Role of T Regulatory Cells in Chronic Infection

National Institute Of Allergy And Infectious Diseases

Investigators

Abstract

We have identified a subpopulation of Treg that rapidly and specifically expand early during chronic (strain Clone 13) infection. At 17 days post infection (DPI), the total number of Vbeta5+ Treg is comparable to the total number of Foxp3+ Treg in a nave mouse. CD4+Vbeta5+ cells do not increase in frequency among Foxp3- cells, suggesting that this response is Treg-specific. A higher percentage of Vbeta5+ Treg express the activation markers CD101 and CD103, but lower levels of the co-inhibitory receptors ICOS and PD-1 compared to Vbeta5- Treg. Expansion of Vbeta5+ Treg is solely related to chronic LCMV infection and not to a difference between the strains of virus that result in acute (Armstrong) and chronic infection. We have examined a number of different sources for the expanded Treg: 1) CD4+Vbeta5+ T cells may escape negative selection in the thymus during the infection and differentiate into Treg. However, the Treg response in adult thymectomized mice infected with LCMV Clone 13 appeared completely normal both in terms Vbeta5 expansion among Foxp3+ cells. 2) Treg differentiation can occur in the peripheral sites in response to TCR stimulation and TGF-beta. To test this hypothesis, we transferred Foxp3- cells into nave congenic C57/BL6 mice one day before LCMV Clone 13 infection. Following infection, endogenous and transferred CD4+ T cells were analyzed to determine the percentage of cells expressing Foxp3. Among the endogenous CD4+ T cells, the percentage of cells expressing Foxp3 increased similar to our previous results. However, transferred CD4+ cells did not express any appreciable amount of Foxp3, suggesting that Treg are not generated de novo following Clone 13 infection. 3) Vbeta5+ Treg may preferentially expand following Clone 13 infection. To test this, we injected bromodeoxyuridine (BrdU) into Clone 13 infected mice 7 days post infection (DPI) and analyzed the percentages of CD4+ T cells incorporating BrdU and expressing the cell cycle marker, Ki-67, 24 hours later. CD4+Foxp3+Vbeta5+ cells had the highest percentage of BrdU+, Ki-67+ cells compared to all other CD4+ T cell populations, suggesting that, in fact, the observed expansion is due to an increased rate of cell division of the endogenous Vbeta5+ Tregs. However, when we injected Foxp3+ cells from congenic mice one day before Clone 13 infection, the percentage of the transferred cells expressing Vbeta5 on DPI 15 did not increase, while expansion of the endogenous Treg occurred normally. It remains possible that that Treg expansion may occur in a microenvironment that is not easily accessible to the transferred Treg. We are currently focusing on the gut as the site of Treg activation and expansion. In preliminary studies, mesenteric lymph node and lamina propria T cells of the small intestine had significantly higher percentages of Foxp3+V&#946;5+ Treg than any other lymphoid tissue examined. We are currently focusing on determining how the microenvironment of the gut may influence Treg expansion in chronic infection.[unreadable] [unreadable] It is likely that the expansion of the Treg is driven by T cell receptor driven stimulation, but the specificity of antigen recognition by Treg remains a controversial issue. Treg have been shown to recognize both autoantigens as well as pathogen-derived antigens. To determine whether the Treg specifically recognize an LCMV-derived antigen, we developed an in vitro proliferation assay in which we stimulated Treg with overlapping peptides that span the entire LCMV proteome. We have assayed 224 peptides that span the entire glycoprotein, nucleocapsid and Z protein, but have not observed a Treg response to an LCMV peptide. In addition, Treg from chronically infected mice (both Vbeta5+ and Vbeta5-) do not bind an immunodominant LCMV peptide-MHC class II tetramer Taken together, these data suggest that the expanded Treg in mice chronically infected with LCMV are not specific to a viral peptide epitope.Because the expansion of the Treg is restricted to a specific Vbeta segment, it is possible that activation of these cells is secondary to recognition a superantigen that binds outside the peptide/MHC binding region. We have sequenced the complementarity-determining region 3 (CDR3) of the TCR from multiple clones derived from Vbeta5+ Treg and observed that the profile is quite polyclonal. Sequence data from three individual mice did not reveal any public clones that were shared among any of the animals and <25% of the sequences from any individual mouse were conserved. We have shown that the expansion of Vbeta5+ Treg in Clone 13 infection is CD4-independent, but MHC class II- dependent. Lastly, the expansion of Vbeta5+ Treg is conserved among a few different inbred strains of mice with different H-2 haplotypes. For example, in BALB/c mice, Vbeta5+ Treg (in addition to Vbeta12+) specifically expand during Clone 13 infection. Taken together, these studies strongly suggest that the expansion of the Treg is superantigen driven. Our main goal over the next year is to determine the source of the superantigen. We are focusing on the shared endogenous retroviral superantigens and/or superantigens that may be present in the gut flora. We are in the process of generating mice with a greatly reduced gut flora and will infect these mice with clone 13 and determine whether expansion of the Treg is inhibited. We believe these studies have potential relevance to other chronic infections such as HIV that also involve infection of the GI tract.[unreadable] [unreadable] The major issue to be addressed in the next year is to determine the impact of the Treg expansion on the course and outcome of the chronic infection. We have had difficulties in the past in the execution of these studies because of the limited tools that can be used to deplete Treg from mice either before or during the course of the chronic infection. We have recently obtained mice that express the human diphtheria toxin receptor under control of the mouse Foxp3 promoter. Treatment of these mice with diptheria toxin results in a profound, but transient, depletion of Foxp3+ Treg. In preliminary studies, depletion of Treg prior to Clone 13 infection resulted in an enhancement of the activation of CD4+ T cells. As CD4+ T cells modulate the level of CD8 cell exhaustion during chronic infection, we also plan to use this depletion model to help tease out the role that Treg may be playing in this process. One possibility is that Treg modulate viral antigen presentation by DC and thereby inhibit the activation of virus-specific CD4+ cells.