The present study underscores the advantages of targeted delivery of IFN-γ to a prescribed tissue microenvironment. Administration of IFN-γ as either a recombinant protein, rIFN-γ, or as a recombinant avipox virus, rF-IFN-γ, induces profound expansions of immune cells within the local, regional LNs. Several distinct advantages are gained, however, by delivering IFN-γ as rF-IFN-γ rather than injecting the rIFN-γ protein. First, the expansion of immune cells, particularly NK1.1+ cells, within the regional LNs was sustained for 5-6 days in comparison to a transient (approx. 24 hrs) expansion after rIFN-γ injection. Second, the enrichment within the regional nodes was achieved without any measurable circulating IFN-γ levels, thus negating any side effects associated with high doses and/or multiple injections of the rIFN-γ protein. And, finally, the combined administration of rF-IFN-γ, not rIFN-γ, with antigen (i.e., highly tumorigenic MC38 tumor cells) induced sufficient tumor-specific adaptive T cell immunity to protect mice against primary tumor growth and subsequent tumor challenge.
In the past, evaluations of rIFN-γ as a single antitumor agent have been predicated on the ability to deliver sufficient amounts of the cytokine to the tumor site. Upregulation of MHC, Fas and tumor associated antigens on tumor cells, inhibition of immunosuppressive factors and potent anti-angiogenic effects have all been document with systemic IFN-γ administration and have provided the argument for the immunomodulatory and, perhaps, antitumor actions of this cytokine. However, clinical studies revealed severe treatment-limiting toxicities that have blunted the use of rIFN-γ in cancer treatment. Yet, IFN-γ remains a potent proinflammatory cytokine as do questions on how best to exploit its actions. Our rationale was to target IFN-γ production to a tissue microenvironment via rF-IFN-γ injection, thus localizing its proinflammatory actions that would induce a controlled migration of host immune cells to the injection site. Under those conditions, with the addition of antigen, in the form of rF-IFN-γ-infected MC38 tumor cells, tumor specific CTL activity was generated in the antigen-stimulated LNs. Furthermore, T cell depletion studies established their requirement to protect mice from the growth of rF-IFN-γ-infected MC38 tumor cells. Thus, the findings of this study might provide the rationale to reexamine IFN-γ as a classic immune adjuvant when delivered with antigen to a selected tissue microenvironment.
The ability of rF-IFN-γ to act as an immune adjuvant was dependent on: (1
) the sustainability of IFN-γ production at the injected site, consistent with using a fowlpox-based vector as a delivery vehicle and (2
) the cellular enrichment (), particularly NK1.1+ cells () within the regional lymph nodes of mice receiving rF-IFN-γ. Several intriguing findings focused our attention on the NK1.1+ cell population in the regional lymph nodes of mice injected with rF-IFN-γ. In vivo NK1.1 depletion studies suggested that they play an important role(s) in (1
) the protection against primary tumor growth () and (2
) the generation of a tumor-specific host immune response (). In contrast, in a tumor challenge experiment of vaccinated [rF-IFN-γ-infected MC32A (CEA-expressing) cells] mice, the ability to reject tumor was not lost with NK1.1+ cell depletion (). Those findings indicate a role(s) for NK1.1+ cells in T cell priming but not for the recall of memory T cells.
The results argue that injection of rF-IFN-γ-infected MC38 initiates signals for the extravasation of NK1.1+ cells from the circulation to the site of inflammation. Traditionally, NK cells function as peripheral effector cells that recognize and kill stressed, transformed and virally infected cells, their principle role in innate immunity (32
). Whether NK1.1+ cells recognize and kill rF-IFN-γ-infected MC38 tumor cells, contributing to the protection of mice against primary tumor growth is arguable. Infection of MC38 tumor cells with rF-IFN-γ enhances MHC class I expression (, ) and might be expected to inhibit NK cell cytotoxicity through engagement of NK cell receptors containing a tyrosine-based inhibitory motif (34
). Yet, when mice were depleted of NK cells, protection from the growth of rF-IFN-γ-infected MC38 tumor cells was lost (). One explanation is that the NK1.1+ cells within the antigen-stimulated LNs are not tumoricidal, but their presence supports the development of tumor-specific adaptive immunity. Indeed, priming of naïve T cells to tumor-specific antigen occurs within secondary lymph sites and requires the presence of mature DCs. Within sites of chronic inflammation (i.e., allergen-induced atopic eczema/dermatitis syndrome), NK cells are in close contact with resident DCs (35
) which results in DC maturation and local IL-12 production (36
) driving a TH
1 cellular response. In the present study, flow cytometric analyses revealed that not only were the antigen-stimulated lymph nodes draining the rF-IFN-γ injection site enriched for NK1.1+
cells, but a significant percentage co-expressed the NKG2D-activating receptor, the primary cytotoxicity receptor for murine NK cells (). Usually, following NKG2D receptor-ligand interaction cytolysis by the NK, NKT and T cells is perforin-mediated (37
), however, no perforin-mediated lysis was detected. Unfractionated LN cells from mice injected with rF-IFN-γ lysed YAC-1 cells in a TNF-α-dependent manner (). A previous study (39
) reported NK-dependent DC maturation/migration through a TNF-dependent mechanism. Absence of that pathway in NK cell-depleted mice would be expected to interrupt DC maturation and severely impair the cross-talk between innate and adaptive immune responses. Whether those findings signal a difference between “helper” (40
) and “effector” NK cells or indicate a particular NK cell subset will be addressed in future studies.
More recent data have shown that NK cells also provide other “helper” function(s) to the adaptive immune system. Their presence in secondary lymphoid sites, particularly, antigen-stimulated LNs, provides a local IFN-γ source that assists in T-cell priming (41
). One could speculate that the local IFN-γ production, by virtue of rF-IFN-γ injection, might substitute some needed “helper” functions in the NK cell-depleted mice, such as IL-12 or IL-18 induction in the microenvironment, and support some measurable T cell priming. However, depletion of NK cells prior to the administration of the cell-based vaccine (i.e., rF-IFN-γ-infected-MC38 tumor cells) led to a complete loss in the ability to generate a primary antigen-specific T cell response (). Those findings suggest that other NK-mediated events are required for T-cell priming which is consistent with recent evidence of an IL-18-dependent soluble factor released by activated NK which promotes DC maturation (45
). Thus, NK cells provide crucial signals which can (1
) impact DC editing and (2
) provide a local source of IFN-γ and the absence of those signals due to NK cell depletion can severely impact T-cell priming (46
). Indeed, patients with deficits in NK cell function have impaired induction of virus-specific immune response and a heightened susceptibility to recurrent viral infection (48
). Therefore, it seems that NK cells are important early innate cells involved at multiple steps leading to the priming of adaptive T cell responses. An understanding of the cellular signals delivered to the innate immune system and how they shape the events upstream of the DC-T helper cell and/or NK-cytotoxic T cell interactions could contribute to purposefully driving a particular adaptive immune response and facilitate the design of novel immunization schema.