Treg have been reported to play diverse roles in regulation of host defense against infection, in the control of autoimmunity, and in anti-tumor responses. How they affect immunity to pathogens and regulate a vigorous effector response in vivo remains undefined. Here we provide evidence that Treg do not globally suppress adaptive immunity in response to a virus, but have a predominant and indeed, near exclusive effect on SLEC in physiological conditions of Treg activation during an emerging immune response. Only in artificial conditions of pre-activation of Tregs do we observe a modest impact on memory cell formation.
These observations of a very selective effect of Treg on one aspect of the evoked adaptive T cell response to infection raised the question of how such selectivity is achieved. Several factors have been shown to contribute to CD8+ Teff generation including IFNγ, IFNα/β, and IL-12 (17
). The latter two have been established to be crucial signal 3 elements with varying relative importance depending on the system analyzed. Absence of IFN α/β receptors or IL-12 receptor on T cells can lead to complete loss of functional CD8+ T cell priming (34
). So while these signals are important for generation of Teff, they also seem to be crucial for the generation of a robust memory response. A more compelling case can be made for a role of IL-2 availability in the effects we observe. Thus, in contrast to IL-12 signals that are required early after T cell priming (d1-3), IL-2 signals seem to be pivotal for optimal Teff generation particularly later during the response (>d3). Indeed, absence of IL-2 sensing due to the absence of CD25 leads to a dramatic reduction of SLEC during primary antigen encounter, but also impacts memory T cell responses (12
). The important conclusion that can be drawn from these latter studies is that low level IL-2 signaling early during the response is sufficient to drive full memory differentiation, while prolonged availability of IL-2 is required for Teff generation. Therefore, late changes in IL-2 availability are expected to impact on Teff generation while sparing the formation of immunological memory.
In agreement with previous research, we found a striking inhibitory influence of Treg on the production of IL-2 by antigen-activated CD8+ T cells and a capacity for late administration of long-lived IL-2 complexes to rescue the SLEC response in the presence of Treg. Therefore, we conclude that Treg limit IL-2 availability and thus specifically inhibit the size of the Teff pool while leaving just enough IL-2 to allow for Tmem generation. In vivo
experiments have shown that Treg are activated via IL-2 derived from primed Teff and that IL-2 seems to be dominantly acting in a paracrine fashion, demonstrating that IL-2 derived from Teff is in principle available to Treg (35
). This argues that besides restricting the production of IL-2, Treg might additionally restrict IL-2 availability by competing with Teff for this critical cytokine. In vitro
experiments and mathematical models derived from those experiments strongly argue that Treg via their high CD25 expression can effectively out-compete developing effector T cells for limited IL-2 in their environment (7
). As stated above, IL-2 is not the only contributor to Teff generation (17
) and therefore, it is likely that Treg act on Teff in additional ways, particularly those which affect Teff survival. While TGF-β did not seem to play a role in our viral infection model, Flavell and colleagues reported opposing roles of TGF-β and IL-15 in regulating survival of Teff in a bacterial infection model; the source of TGF-β was not analyzed, but could have been from Treg (30
). Finally, Treg-derived IL-10 that seems to directly act on CD8+ T cells () (28
) has a potential role in Treg mediated suppression. The relative contribution of these apparently auxiliary mechanisms may vary depending on the analyzed model. Yet, the common ground is that the primary target of Treg seems to be Teff rather than Tmem cells.
Using anti-CD25 antibodies to inhibit Treg function, previous studies came to the conclusion that Treg control the magnitude of both the primary and memory CD8+ T cell response. (39
). This is in contrast to our finding showing no change in memory responses after depletion of Treg using diphtheria toxin based depletion of Foxp3 positive cells (). There are several differences between our model and previous studies. First, antibody-mediated Treg depletion is not fully effective because not all Treg express CD25. Second, effects of anti-CD25 antibody treatment seem to be rather long-lasting compared to the transient depletion using diphtheria toxin-based mouse models. This latter point is of importance since it has been shown that anti-CD25 antibody treatment influences T cell contraction and memory CD8+ T cell homeostasis (40
). Third, anti-CD25 antibody treatment doesn’t actually cause depletion of Treg but rather functional inhibition by blocking IL-2 signaling (44
). Most importantly, the IL-2 receptor alpha chain (CD25) is not specific for Treg and is also found on activated CD8+ T-cells, CD4+ T cells and B cells. Given the central role of IL-2 for CD8+ T cell differentiation, anti-CD25 antibody treatment most likely has significant direct effects on CD8+ T cells beyond blocking Treg function. In a very recent study also using anti-CD25 treatment, the authors concluded that availability for IL-2 plays a central role in regulating the size of the CD8+ T cell response, yet a possible differential effect of Tregs on subpopulations of CD8+ T cells was not addressed (45
). That study found that anti-CD25 treatment increased the frequency of polyfunctional CD8 T cells and the “memory” response on d21. Careful elucidation of relative vs. absolute numbers of CD8+ T cell subpopulations as well as analysis of immune responses at later time-points (d60) was not investigated in that study.
