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We have examined the formation, participation and functional specialization of virus-reactive Foxp3+ regulatory T cells (Tregs) in a mouse model of influenza virus infection. “Natural” Tregs generated intra-thymically based on interactions with a self-peptide proliferated in response to a homologous viral antigen in the lungs, and to a lesser extent in the lung-draining mediastinal LN (medLN), of virus-infected mice. By contrast, conventional CD4+ T cells with identical TCR specificity underwent little or no conversion to become “adaptive” Tregs. The virus-reactive Tregs in the medLN and the lungs of infected mice upregulated a variety of molecules associated with Treg activation, and also acquired expression of molecules (T-bet, Blimp-1 and IL-10) that confer functional specialization to Tregs. Notably, however, the phenotypes of the T-bet+ Tregs obtained from these sites were distinct, since Tregs isolated from the lungs expressed significantly higher levels of T-bet, Blimp-1 and IL-10 than did Tregs from the medLN. Adoptive transfer of antigen-reactive Tregs led to decreased proliferation of anti-viral CD4+ and CD8+ effector T cells in the lungs of infected hosts, while depletion of Tregs had a reciprocal effect. These studies demonstrate that thymically-generated Tregs can become activated by a pathogen-derived peptide and acquire discrete T-bet+ Treg phenotypes while participating in and modulating an antiviral immune response.
Foxp3+ regulatory T cells (Tregs) are a subset of CD4+ T cells with a unique ability to exert dominant suppression of adaptive immune responses (1, 2). The clearest manifestation of their activity in vivo is the severe lymphoproliferative inflammatory disease that develops in mice and humans that lack Foxp3 expression, and because they are required to control a latent auto-aggression that exists in the normal immune repertoire, much attention has focused on the ability of Foxp3+ Tregs to control immune responses to self-antigens (3). However, Tregs also participate in immune responses to pathogens, where they can modulate how the immune system reacts to the pathogen itself, and may also play a role in limiting immune-mediated damage to the infected host’s own cells and tissues (4).
There are presently thought to be two main sources of Foxp3+ Tregs that can participate in anti-pathogen immune responses (5). Thymically-generated Foxp3+ Tregs (termed “natural” Tregs) appear to comprise the bulk of the peripheral Tregs that are present in naïve mice, and are generated based on their specificity for self-peptides (6, 7). This bias toward self-reactivity may play an important role in directing the activity of Tregs toward tissue-specific antigens in the periphery, and it may allow Tregs to recognize self-peptides expressed by cells in the infected site. It is also possible, however, that Tregs that were formed in response to self-peptides can become activated by recognizing virus-derived peptides with which they can crossreact. A second possible source of Tregs at infection sites could be “adaptive” Foxp3+ Tregs that can develop from conventional CD4+ T cells in response to signals such as TGF-β and retinoic acid (8, 9). Inasmuch as CD4+ T cells with identical TCR specificity can be induced to become either adaptive Tregs or differentiated cytokine-secreting effector cells (e.g. Th1 cells) in response to different cytokines (e.g. TGF-β vs. IL-12), it has been thought that the formation of adaptive Tregs from conventional CD4+ T cells may be a typical source of Foxp3+ T cells during immune responses (10). However, the extent to which this process actually occurs during infections remains poorly understood, and in one infectious setting appeared not to occur (11).
Recently, it has become apparent that Foxp3+ Tregs can themselves differentiate to acquire new properties and phenotypes during the course of an immune response (12). This process has been termed “functional specialization”, and interestingly, transcription factors that have been shown to play important roles in promoting effector T cell differentiation appear to be utilized by Foxp3+ Tregs to acquire phenotypes that are specialized to control the corresponding effector T cell function. For example, T-bet plays a major role in promoting the development of a Th1 effector phenotype during an infection and directly influences the production of IFN-γ by both CD4+ and CD8+ T cells (13, 14). Foxp3+ Tregs have been shown to respond to IFN-γ by upregulating T-bet, which in this case induces expression of a homing receptor (CXCR3) and a cytokine (IL-10) that confer on these T-bet+ Tregs the ability to migrate to sites of Th1-mediated inflammation and inhibit Th1 effector cell activity (15). Similarly, mice in which Foxp3+ Tregs selectively lack expression of transcription factors associated with the development of Th2 or Th17 effector cell phenotypes spontaneously develop diseases associated with the corresponding Th2 or Th17 cell hyperactivity (16, 17). The extent to which Foxp3+ Tregs typically undergo this kind of functional specialization during an infection is not currently understood, however, and whether Tregs might be able to adopt distinct phenotypes while responding to a pathogen has not been described.
