The results described here using cytokine receptor-deficient OT-I T cells demonstrate that both IL-12 and Type I IFNs contribute to programming for memory in CD8 T cells responding to vaccinia virus or LM, and that signals from one or the other must be available to obtain a memory population in a normal host. For vaccinia infection, the predominant cytokine that supports memory is IL-12, with IFN-α/β making a relatively small contribution (). For LM infection, the memory population is substantially reduced when the receptor for either cytokine is absent (), suggesting that when the OT-I T cells are initially responding to Ag the levels of IL-12 and IFN-α/β may be relatively low and that many of the cells only receive sufficient signaling to program for memory when they can respond to both cytokines. Thus, IL-12 or IFN-α/β signals are not only important for differentiation of naïve CD8 T cells to effectors (8
), they are required for development of a memory population following responses to pathogens in a normal host. Although Ag and costimulation levels, as well as numerous cytokines and surface ligands, can influence the magnitude of primary and memory CD8 T cell responses, it appears that IL-12 and IFN-α/β are uniquely able to act as the switch that determines whether the outcome of a response to Ag is memory versus tolerance in a normal host.
Although required for memory development, signals from IL-12 and IFN-α/β made only modest contributions to the magnitude of primary clonal expansion to vaccinia or LM, with at most a three-fold reduction in expansion in response to LM when the cells lacked both cytokine receptors. This contrasts with LCMV infection, where primary expansion of TCR transgenic P14 CD8 T cells deficient for Type I IFNR1 is reduced by more than 99% compared to WT cells (6
). Because primary expansion was severely compromised, the importance of IFN-α/β signals for memory programming could not be distinguished in those studies. However, a small number of Type I IFNR1-deficient cells did persist long term following LCMV infection (<1%), and were likely cells that received an IL-12 signal. Consistent with this, Type I IFNR1-deficient mice produce more IL-12 in response to LCMV infection than do WT mice, and can make a strong CD8 T cell response to the virus (16
It is not clear why primary expansion of OT-I CD8 T cells to VV-OVAp () is affected relatively little when signals from these cytokines are absent (Figs. and ), while the expansion to LM-OVA is substantially reduced (), and the expansion of P14 cells to LCMV is severely compromised in the absence of IFN-α/β signaling (6
). In a peptide immunization model, co-administration of IL-12 was shown to strongly enhance primary clonal expansion at low peptide doses by enhancing survival, but had less effect at high peptide doses where clonal expansion was strong in the absence of the cytokine (7
). It may be the case that signal three cytokines can promote survival to enhance primary expansion when Ag levels are low, but contribute less when strong early survival signals are available due to high levels of Ag, high TCR affinity, or possibly high levels of costimulatory ligands. Differences in these parameters for VV, LM and LCMV infections may account for the varied dependence of primary expansion on IL-12 and Type I IFN signals. In all cases, however, formation of a responsive memory population is critically dependent on IL-12 or IFN-α/β signals.
Although CD8 T cells require IL-12 or IFN-α/β signals to develop memory in a normal host environment, where both IL-12 and IFN-α/β are present and the endogenous host cells express receptors for the cytokines, this is not the case in an IL-12-deficient host. In the absence of IL-12, both OT-I.IFNARKO and OT-I.DKO cells form memory populations following infection with VV-OVAp (), consistent with the results of Orgun et.al. (28
) showing that mice deficient in both IL-12 p40 and Type I IFNR1 expression develop CD8 T cell memory populations comparable to those of wild type mice following infection with LM ΔactA, an attenuated strain of LM. Host deficiency for a given cytokine can have multiple effects, including altered production of other cytokines. For example, CD8 T cell responses to LCMV infection in WT mice depend almost completely upon IFN-α/β (6
), but a strong CD8 T cell response occurs in Type I IFNR-deficient mice where IL-12 production is increased (16
). Our results suggest that an alternate third signal is present in the IL-12-deficient environment, one that is not normally produced in sufficient amounts in an intact host to support a memory response. Further work will be needed to determine the identity of this alternate signal, but IL-21 is a candidate given its ability to support development of cytolytic function in vitro (19
The ability of OT-I.DKO cells to form memory in IL-12-deficient hosts makes the important point that most of the experiments described here examine responses of WT and receptor-deficient CD8 T cells responding in a normal host environment, where both IL-12 and IFN-α/β can be present and the endogenous host lymphocytes express receptors for the cytokines. Thus, conclusions can be drawn regarding the signals directly needed by the transferred cells to respond and develop memory. However, the results cannot be extrapolated to what might be seen upon infection of mice that are deficient in the cytokine or receptor, where there may be effects on numerous lymphocyte and dendritic cell subsets, levels of production of other cytokines, rates of Ag clearance, etc.
