TCR and CD28 signals stimulate multiple rounds of CD8 T cell division (15
) but in the absence of a signal from IL-12 or IFNα/β survival is compromised, effector functions and memory do not develop, and the small number of cells that do survive are tolerant (4
). Use of aAPC provides a powerful approach for studying gene regulation by IL-12 and IFNα; the signals are well defined and all of the cells become activated in a narrow time frame, thus allowing analysis of the time course of changes in gene expression levels. In contrast, this level of definition would not be feasible for cells responding in vivo due to the redundancy of the effects of IL-12 and Type I IFNs, potential modifications of the genetic program due to other signals present in vivo, and the fact that all of the T cells will not be stimulated simultaneously, instead being recruited over time as the infection progresses. Thus, the in vitro studies described here provide a baseline definition of the signal 3-dependent differentiation program, and will form the basis for studying in vivo responses in more detail by examining expression of gene products known to be regulated as part of this program. The program includes about 355 genes regulated in common by IL-12 and IFNα (, Table SIV
). This regulation is superimposed on the regulation of over 3,000 genes by TCR and CD28 signals, many of which are involved in cell cycle regulation, DNA synthesis and repair, protein translation and metabolism (Table SI
). Much of the signal 3-dependent program involves regulation of genes that encode proteins of obvious relevance to the differentiation process that leads to effector and memory cells.
Once CD8 T cells have undergone an initial burst of proliferation over about three days, expansion and survival can be further increased by additional signals. IL-2 produced by CD4 helper T cells can sustain and expand effector CD8 T cell populations (39
). IL-12 increases the expression of CD25
(), thus increasing IL-2Rα protein expression and making the effector cells more responsive to this signal (23
). Receptors belonging to the tumor necrosis factor receptor super family (TNFRSF) can also increase the survival of effector cells by increasing expression of anti-apoptotic genes (41
). Expression levels of genes for several of these receptors, including OX-40, 4-1BB and GITR, are upregulated IL-12 and IFNα ( and ), and this has been confirmed at the level of cell surface protein expression for OX-40 and 4-1BB (42
). In contrast CD27, a member of the TNFRSF that acts earlier in the response (43
), was upregulated with just two signals, and expression decreased in the presence of IL-12 or IFNα (). IL-12 and IFNα/β also upregulate a number of cell intrinsic defense genes including cysteine protease inhibitors (CTLA-2α and 2β
) and serine protease inhibitors (spi6, spi2e, serpin5, serpin1
) ( and ). Spi6 is important for protecting effector CD8 T cells from death induced by their own lytic machinery (44
). In addition, IL-12 (45
) and IFNa (our unpublished results) also upregulate expression of Bcl-3, a member of the IkB family of proteins that can enhance T cell survival (46
). Thus, a critical part of the signal 3-dependent differentiation program involves increased expression of numerous genes that encode for proteins important for clonal expansion and survival of the effector and memory cells that develop.
Major functions of effector CD8 T cells include production of cytokines and killing of target cells by the perforin/granzyme-dependent degranulation mechanism or the FasL pathway, and IL-12 and IFNα regulate expression of genes for several of the critical proteins of these pathways. GrzB is expressed weakly and transiently in response to two signals, and is increased to high, sustained levels by IL-12 or IFNα (, ). While IL-12 and IFNα both support development of cytolytic activity (), there may be differences in functional capacities depending on which signal a cell receives, since grzC and grzF mRNAs were upregulated by IL-12 but not by IFNα ( and ). Unlike granzymes, perforin mRNA and protein are strongly upregulated by two signals alone, and only marginally increased in the presence of IL-12 or IFNα (22
). The Fas-dependent killing pathway also appears to require signal 3, as FasL mRNA expression is not upregulated by two signals but is strongly upregulated by IL-12 or IFNα (). IFNγ mRNA and protein are also strongly upregulated by both IL-12 and IFNα (, ). In contrast, TNFα mRNA is highly expressed in naïve cells, and is initially downregulated (24 hr) but later increased (72 hr) to naïve levels in response to two signals (), and expression does not significantly change in response to IL-12 or IFNα, and we have confirmed this at the protein level by intracellular staining (our unpublished results). This is consistent with the fact that naïve CD8 T cells rapidly produce TNFα upon Ag stimulation, and this capacity declines as the cells become effectors (47
). Thus, unlike cytolytic activity and IFNγ production, the capacity to produce TNFα does not require differentiation of the naïve cells nor does it depend upon a third signal.