We have examined these questions in a mouse model of influenza virus infection using transgenic mice with which the role that TCR specificity for self- and/or viral peptides in Foxp3+ Treg cell formation can be addressed, and with which the fate of virus-activated Tregs in infected mice can be followed. We show that Foxp3+ Tregs that were generated intrathymically in response to a self-peptide derived from the influenza virus hemagglutinin (HA) proliferate and differentiate into T-bet+ Tregs in response to the viral HA in influenza virus-infected mice. Moreover, the phenotypes that these T-bet+ Tregs display in the infected lungs versus the lung-draining medLN are distinct, providing evidence for the formation of discrete subsets of virus-reactive T-bet+ Tregs in an infected host.
TS1 and HA28 mice have previously been described and are on a BALB/c background (7). BALB/c.Foxp3eGFP mice (18) were obtained from Jackson Laboratories. Mice were intermated to produce TS1.Foxp3eGFP and TS1xHA28.Foxp3eGFP mice. All mice were maintained in specific pathogen-free facilities under protocols approved by the Wistar IACUC.
Influenza virus PR8 (A/Puerto Rico/1934 (H1N1)) and the reassortant influenza virus J1 (H3N1) (19) were propagated in 10-d hen’s embryonated eggs. For infection mice were anesthetized with ketamine/xylazine and 50 μl (200 TCID50) of virus was administered intranasally (i.n.).
6.5+CD4+CD25+Foxp3eGFP+ cells or 6.5+CD4+CD25−Foxp3eGFP− cells were obtained from freshly isolated LN and spleens from TS1xHA28.Foxp3eGFP and TS1.Foxp3eGFP mice, respectively, by staining with anti-CD4, anti-CD25 and 6.5-biotin (20). GFP fluorescence was also used for detection of Foxp3+ cells and cells were FACS-sorted using a high-speed MoFlo XDP cell sorter (Beckman Coulter) and were adoptively transferred into mice (106 cells in 100 μl/mouse) by tail vein injection.
Spleen, pLN, medLN, lung and BAL cells were collected from infected mice by tissue homogenization followed by resuspension in FACS buffer (PBS, 2% fetal bovine serum). Lungs were first perfused by injecting 3 ml PBS in the right ventricle of the heart, and lung tissue was incubated in complete DMEM media supplemented with collagenase D (700 units/lung, Roche Diagnostics) and DNase I (350 units/lung, Roche Diagnostic) for 1 h at 37°C before generation of a single cell suspension. A combination of PMA (50 ug/ml), Ionomycin (1 uM) and Brefeldin A (1X; eBioscience) was used for ex vivo stimulation of cells for 5 h at 37°C. Intracellular cytokine staining was performed according to the instructions in the eBioscience kit and all antibodies were purchased from BD Biosciences or eBioscience. APC-conjugated H2-Kd:NP147 tetramer was directly added to the primary antibody dilution and cells were incubated for 30 min at 4°C. Stained cell samples were analyzed using a LSRII flow cytometer (BD Bioscience) and collected data analyzed with FlowJo software (Treestar).
Mice were injected i.p. with 0.5 mg of anti-CD25 mAb (PC-61) or control rat IgG antibodies in sterile PBS solution 3 d before and 2 d after PR8 virus infection. Organs and tissues were processed at 8 d p.i., and cells were analyzed by FACS.
Lungs of PC-61-treated PR8-infected mice and PR8-infected mice that had or had not received 6.5+ Treg cells were isolated on d 8 p.i., perfused with 3 ml of PBS and fixed in a 4% paraformaldehyde:PBS solution. Fixed lungs were embedded in paraffin and sectioned using a Reichert-Jung 2065 rotary microtome at a width of 7 μm. Sections were stained with hematoxylin and eosin, and samples were photographed using a Nikon E600 inverted bright-field microscope. Analysis of leukocyte-infiltrated areas of lung sections was performed using Image Pro Plus software (Media Cybernetics Inc.).
RNA was isolated using the Arcturus® PicoPure® RNA Isolation Kit (Applied Biosystems) and cDNA was synthesized with the TaqMan® Gene Expression Master Mix (Applied Biosystems). Quantitative real-time PCR for detection of T-bet (Tbx21), IL-10, Blimp-1 (Prdm1), IL-27Rα, IFN-γ, IL-27, IRF4, GATA3, Bcl-6 and ROR-γT (Rorc) mRNA was performed with the 7500 Fast Real-Time PCR System thermal cycler (Applied Biosystems) and the TaqMan® Gene Expression Assays with validated primers (Applied Biosystems).
Serum samples were analyzed using the Milliplex Cytokine-Bead Array Kit (Millipore) according to manufacturer’s directions by the Human Immunology Core at the University of Pennsylvania.
Lungs were obtained from uninfected and PR8-infected mice at 3, 5 and 8 d p.i., homogenized, and 50 μl of supernatant were added in serial dilution to MDCK cells. The cells were then incubated in 0.1% BSA containing trypsin for 2 d at 37°C and supernatant from cultures was assayed for virus based on hemagglutination.
