In this study, we show that IFN-αβ can have a direct role in linking innate and adaptive responses by providing the “third signal” needed by naive CD8 T cells responding to Ag and costimulatory ligands. Stimulation of CD8 T cells in the absence of a third signal leads to proliferation, but clonal expansion is limited by poor survival and effector functions do not develop. We show that IFN-αβ can provide the third signal directly to CD8 T cells via a STAT4-dependent pathway to stimulate survival, development of cytolytic function, and production of IFN-γ. Provision of the third signal by either IFN-αβ or IL-12 results in regulation of the expression of a number of genes, including several that encode proteins critical for effector function.

Activation of naive CD8 T cells to undergo clonal expansion and develop effector function requires three signals: (a) Ag, (b) costimulation, and (c) IL-12 or adjuvant. The requirement for the third signal to stimulate Ag-dependent proliferation is variable, making the greatest contribution when Ag levels are low. At high Ag levels, extensive proliferation can occur in vitro or in vivo in the absence of a third signal. However, despite having undergone the same number of divisions, cells that expand in the absence of a third signal fail to develop cytolytic effector function. Thus, proliferation and development of cytolytic function can be fully uncoupled. Furthermore, these cells are rendered functionally tolerant in vivo, in that subsequent restimulation with a potent stimulus results in limited clonal expansion, impaired IFN-γ production, and no cytolytic function. Thus, the presence or absence of the third signal appears to be a critical variable in determining whether stimulation by Ag results in tolerance versus development of effector function and establishment of a responsive memory population.

Autocrine IFN-γ signaling is important for CD4 differentiation to T helper 1 effector cells, but it has been unclear whether it contributes to CD8 T cell differentiation. We show here that naïve murine CD8 T cells rapidly and transiently produce low levels of IFN-γ upon stimulation with Ag and B7-1, with production peaking at about 8 hours and declining by 24 hours. The autocrine IFN-γ signals for upregulation of expression of Tbet and GrzB, and induces weak cytolytic activity and effector IFN-γ production. IFN-α acts synergistically with IFN-γ to support development of strong effector functions, while IL-12 induces high T-bet expression and strong function in the absence of IFN-γ signaling. Thus, IFN-γ is not only an important CD8 T cell effector cytokine, it is an autocrine/paracrine factor whose contributions to differentiation vary depending on whether the response is supported by IL-12 or Type I IFN.

Naive CD8 T cells that respond in vivo to Ag and costimulation in the absence of a third signal, such as IL-12, fail to develop cytolytic function and become tolerized. We show in this study that CD8 T cells purified from TCR transgenic mice and stimulated in vitro in the presence or absence of IL-12 form conjugates with specific target cells, increase intracellular Ca2+, and undergo degranulation to comparable extents. Perforin is also expressed at comparable levels in the absence or presence of a third signal, but expression of granzyme B depends upon IL-12. Levels of granzyme B also correlate strongly with the cytolytic activity of cells responding in vivo. In contrast, an increase in CD107a (lysosomal-associated membrane protein 1) expression resulting from degranulation cannot distinguish in vivo generated lytic effector cells from tolerized, noncytolytic cells. Thus, it appears that cells rendered tolerant as a result of stimulation in the absence of a third signal fail to lyse target cells because they are “shooting blanks.”

CD8 T cells need a third signal, along with Ag and costimulation, for effective survival and development of effector functions, and this can be provided by IL-12 or type I IFN. Adoptively transferred OT-I T cells, specific for H-2Kb and OVA, encounter Ag in the draining lymph nodes of mice with the OVA-expressing E.G7 tumor growing at a s.c. site. The OT-I cells respond by undergoing limited clonal expansion and development of effector functions (granzyme B expression and IFN-γ production), and they migrate to the tumor where they persist but fail to control tumor growth. In contrast, OT-I T cells deficient for both the IL-12 and type I IFN receptors expand only transiently and rapidly disappear. These results suggested that some signal 3 cytokine is available, but that it is insufficient to support a CTL response that can control tumor growth. Consistent with this, administration of IL-12 at day 10 of tumor growth resulted in a large and sustained expansion of wild-type OT-I cells with enhanced effector functions, and tumor growth was controlled. This did not occur when the OT-I cells lacked the IL-12 and type I IFN receptors, demonstrating that the therapeutic effect of IL-12 results from direct delivery of signal 3 to the CD8 T cells responding to tumor Ag in the signal 3-deficient environment of the tumor.