To carry out its effector functions, a CD8 activated in a draining lymph node must migrate to the site of the foreign Ag. Two signals (Ag-B7) are sufficient to increase expression of a number of genes for cell adhesion and homing receptors, including CD44, MAC-2, and ICAM-I, CCR5, and CXCR3 (), and to downregulate genes for secondary lymphoid homing receptors, including CD62L and CCR7. Regulation of these receptors by just two signals is consistent with the observation that CD8 T cells activated in the absence of a third signal can nevertheless migrate to peripheral sites of Ag, but fail to mediate autoimmunity (38
), graft rejection (12
), or elimination of infected cells (48
). IL-12 and IFNα/β do regulate expression of genes for several of the other receptors and chemokines involved in controlling migration, but differing patterns of regulation were observed for IL-12 versus IFNα ( and ). IL-12 upregulated CCR2 and CCR5, chemokine receptors that promote migration to inflammatory and allergic response sites, while IFNα did not increase CCR2 expression and only weakly increased CCR5 expression. IL-12 also induced chemokines that may aid recruitment of CD4 TH
cells, DCs and monocytes, including CCL9, MIP-1α,β and XCl-1, while IFNα increased expression of only XCl-1. CXCL-10, a chemokine that helps attract NK cells, B cells, neutrophils and Type I T cells, was induced by IFNα, but not by IL-12. Galectin-3, an adhesion molecule, was strongly upregulated by IL-12, while galectin-9, an eosinophil attractant, was upregulated by IFNα. Finally, integrin β7 that affects migration to mucosal regions was downregulated by both IL-12 and IFNα. Thus, the signal 3-dependent differentiation program includes alterations in expression of genes for a number of receptors and chemokines important for migration into sites of foreign Ag and recruitment of additional effector cells to the sites, but the migration phenotypes of the effector cells may differ depending upon whether the third signal was provided by IL-12 or Type I IFN.
Numerous genes encoding proteins involved in signal transduction pathways are regulated by IL-12 and/or IFNα, including both cell surface receptors (e.g. IL-18R1, IL-18Racp, IL-12Rβ1 and β2, IL-2Rα) and intracellular signaling intermediates (e.g. MyD88, Traf4, Traf6, Gadd45β and Gadd45γ). This may contribute to enhanced signaling for effector responses. IL-12 and IFNα also up- or down-regulate expression of a number of genes for transcriptional factors, including ATF-3,- 4, -5, Jun-B, CREM, STAT5β, Map2k1, Sos2, regulators of G-protein signaling, and others. Two particularly interesting transcription factors regulated by IL-12 and IFNα/β are T-bet and Eomes, both of which have are involved in CD8 T cell acquisition of effector functions upon stimulation with Ag (26
), in part through a role in up-regulating grzB expression. T-bet expression is weakly induced in response to just two signals, and increases in response to IL-12 or IFNα. In contrast, Eomes is expressed in naïve cells and decreases in response to Ag and B7-1, but is sustained at a high level when either IL-12 or IFNα are present (). Thus, it appears likely that these signal 3 cytokines are promoting differentiation in part by maintaining these critical transcription factors at high levels. Many additional transcription factors and regulators are also positively or negatively regulated by IL-12 and IFNα/β (), and are likely to have important roles in the differentiation program.
