CD8+ T cell persistence by increasing the efficiency of IL-15 trans-presentation by mDCs. Interestingly, we found that IFN-α also upregulated IL15Rα expression on total CD8+ T cells (data not shown) and pmel-1 T cells. The upregulation of IL-15Rα on antigen-specific CD8+ T cells has been previously demonstrated in a study of the ability of IFN-α to promote CD8+ T cell expansion through cross-priming, in which IL-15Rα expression on CD8+ T cells peaked on day 3 after immunization in the presence of IFN-α (9
). There is also evidence that IL-15Rα expression can enhance the response of CD8+ T cells to limiting amounts of IL-15 (15
). At present the role, if any, of enhanced IL-15Rα expression on CD8+ T cells after IFN-α treatment remains unclear.
Collectively, our data support a model in which IFN-α, when used as an adjuvant for anti-tumor peptide vaccination, acts on mDCs and vaccine-induced CD8+ T cells to increase the height, longevity and anti-tumor effect of the antigen-specific CD8+ T cell response. During the initial expansion and contraction of the antigen-specific CD8+ T cell compartment, in addition to the previously-described ability of Type I IFNs to enhance antigen presentation and costimulation by DCs, enhanced proliferation and survival requires direct stimulation of the IFNAR on the T cells themselves. Once IFN-α-enhanced CD8+ T cell levels decline to a plateau (in our studies, typically 50-80% of the peak response by day 12-15) their continued maintenance depends on the expression of IL-15 and probably IL-15Rα by host cells. Further studies will investigate whether the IFN-α-mediated increase in IL-15Rα on DCs and other cell types seen on day 4 post-vaccination continues to longer timepoints, as suggested by the ability of IFN-α to support IL-15-dependent long-term persistence of antigen-specific CD8+ T cells. Experiments are also ongoing to determine whether persistence of antigen-specific CD8+ T cells during the plateau phase requires continued delivery of exogenous IFN-α such as achieved during HGT with IFN-α expression plasmid. Alternatively, IFN-α and IL-15/IL-15Rα signaling in the early phase of the vaccine response may initiate a developmental program in antigen-specific CD8+ T cells which favors their persistence even after levels of exogenously-supplied IFN-α have waned.
In summary, these data demonstrate the activity of IFN-α as an adjuvant for anti-tumor peptide vaccination, and provide a model system for further dissecting the mechanisms of adjuvant activity and optimizing IFN-α-enhanced vaccination in pre-clinical studies. Since IFN-α is already an approved treatment for melanoma, our data point towards a potentially promising role as vaccine adjuvant in human clinical trials. In particular, patients with surgically resected stage III melanoma that are receiving IFN-α as part of their standard-of-care treatment might benefit from concurrently receiving a proven safe and non-toxic peptide vaccination to potentially reduce their risk of disease recurrence through the induction of tumor-specific T cell responses.