IL-12 and Type I IFN, and other inflammatory cytokines, are produced early in response to infections and levels decline within a few days, suggesting that early IL-12 and IFN-α/β signaling, during the time the cells are initially responding to Ag, may program the CD8 T cells to subsequently form a memory population. Consistent with this, in vitro stimulation with Ag, B7-1 and IL-12 for 72hr was sufficient to program development of a functional memory population upon transfer into a normal host mouse (). Thus, during the period that the signal 3 cytokines are supporting differentiation to develop effector functions (29
), they also initiate the gene regulation program required for survival and formation of long-lived memory cells. Furthermore, despite presumably uniform delivery of signals in vitro, the memory cells include both CD62Lhi
populations, a phenotype consistent with both effector and central memory cells being present.
While our results show that IL-12 can provide a critical third signal, along with Ag and B7-1, to program memory development, there is also some evidence that IL-12 can hinder development of long-term memory by promoting formation of relatively short-lived, fully activated effector cells (13
). Joshi et.al. (13
) characterized a short-lived effector population that arises during a response to LCMV that could be identified based on increased expression of KLRG1 on the surface, and suggested that formation of this population was driven by high T-bet expression induced by high levels of inflammatory cytokines, including IL-12. In contrast, a more recent report by Sarkar et.al. (32
), also examining responses to LCMV, has suggested that formation of this KLRG1hi
terminal effector population is driven by continuing Ag stimulation during the late stages of infection. Our results clearly show that IL-12 can provide a critical third signal to program for development of memory under conditions where a KLRG1hi
population is not induced (). The system described here, employing in vitro stimulation under well-defined conditions followed by adoptive transfer to monitor the transition to memory, should provide a means of determining how signals present during the post-programming phase will affect the size and phenotype of the memory pool, and such experiments are in progress.
CD4 T helper cells can be necessary for development of CD8 T cell memory, and one way in which they are likely to contribute is by stimulating DC to produce IL-12 and/or IFN-α/β that can then program the CD8 T cells to develop memory. CD4 T cell help to ‘condition’ or ‘license’ DC to effectively activate CD8 T cells requires CD40 engagement on the DC by CD40L on the CD4 T cell (34
), and ligation of CD40 induces DC to produce IL-12 (33
). In an ectopic heart transplant model requiring CD4 T cell help for CD8-mediated rapid graft rejection, Filatenkov et.al. (38
) demonstrated that CD4 T cells stimulated IL-12 production by DC in a CD40-dependent manner, and that IL-12 was necessary for the CD8 T cells to develop effector functions and mediate rejection. In responses to pathogens, this role for CD4 help may not be critical since viral or bacterial components provide TLR ligands that can activate DC and induce inflammatory cytokines, including IFN-α/β and IL-12 (33
). CD4 T cells may still be important, however, for long-term maintenance of CD8 memory T cells (39
), and/or for producing IL-2 that can play an important early role in programming for memory (3
There are reports in the literature of a number of proteins whose expression by CD8 T cells is regulated by IL-12, including Bcl-3 which can promote survival of activated CD8 T cells (40
), cellular FLIPs which may protect against fas-mediated apoptosis (42
), and CD25 which can increase sensitivity to IL-2 signaling (43
). In fact, it is likely that regulation of expression of numerous proteins is involved in IL-12 and IFN-α/β-dependent programming for memory. Oligonucleotide micro array analysis of IL-12 and IFN-α gene regulation in naïve CD8 T cells responding in vitro to Ag and B7-1 has revealed that each cytokine initiates a complex program of altered gene expression during the first seventy-two hours of the response as effector functions develop and programming for memory occurs. Over 350 genes, including those for numerous transcription factors, are regulated in common by the two cytokines (Agarwal et.al. manuscript in preparation
). The realization of the critical role that these cytokines play in determining whether Ag encounter leads to CD8 T cell memory, when they are present, or tolerance, in their absence, and the further elucidation of the molecular pathways that determine the differentiation process, should contribute substantially to the development of improved strategies for optimizing vaccines.