The mouse model of infection with lymphocytic choriomeningitis virus (LCMV) allows one to differentiate between the effects of acute and chronic viral infection on Treg activation. We compared the frequency and phenotype of Treg in C57BL/6 mice during the course of Armstrong and Clone 13 LCMV infection. Treg dramatically increased in frequency among splenic CD4+CD8- T cells during Clone 13, but not Armstrong, infection. A marked increase in the frequency of Foxp3+ Treg expressing TCR Vbeta5 was observed during Clone 13 infection. Because the expansion of the Treg in mice chronically infected with LCMV was restricted to a specific Vbeta segment, we also considered the possibility that a superantigen (Sag) may be driving proliferation. It appeared likely that expansion of the Vbeta5+ Treg was secondary to stimulation by an endogenous Sag such as mouse mammary tumor virus-(Mtv) encoded Sags. We infected BALB/c, that normally express Mtv-6, -8, and -9 and delete Vbeta-5, -11, and -12 T cells in the thymus, with Clone 13 and analyzed the effects of infection on the Vbeta repertoire. We observed a significant increase in the percentage of CD4+Foxp3+Vbeta5+ and a dramatic increase in Foxp3+Vbeta12+ Treg, but no change in the percentage of CD4+Foxp3-Vbeta5+ or Foxp3-Vbeta12+ T cells, following Clone 13 infection. When we infected BALB/c mice lacking all three of the endogenous Mtv proviruses (Mtv-null mice)which express Vbeta5 or Vbeta12 Foxp3+ T cells, we observed no change in the frequencies of either Vbeta5+ or Vbeta12+ Treg. Thus,the observed Vbeta-specific Treg expansion was due to stimulation by an endogenous retroviral superantigen. We have sought to identify the antigen presenting cell (APC) responsible for Sag-dependent Treg expansion. First, we asked whether LCMV infection resulted in increased expression of the endogenous Sag genes. At 8 days post infection, Mtv8 and/or Mtv9 Sag expression was up regulated 25-fold in clone 13 infected mice. Infection of B cell deficient mice resulted in normal Treg expansion similar to wild-type mice. However, Flt3L-/- mice (which are defective in several hematopoietic cell lineages, but primarily dendritic cells) infected with clone 13 displayed only a modest increase in frequency of Vbeta-specific Treg, suggesting that Sag presentation by dendritic cells was primarily responsible for the Treg expansion observed following infection. Because dendritic cells have the potential to produce a plethora of pro-inflammatory cytokines, we further hypothesized that mice unable to make individual pro-inflammatory cytokines may display a defect in Treg expansion following infection. To test this, we infected IL-1Ralpha-/-, IL-6-/-, IL-12 (p35)-/-, TNFalpha-/-, or IFNalpha-betaR-/- mice with clone 13 and analyzed the effect on Vbeta-specific Treg expansion. Only IFNalpa-betaR-/- mice infected with clone 13 showed a lack of Vbeta-specific Treg expansion following infection. We have also found that mice lacking MyD88, a molecule expressed by primarily by dendritic cells and important in innate immune activation, do not show Sag-mediated expansion of Treg following clone 13 infection. Because MyD88 is an essential signal transducer in the IL-1 and Toll-like receptor signaling pathways, we hypothesized that either of these pathways may be required for Sag-mediated Treg expansion. Surprisingly, mice lacking many of the receptors upstream of MyD88 signaling (TLR2, 4, 7, and 9, as well as IL-1 and IL-18) showed normal Sag-mediated expansion of Treg following infection. Recent reports have demonstrated MyD88 also functions in a T cell-intrinsic mechanism to control effector T cell activation following LCMV infection.We are currently exploring the possibility that T cell expression of MyD88 is required for Sag-mediated Treg expansion. Conditional knockouts lacking MyD88 expression in total T cells or only Treg will allow the differentiation between the effeects of MyD88 in the different cell populations. We believe that a complete understanding of the cell type (s) involved in regulation Sag expression and subsequent Treg expansion will provide major insights to the role of the Sag-expanded Tregs in potentially potentiating chronic viral infection. To further define the role of Treg in immune homeostasis in general and how disruption of Treg function might modulate the immune responses to viral infections, we have used diptheria toxin to deplete Treg from normal adult mice expressing the diptheria toxin receptor under control of the Foxp3 promoter. Treg depletion in adult mice rapidly results in an activated phenotype (CD44hi) on the vast majority of conventional (CD4+Foxp3-) T cells. Because CD44 expression is a characteristic of newly activated as well as memory phenotype (MP) T cells, we sought to determine whether the increase in CD44hi cells was the result of nave T cell activation or expansion of MP cells. In an adoptive transfer model, the activation of nave T cells was markedly enhanced by Treg depletion in an antigen-dependent manner, and was inhibited by anti-MHC class II mAbs. This mechanism appeared to play a minor role in the increase of CD44hi cells in non-transferred animals, as their expansion was completely unaffected by the administration of anti-MHC Class II. Thus, it is unlikely that they were generated from the nave pool. Consistent with this finding, the proliferative rate of CD44hi cells increased >3-fold during the 72-hour period following Treg depletion and was also not inhibited by blocking MHC Class II. This increase in proliferative rate was also observed in a model where true memory T cells could be assessed by MHC Class II tetramer staining following acute LCMV infection. Collectively, these results demonstrate that Treg control the size of the MP T cell pool in an antigen-independent manner by controlling their cytokine-driven proliferative rate. We have also begun to explore the potential role of Mtv infection on other aspects of immune responses and have focused our studies on NK T cells and innate CD8+ T cells. Normal mice possess a population of CD8+ T cells that share functional and phenotypic similarity to innate-like or memory-like T cells.Such innate like CD8+ T cells have increased expression of CD44, CD122, and Eomesodermin (Eomes), as well as the capability to rapidly produce interferon-gamma upon stimulation. The development of innate like CD8+ T cells was shown to be dependent on the production of IL-4 from NKT cells expressing the transcription factor promyelocytic leukemia zinc finger (PLZF). The number of PLZF+ cells is greatly increased within the thymus of BALB/c compared to C57Bl/6. The fact that BALB/c express I-E, while C57Bl/6 do not,suggested that a SAg-mediated process may influence the generation of this innate-like CD8+ T cell population. CD8+ T cells in thymi of Mtv-null BALB/c mice had greatly reduced expression of CD44, CD122, CXCR3, and Eomes,and were deficient in IFN-gamma production. The loss of this innate-like CD8+ T cell population in Mtv-null mice correlated with a reduction of PLZF+, IL-4 secreting NKT cells within the thymus compared to BALB/c wilkd type mice. Our findings suggest a role for Mtv SAg in modulating the expression of PLZF among NKT cells and thereby indirectly influencing the presence of innate-like CD8+ T cells.