It is very likely that the findings and underlying mechanism we describe here for CD8+ T cells similarly apply to CD4+ T cells. Indeed, Treg mediated inhibition of IL-2 production in CD4+ T cells in vivo
has been noted recently (46
). In our experiments we saw a similar impact on the size of antigen-specific CD4+ T cell responses on d8 after priming, again with a dominant effect on the magnitude of the CD4+ Teff response, when manipulating the Treg compartment (data not shown). Notably, recent evidence indicates opposing roles of IL-2 on the differentiation of various T helper cell subsets. IL-2 seems important for Th-1 cells, as in our study, yet inhibits generation of Th-17 cells (47
While these studies underline the general importance of IL-2 for Th-1 responses, our study demonstrates how Treg-controlled IL-2 signals regulate effector differentiation within such a response. The key platform upon which regulation takes place is the population of dendritic cells. They are the central interface linking innate and adaptive immunity and they also contribute to the control of autoimmunity. They integrate antigen presentation and inflammatory cues and transfer that information to CD4+ and CD8+ T cells via MHC/TCR interaction and costimulatory receptors. Therefore, it seemed likely that Treg would regulate an immune response by manipulating the DC. In vitro
experiments by Sakaguchi et al. showed that Treg are able to change CD80 and CD86 surface expression on DC and that this depended on CTLA-4 expression on Treg (49
). It was further shown that CTLA-4 expression on Treg and not Teff is crucial to prevent fatal autoimmunity (5
). In line with this we found that in vivo
, even under highly inflammatory conditions, Treg decrease costimulatory molecule expression by DCs, in particular CD86. This effect could be partially counter-acted by blocking CTLA-4 () but did not seem to require MHCII/TCR interactions between the affected DC and Treg (). Possibly, high-level expression of LFA-1 on Treg might be sufficient to engage DC to execute that function (Fig. S1
). In an elegant model, it was recently demonstrated that Treg also regulate the extent of CD80 and CD86 expression in the steady-state in vivo
, thereby controlling peripheral tolerance (51
). The central importance of CTLA-4 expression on Tregs and its function to reduce CD80/CD86 from DC via transendocytosis has recently been demonstrated in vivo
). Interactions between CD80/CD86 on DC and CD28 on T cells are important for effective IL-2 production by CD4+ and CD8+ T cells () (53
). In vitro
, Thornton et al. found a marked reduction of IL-2 mRNA transcripts in CD4+ CD25- when cocultured in the presence of Treg. The suppressive activity of Treg could be enhanced by blocking CD86 and was reversed after costimulation via CD28 (6
). Apart from IL-2, blocking of CD86 during MVA infection also resulted in diminished IFNγ production by OT-1 T cells (), which has been shown to contribute to the size of the Teff pool (54
). Other costimulatory molecules like CD70 seemed resistant to Treg suppression.
Our data thus point to two central components of Treg-mediated suppression: CTLA-4 that interferes with DC costimulation via reduced CD80/CD86 expression and CD25 to allow for Treg survival, activation, and effective competition for limited IL-2 during infection. Interestingly, studies concerning autoimmunity also identified these two pathways as being crucial components of Treg function (besides the central role of the transcription factor Foxp3). Based on these observations it has been proposed that IL-2 and CTLA-4 are core mediators of Treg suppression while other mechanisms might represent auxiliary means of Treg-mediated regulation or have varying importance depending on the conditions/infections or tissues being analyzed (55
). For example, Treg derived IL-10 is important to control inflammation at mucosal surfaces (2
) and the inflammatory pathology associated with IL-10 deficiency is largely restricted to the intestines and is eliminated in gnotobiotic mice lacking intestinal microbiota (29
Importantly, our work provides evidence that provision of IL-2 immune complexes can override Treg mediated suppression (). In a clinical setting administration of such complexes could be a more feasible approach to boost CD8+ T cell immunity than trying to deplete the Treg compartment as a whole with the risk of subsequent autoimmunity. It should be noted that IL-2 treatment was detrimental when applied during acute LCMV infection (56
). However, neither Treg depletion, using the DEREG mouse model (Punkosdy G personal communication), nor CTLA-4 blockade, has an effect on the CD8+ T cells response in that model (57
). Since LCMV induces a massive expansion of T cells and high-level IL-2 production during the course of infection, one may conclude that LCMV possibly exceeds the limits of Treg mediated suppression and that excess IL-2 further aggravates this condition. Consequently, the immune system would rely on other mechanism to control immoderate Teff responses (57
As a consequence of Treg primarily regulating Teff over Tmem, manipulation of the Treg compartment might be highly beneficial in therapeutic settings aiming at the efficient induction of effector T cells, such as cancer therapy (58
), but of little value for prophylactic vaccination, since numbers of polyfunctional T cells (which correlate with protective immunity) remained unaltered and antibody titers were not affected after Treg depletion (data not shown) (11
In summary, we have used a viral vaccine model to explore the role of Treg during anti-viral responses. Rather than broadly blunting the immune response, we find that Treg selectively limit the number of Teff generated while preserving the memory response. They do so by changing the amount of CD80 and CD86 displayed on DC and the availability of IL-2, which is required for the generation of short-lived effector cells. These results have important implications for vaccination and therapies against infectious diseases and cancer designed to target the Treg compartment and manipulate cell-mediated immunity for host benefit.