We have directly enumerated ex vivo cytotoxic T cells responding to infection with Listeria monocytogenes
. We show that early after infection with live bacteria a very high percentage of responding antigen-specific CD8 T cells gain cytotoxic effector functions. This portion of cytotoxic, antigen-responsive CD8 T cells, in relation to the IFN-γ-secreting population, steadily declines over the course of the response. These findings follow the same trends as the expression of surrogate markers of cytotoxicity, such as perforin and granzyme A/B, at the protein (31
) and mRNA levels (12
), although differences in sensitivities of particularly the mRNA detection methods may account for quantitative differences in the frequencies observed. Thus the early regulation of cytotoxicity that we describe may be mediated at the level of effector molecule expression, although we cannot exclude the possibility that changes in the threshold required to trigger degranulation also occur.
Our data further suggest that IFN-γ secretion, cytotoxicity, and proliferation are all regulated independently during the acute phase of CD8 T-cell responses to infection. In a set of very elegant experiments, it was shown before that proliferation and differentiation of CD8 T cells are regulated in different ways (16
). Immunization of mice with heat-killed L. monocytogenes
induced the expansion of antigen-specific CD8 T cells, which, however, did not acquire cytotoxic effector function. Most remarkably, immunization with a mixture of live bacteria and heat-killed L. monocytogenes
also induced a mixed CD8 T-cell response, indicating that (i) the stimulus for differentiation is given already at a very early time point during priming and (ii) this stimulus must be given at a compartmentalized level, perhaps even at the single-cell level, during interaction between the priming dendritic cells and the naive CD8 T cells. Further, it has been shown that the early inflammatory environment can influence clonal expansion (2
). However, additional signals later during the response, as created, for example, by extended pathogen presence (18
) or by reinfection with the pathogen (5
), can further influence the magnitude of the response. Our results suggest a similar form of regulation for the cytotoxic effector function. At early time points of infection, the responding CD8 T cells gain cytotoxic effector function. However, they rapidly lose this again with a characteristic dynamic and independent of the presence of antigen (Fig. ) or CD4 T cells (Fig. ). At the same time, strong stimuli can increase the portion of cytotoxic T cells (Fig. ).
Most interestingly, the percentage of cytotoxic cells followed a consistent pattern during the immune response and was well coordinated with T-cell expansion: the further the antigen-specific response had expanded within an individual mouse, the further the decline of the cytotoxic proportion had progressed (Fig. ). This trend occurred even in groups with considerable heterogeneity at the peak of the response. This suggests that in each mouse the CD8 T-cell response unfolds in a consistent manner regardless of the precise kinetics of the response within individual mice.
Taken together, our data suggest that, similar to the clonal expansion of the CD8 T-cell response after priming, the dynamics of cytotoxic effector function are determined at a very early time point and then follow a specific program.
These findings raise the question of why the cytotoxic effector function is restrained already when the overall antigen-specific CD8 T-cell response is still expanding. One obvious advantage can be seen in the fact that the cytotoxic effector function causes direct damage to the host. In situations in which a very high percentage of the tissue is infected, tight regulation of this effector function could potentially prevent excessive, life-threatening tissue damage to the host. An additional advantage might be related to preservation of professional antigen-presenting cell activity. The expanding CD8 T-cell response limits the priming of novel pathogen-specific T-cell responses by a feedback loop that kills professional antigen-presenting cells that present the original epitope (34
). Thus, the early loss of cytotoxic effector function might allow an accelerated renewed priming of potentially more protective CD8 T-cell responses in case the original response failed to do so.
How cytotoxic effector function in vivo is regulated precisely, on a molecular level, remains unknown. In order to kill a target cell, a CD8 T cell has to express multiple different effector molecules (12
) and focus these in the immunological synapse (8
). It is possible that there are two independent CD8 T-cell populations, one cytotoxic and one not, and that the observed selective loss of cytotoxic effector function is explained by different rates of expansion or contraction of these two populations within the spleen. Nevertheless, we consider it far more likely that a change in phenotype of individual T cells occurs within the entire CD8 T-cell population, which may explain the observed selective reduction in cytotoxicity during the immune response. Loss of cytotoxicity could precede the depletion of the CD8 T cells in the contraction phase. This could also explain why cells retain increased cytotoxicity levels in this phase if they receive additional survival signals, such as through interleukin-7/interleukin-7 receptor interaction (10
), or activation signals, such as through CD70/CD27 interaction during an influenza virus infection (1
). These reports fit well with our observation that reinfection with a very high load of pathogen, which is likely to have provided a novel pulse of additional antiapoptotic, inflammatory stimuli, just before the onset of the contraction phase led to an increased proportion of antigen-specific CD8 T cells expressing cytotoxic effector function (Fig. ).
In conclusion, our data shed new light on the different regulations to which different CD8 T-cell effector functions are subjected during an immune response. Our data indicate that, upon infection, individual CD8 T cells rapidly gain cytotoxic effector functions and then progressively loose them, according to a set internal regulation.