Histone-dependent chromatin remodeling appears to be a major mechanism by which IL-12 and IFNα/β regulate the differentiation program. Expression of many of the IL-12/IFNα-regulated genes increases early in response to Ag and B7-1 but then declines by 72 hr (, S1
). Expression is increased and sustained by IL-12 or IFNα, suggesting that these loci might initially be accessible to transcription factors induced by Ag and B7-1, but that alterations in chromatin structure then render them inaccessible unless a signal 3 cytokine is present. The ability of trichostatin A, a class I and II HDAC inhibitor, to substitute for IL-12 or IFNα/β () indicated that the cytokines might act by increasing the level of histone acetylation at critical gene loci to allow their continued transcription. We directly demonstrated this for the grzB
genes, where IL-12 or IFNα caused increased acetylation of histones H3 and H4 at the proximal promoter regions, and at the distal exon region of grzB (). In a similar approach using naïve CD8 T cells stimulated with anti-TCR and anti-CD28 mAb, IL-12 was shown to cause long-range hyperacetylation in the promoter and exon regions of the IFNγ gene (31
). Thus, it appears that IL-12 and IFNα/β enforce the gene regulation program, at least in part, by promoting chromatin remodeling to allow sustained expression of critical genes. Both cytokines upregulate expression of mRNA for cyclin-dependent kinase inhibitor p21, (Cdkn1A, p21), a G1/S arrest gene, by 24h, and it continued to be expressed at 72 hr (). This may contribute to chromatin remodeling by prolonging the G1/S phase, since CD4 TH cells in this phase of the cell cycle have been shown to be the most susceptible to epigenetic modifications of chromatin (49
There is increasing evidence that chromatin remodeling involving histone acetylation may play an important role in differentiation of CD8 T cells to develop memory. Memory CD8 T cells exhibit increased histone acetylation levels for many of the genes that are expressed more rapidly than in naïve cells following activation, including Eomes
). The results described here, together with the evidence that a signal from IL-12 or Type I IFN is required to program for memory development (13
), suggest that much of the chromatin remodeling may be initiated by these cytokines. Epigenetic memory of the remodeling is likely to account for the more rapid responses of memory cells upon re-encountering Ag, and would be consistent with the fact that memory CD8 T cells do not require a third signal in order to efficiently respond to Ag and costimulation (10
CD4 T cells can provide help to CD8 T cells through CD40-dependent stimulation of DC to produce IL-12 (12
), thus making this critical third signal available to the responding CD8 T cells. In experimental models that require CD4 T cell help for CD8 memory formation, increased acetylation in the memory cells has been shown to be CD4 T cell-dependent (35
). CD8 T cells activated with anti-TCR and anti-CD28 mAb in splenocyte cultures in the absence of CD4 T cells remain hypoacetylated and do not develop into functional memory cells upon transfer into mice, but addition of trichostatin A to these cultures to inhibit histone deacetylation results in generation of cells that survive and become functional memory cells upon transfer (36
). It would seem reasonable to speculate that CD4 T cells are providing help, at least in part, by stimulating production of IL-12 and/or IFNα/β, and that trichostatin A can bypass this requirement for memory formation as it does for induction of effector functions ().
Kaech et.al. (52
) compared gene expression levels in naïve, effector and memory CD8 T cells responding to LCMV infection, a response that depends almost completely on Type I IFNR (7
). Comparison of our results to those shows that of the 355 genes regulated by both IL-12 and IFNα, about twenty-five percent were similarly up or down regulated in memory versus naïve cells. This suggests that once these genes are up or down regulated by a signal 3 cytokine during the primary response they continue to be expressed at similar levels in the memory cells. For transcription factors upregulated by IL-12 and IFNα ( and SII
), several were also seen to be increased in the effector cells (day 8) responding to LCMV, including Blimp-1, BhlhB2, Eomes (Tbr2), and nfil3/E4BP4, while Lef-1, Tcf-7, Pou2af1, Idb3, and others were also downregulated. Nfil3/E4BP4 and BhlhB2 genes continued to be expressed at increased levels in memory cells (day 40), while Idb3 continued to be repressed. Thus, the results of our analysis of in vitro stimulated cells agree well with those for cells responding in vivo, and the comparison suggests that many of the changes in expression level that occur in response to IL-12 and IFNα/β during primary stimulation persist in resting memory cells. When naïve cells respond to Ag and costimulation in the absence of a signal 3 cytokine, survival is compromised, effector functions do not develop, and the cells that do survive long term are tolerant. This raises the possibility that the response to Ag and B7 alone may lead to permanent silencing of critical genes to result in tolerance.