Unpaired two-tailed Student’s t test was performed when two independent groups within one set of samples were analyzed. The Mann-Whitney U test was performed for lung viral titers analysis for which non-parametric distribution was assumed. Comparisons between groups within the same datasets were analyzed using one-way ANOVA test followed by either Dunnett’s or Bonferroni’s post-test for multiple comparisons.
To investigate how Tregs participate in an acute influenza virus infection, BALB/c mice were infected with PR8 virus and the representation of CD4+CD25+Foxp3+ cells in the lungs, bronchoalveolar lavage (BAL), medLN, spleen, and peripheral, non-lung draining peripheral LN (pLN) was determined by flow cytometry. Five days post-infection (p.i.) the number of CD4+CD25+Foxp3+ cells in the medLN had increased ~100-fold relative to uninfected mice (Fig. 1A, 1B). The numbers of CD4+CD25+Foxp3+ cells in the medLN, lungs and BAL were also significantly higher in infected than in uninfected mice at 5, 8 and 12 d p.i., although in each case there was a decline in these numbers between d 8 and 12 p.i. (Fig. 1B). There were also substantial increases in the frequencies of CD4+CD25+Foxp3− cells in the lungs and BAL of infected mice, most likely reflecting the mobilization of CD4+ effector cells to the site of infection (21, 22) (Fig. 1A). Conversely, the number of CD4+CD25+Foxp3+ cells in pLN decreased during infection (Fig. 1B). Collectively, these data indicate that CD4+CD25+Foxp3+ T cells accumulate in the lungs, airspaces and medLN of PR8-infected mice.
CD4+Foxp3+ Tregs can be generated intrathymically based on interactions between developing thymocytes and self-peptides, or by conversion from conventional CD4+ T cells in response to peptide stimulation and/or cytokines such as TGF-β (23). To determine how these different pathways contribute to Treg participation during influenza virus infection, we seeded naïve BALB/c mice either with thymically-generated CD4+Foxp3+ cells expressing a PR8 HA-specific TCR, or with conventional CD4+Foxp3− cells expressing the same TCR, and analyzed their response to PR8 virus infection. As a source of thymically-derived virus-reactive Tregs we used 6.5+CD4+CD25+eGFP+ cells from TS1xHA28.Foxp3eGFP mice; these mice express the PR8 HA-specific TS1 TCR (which was obtained from a PR8-infected BALB/c mouse, and which can be detected with the anti-clonotypic mAb 6.5) (20), they co-express the PR8 HA as a self-peptide (which induces the formation of thymically-generated HA-specific 6.5+CD4+CD25+Foxp3+ Tregs)(7, 24), and they also co-express a Foxp3eGFP reporter allele (which allows purification of Foxp3+ cells based on eGFP expression) (25) (Fig. 2A). At 8 d p.i., more than 10% of the CD4+ T cells in the lungs and BAL of mice that had received virus-reactive Tregs were 6.5+eGFP+ cells, indicating that they had been derived from the donor cell population (Fig. 2B). Lower percentages of these cells were also found in the medLN, pLN and spleens of infected mice, while no such cells were detected in the lungs of mice that had received virus-reactive Tregs but were not PR8-infected. Notably, very few of the 6.5+CD4+ cells in recipient mice were eGFP−, indicating that there was little loss of Foxp3 expression by donor-derived virus-reactive Tregs. To establish that Tregs that were generated intrathymically can respond to the viral peptide, we also transferred 6.5+CD4+CD8−eGFP+ thymocytes from TS1xHA28.Foxp3eGFP mice into BALB/c mice, and at 8 d p.i., the accumulation of 6.5+CD4+eGFP+ cells in the lungs and BAL was similar to mice that had received 6.5+CD4+CD25+eGFP+ LN cells (Fig. 2C). Thus, Tregs expressing the TS1 TCR and that were generated in response to the HA as a self-peptide can accumulate in the lungs and medLN of PR8-infected mice.
To determine whether conventional CD4+ T cells expressing the TS1 TCR can undergo conversion to become Foxp3+ Tregs in response to influenza virus infection, we transferred 6.5+CD4+eGFP− cells from TS1.Foxp3eGFP mice into BALB/c mice, and again infected the mice with PR8 virus (Fig. 2A). Infected mice that had received 6.5+CD4+eGFP− cells from TS1.Foxp3eGFP mice contained large percentages of donor-derived cells in the lungs and BAL (in this case, more than 50% of the CD4+ cells were donor-derived), and lower percentages were found in the medLN, pLN, and spleens (Fig. 2B). Significantly, however, very few of the donor-derived 6.5+CD4+ cells in the infected mice were eGFP+, indicating that little or no up-regulation of Foxp3 expression occurred among conventional 6.5+CD4+ T cells in PR8-infected mice. Altogether, these results demonstrate that Tregs that were generated intrathymically in response to a self-peptide can accumulate in the lungs and medLN in response to influenza virus infection, and that conventional CD4+ T cells with identical specificity for the viral antigen undergo little or no conversion into CD4+Foxp3+ Tregs.
