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.

Stimulation of naïve CD8+ T cells with antigen and costimulation results in proliferation and weak clonal expansion, but the cells fail to develop effector functions and are tolerant long term. Initiation of the program leading to the strong expansion and development of effector functions and memory requires a third signal that can be provided by interleukin-12 (IL-12) or interferon-alpha (IFN-alpha). CD4+ T cells condition dendritic cells (DCs) to effectively present antigen to CD8+ T cells, and this conditioning involves, at least in part, CD40-dependent upregulation of the production of these signal 3 cytokines by the DCs. Upon being fully activated, the cytotoxic T lymphocytes develop activation-induced non-responsiveness (AINR), a form of split anergy characterized by an inability to produce IL-2 to support continued expansion. If antigen remains present, IL-2 provided by CD4+ T cells can reverse AINR to allow further expansion of the effector population and conversion to responsive memory cells following antigen clearance. If IL-2 or potentially other proliferative signals are not available, persistent antigen holds cells in the AINR state and prevents the development of a responsive memory population. Thus, in addition to antigen and costimulation, CD8+ T cells require cytokine signals at distinct stages of the response to be programmed for optimal generation of effector and memory populations.

Summary CD8 T cells require a third signal, along with Ag and costimulation, to make a productive response and avoid death and/or tolerance induction. Recent studies indicate that IL-12 and Type I IFN (IFNα/β) are the major sources of signal 3 in a variety of responses, and that the two cytokines stimulate a common regulatory program involving altered expression of about 350 genes. Signal 3-driven chromatin remodeling is likely to play a major role in this regulation. Although less well studied, there is emerging evidence that CD4 T cells may also require a ‘third signal’ for a productive response and that IL-1 can provide this signal. Signal 3 cytokines can replace adjuvants in supporting in vivo T cell responses to peptide and protein antigens, and a better understanding of their activities and mechanisms should contribute to more rational design of vaccines.

SUMMARY Immune cells sense microbial products through Toll-like receptors (TLR), which trigger host defense responses including type 1 interferons (IFNs) secretion. A coding polymorphism in the protein tyrosine phosphatase nonreceptor type 22 (PTPN22) gene is a susceptibility allele for human autoimmune and infectious disease. We report that Ptpn22 selectively regulated type 1 IFN production after TLR engagement in myeloid cells. Ptpn22 promoted host antiviral responses and was critical for TLR agonist-induced, type 1 IFN-dependent suppression of inflammation in colitis and arthritis. PTPN22 directly associated with TNF receptor-associated factor 3 (TRAF3) and promotes TRAF3 lysine 63-linked ubiquitination. The disease-associated PTPN22W variant failed to promote TRAF3 ubiquitination, type 1 IFN upregulation, and type 1 IFN-dependent suppression of arthritis. The findings establish a candidate innate immune mechanism of action for a human autoimmunity “risk” gene in the regulation of host defense and inflammation.

Inflammation can have both positive and negative effects on development of CD8 T cell memory, but the relative contributions and cellular targets of the cytokines involved are unclear. Using CD8 T cells lacking receptors for IL-12, type I IFN, or both, we show that these cytokines act directly on CD8 T cells to support memory formation in response to vaccinia virus and Listeria monocytogenes infections. Development of memory to vaccinia is supported predominantly by IL-12, whereas both IL-12 and type I IFN contribute to memory formation in response to Listeria. In contrast to memory formation, the inability to respond to IL-12 or type I IFN had a relatively small impact on the level of primary expansion, with at most a 3-fold reduction in the case of responses to Listeria. We further show that programming for memory development by IL-12 is complete within 3 days of the initial naive CD8 T cell response to Ag. This programming does not result in formation of a population that expresses killer cell lectin-like receptor G1, and the majority of the resulting memory cells have a CD62Lhigh phenotype characteristic of central memory cells. Consistent with this, the cells undergo strong expansion upon rechallenge and provide protective immunity. These data demonstrate that IL-12 and type I IFN play an essential early role in determining whether Ag encounter by naive CD8 T cells results in formation of a protective memory population.

Full activation of naive CD8 T cells requires Ag, costimulation, and a third signal that can be provided by IL-12. Brief exposure (6 h) to Ag and B7-1 is sufficient to stimulate multiple rounds of cell division, but clonal expansion and development of effector function are minimal even when signal 3 is present. Full activation instead requires concerted signaling by Ag, B7-1, and IL-12 for greater than 40 h. Thus, the gene expression program required for cell division can be initiated by brief interaction with Ag and costimulation, but maintaining the expression of the genes needed for survival and effector function requires prolonged signaling by a signal 3 cytokine in concert with Ag and costimulation.

A tumor-specific CD8+ T cell response was studied using adoptive transfer of OT-I TCR transgenic cells. Upon i.p. challenge with E.G7 tumor, OT-I cells undergo CD4+ T cell-independent expansion at the tumor site and develop lytic function. Before tumor elimination, however, they leave the peritoneal cavity (PC) and appear in the LN and spleen where they exhibit "split anergy" and cannot further proliferate to antigen. Administering anti-CTLA-4 mAb early caused sustained OT-1 expansion in the PC, and late administration caused the OT-I cells to return to the PC and further expand; in both cases, tumor was controlled. These effects required CD4+ T cells and IL-2 and appear to result from reversal of the nonresponsive state of the CD8+ T cells.

A third signal that can be provided by IL-12 or type I IFN is required for differentiation of naive CD8 T cells responding to Ag and costimulation. The cytokines program development of function and memory within 3 days of initial stimulation, and we show here that programming involves regulation of a common set of ∼355 genes including T-bet and eomesodermin. Much of the gene regulation program is initiated in response to Ag and costimulation within 24 h but is then extinguished unless a cytokine signal is available. Histone deacetylase inhibitors mimic the effects of IL-12 or type I IFN signaling, indicating that the cytokines relieve repression and allow continued gene expression by promoting increased histone acetylation. In support of this, increased association of acetylated histones with the promoter loci of granzyme B and eomesodermin is shown to occur in response to IL-12, IFN-α, or histone deacetylase inhibitors. Thus, IL-12 and IFN-α/β enforce in common a complex gene regulation program that involves, at least in part, chromatin remodeling to allow sustained expression of a large number of genes critical for CD8 T cell function and memory.