A number of studies examining this concept, demonstrated the contribution of Tregs in promoting suppression of pathogen specific effector responses [
7‐
10,
45‐
48], while others showed beneficial effects of Tregs upon infections [
12,
49‐
52]. In this study, we set out to primarily examine the role of Tregs in modulating the MCMV-specific NK cell responses during the acute phase of infection, which until now remained incompletely defined. We observed elevated Treg frequencies among CD4
+ T cells in the spleen early upon infection, indicating that MCMV infection may preferentially support differentiation of naïve T cells into Tregs, similarly described in a hepatitis virus infection model [
53] where TGF-β induced by infection controlled this phenotypic change. To specifically address the question of whether the increase in Tregs influences the ongoing activation of innate and adaptive immune responses, we utilized DEREG mice to facilitate specific Treg depletion by Diphtheria toxin (DT) administration [
5]. The advantage of the rapid and efficient depletion of Tregs in our model provided us the opportunity to infect mice on the day of first DT injection to really assess the influence of Tregs on NK cells during virus replication and thus minimize effects occurring prior to the onset of infection. This fact could account for the contrasting results Sungur et al. reported in terms of enhanced viral clearance upon CD25 antibody-mediated Treg depletion starting 2 days before infection [
54]. In respect to these findings, we observed that under homeostatic conditions, depletion of Tregs significantly increased NK cell numbers and NK cell CD69 expression. Thus, depletion prior to infection could contribute to this discrepancy between both studies by conferring enhanced anti-viral defense already before infection. As Tregs return to baseline levels by day 7 p.i. in uninfected mice, our experimental mouse model avoids the development of artificial autoimmunity [
55] and hence provides an unbiased approach to examine the phenotypes observed here upon infection. To further elucidate the interaction of Tregs with NK cells, and its influence on control of MCMV replication in C57BL/6 mice, we examined NK cell numbers and activation in the absence of Tregs. We detected elevated NK cell frequencies in uninfected DEREG mice depleted of Tregs consistent with findings reported in Scurfy mice and FoxP3 DTR knock-in mice [
28,
32]. These cells additionally showed notably higher CD69 expression. In contrast, upon infection, we observed comparable NK cell responses between Treg-depleted and non-depleted mice. Studies by Fulton et al. and Lee et al. reported concordantly increased NK cell numbers in the lungs of Respiratory Syncytial Virus infected-BALB/c mice upon Treg depletion, which was carried out again by CD25 antibody administration starting already 3 days prior to infection [
56,
57]. Using uninfected FoxP3 DTR knock-in mice, Gasteiger et al. pointed out that the increase of NK cell numbers upon Treg depletion corresponds to elevated CD127
+ NK cell frequencies, expressing higher amounts of high affinity IL-2 receptor CD25 [
31]. Therefore, enhanced IL-2 production by effector CD4
+ T cells in the absence of Tregs may represent the likely mechanism underlying this phenomenon. This hypothesis was further substantiated by experiments that showed abrogation of this effect by blocking the IL-2 pathway or depleting the CD4
+ T cell compartment [
32] and similarly reported by Sitrin et al. in an autoimmune diabetes mouse model [
58]. Our results in uninfected mice corroborate these findings as we similarly detected higher activation of CD4
+ T cells upon Treg depletion. Although we observed a boosted CD4
+ as well as CD8
+ T cell response in DEREG MCMV + DT-treated mice as compared with WT MCMV + DT-treated mice, we could not detect differences in NK cell frequencies in infected mice suggesting that this proposed mechanism will need further clarification under a more infectious setting such as a salivary gland infection, where the demand for Ly49H
+ NK cells would be further exemplified. A possible reason for this discrepancy could be that NK cells already achieve maximal proliferation upon tissue cultured MCMV infection and thus, fail to benefit from Treg depletion or elevated IL-2 levels. Treg depletion in MCMV-infected mice leads to higher proliferation of effector T cells, predominantly CD8
+ T cells which represent the majority of T cells at day 7 p.i. Thus, consumption of IL-2 by proliferating CD8
+ T cells which is not seen upon Treg depletion under homeostatic conditions, may offer another potential explanation. Treg ablation leads to similar frequencies of CD62
low CD4
+ T cells as compared with those induced by MCMV infection alone. However, CD8
+ T cells are significantly more activated upon MCMV infection than upon Treg depletion of naive mice and thus could abrogate IL-2 mediated effects. The insensitivity of the viral clearance to a boosted T cell response highlighted the importance of NK cells in limiting WT MCMV replication in C57BL/6 mice emphasized by a rapid clearance until day 7. The implications of Treg control over effector CD8
+ T cell response would prove critical if the Ly49H receptor engagement was somehow abrogated as observed in the case of mice challenged with Δ
m157-strain of MCMV, where CD8
+ effector T cells critically governed the outcome of viral replication in infected organs [
42]. In Ly49H
+ NK cell competent C57BL/6 mice, we observed an initial viral burden that was already 100-fold reduced and close to the detection limit when the T cell response peaked. Our findings provide further support to the multi-functional importance of NK cells spanning the innate and adaptive arms of the immune system [
59‐
61]. Furthermore, since MCMV infection primarily induces stronger CD8
+ T cell responses, the contribution of the enhanced CD4
+ T cell activation we observe upon infection in Treg depleted mice would require further investigation. CD4
+ T cells are key players in establishing immunological memory and moreover known to develop cytotoxic abilities to directly attack infected cells under certain circumstances [
62‐
64]. This makes them an important factor during MCMV infection and their importance may be further enhanced upon their suppression by Tregs. Thus, our results provide new evidence that Tregs play a role in modulating the immune response to MCMV infection, but this effect seems to be restricted to the suppression of adaptive immune cell activation. Our results suggest that Tregs enhance the general effector T cell response while NK cell function remains unaltered. This expansion in the CD8 T cell pool would warrant for further investigation into the contribution of Treg depletion on the antigen-specific effector T cell compartment after infection. The importance of Treg regulation on CD8 T cells in the absence of Ly49H-NK cell recognition has been recently analyzed in an independent study described by us in collaboration with Hansen and colleagues, showing enhanced activation, cytotoxicity and improved viral clearance in DEREG Balb/c mice depleted of Tregs [
65]. Thereby, suggesting an important regulatory role by which NK-Ly49H function in concert with Tregs modulate anti-MCMV T cell effector responses [
65]. This could be further extended into infection models in C57BL/6 mice employing a Δm157 MCMV strain, where the requirement for antigen specific T cells in viral clearance is further exemplified. Overall, our findings provide a foundation for the development of future Treg-mediated therapeutics in viral infections and in a broader context, in Treg-modulating strategies to overcome transplant rejection.