To assess whether Tregs were accumulating in the lungs and medLN due to specific recognition of the HA viral antigen, or more generally in response to inflammatory signals, we transferred 6.5+CD4+Foxp3+ Tregs into BALB/c mice, and infected the recipient mice either with PR8 virus, or with a reassortant virus (J1) containing an antigenically distinct H3 subtype HA molecule that lacks the S1 peptide recognized by the 6.5 TCR (19). Unlike mice that were PR8-infected, there was no accumulation of 6.5+CD4+Foxp3+ T cells in the medLN, lungs or BAL of J1-infected mice 8 d p.i. (Fig. 2D). To determine whether 6.5+CD4+Foxp3+ Tregs actively proliferate in response to PR8 infection, we examined levels of the cellular division marker protein Ki-67. More than a quarter of the CD4+Foxp3+ cells in the medLN, and more than half of CD4+Foxp3+ cells in the lungs of PR8-infected BALB/c mice were Ki-67+ (Fig. 2E). Moreover, in mice that had received 6.5+CD4+Foxp3+ T cells from TS1xHA28 mice, the large majority of 6.5+ cells in the medLN, and roughly half of those in the lungs, were Ki-67+. This increase in Treg division reflected activation by the viral antigen, as only a small fraction of the 6.5+CD4+Foxp3+ T cells isolated from the lungs of uninfected donor TS1xHA28 mice were Ki-67+ (Fig. 2E).
The virus-reactive 6.5+ Tregs localizing to the lungs and medLN also exhibited higher levels of expression of CTLA-4, GITR, CD44, CD69 and ICOS than “conventional” CD4+Foxp3− T cells in both the medLN and the lungs, and these higher expression levels were again reflective of activation by viral antigen because they were also elevated relative to input 6.5+CD4+Foxp3+ cells from TS1xHA28 mice (Fig. S1). The non-6.5-expressing endogenous CD4+Foxp3+ cells in the lungs and medLN also included cells expressing higher levels of these molecules than were expressed by CD4+Foxp3− “conventional” CD4+ T cells, although only a subset of the cells expressed levels equivalent to those expressed by 6.5+CD4+Foxp3+ T cells, and for CD69 and ICOS, upregulation occurred predominantly in the lungs (Fig. 2F). Collectively, these data demonstrate that thymically-derived virus-reactive Tregs can accumulate, proliferate and acquire activation-associated phenotypes in the medLN and lungs of influenza-infected mice in response to the recognition of viral peptide.
To understand how virus-reactive Tregs can modulate the immune response to influenza virus infection, we analyzed the accumulation of effector CD4+ and CD8+ T cells in the lungs and medLN of mice that had or had not received Tregs from TS1xHA28 mice. The introduction of Tregs from TS1xHA28 mice led to significant decreases in the numbers of CD4+ and CD8+ T cells in the lungs of infected mice, although notably, accumulation in the medLN was not affected (Fig. 3A). The frequencies and numbers of viral antigen-specific CD8+ T cells detected with a H2-Kd:NP147+ tetramer were similarly decreased in the lungs, but not in the medLN (Fig. 3B). The decreased accumulation of effector CD4+ and CD8+ T cells had an apparent effect on virus replication, since virus titers in the lungs were significantly increased at 5 dpi in the presence of virus-reactive Tregs, although virus titers at days 3 and 8 were unaffected (Fig. 3C).
We also analyzed the effect of depleting Tregs on the numbers of CD4+ and CD8+ T cells that accumulated in the lungs of infected mice. Treatment of BALB/c mice with the anti-CD25 mAb PC-61 resulted in a decreased frequency of CD4+Foxp3+ cells (Fig. S2), and this reduction in Tregs was accompanied by a significantly higher accumulation of effector CD4+ and CD8+ T cells in the lungs, and a modest (but not significant) increase of these cells in the medLN of infected mice (Fig. 3D). Depletion of CD4+Foxp3+ cells also led to a significant increase in inflammatory processes involving bronchioles and alveoli in the lungs of infected mice, whereas the presence of adoptively transferred virus-reactive Tregs reduced the accumulation of inflammatory leukocytes at these sites (Fig. 3E). Together, these data show that virus-reactive Tregs can control the number of effector cells at the infected site during influenza virus infection. Moreover, since increasing the frequency of virus-reactive Tregs could suppress the accumulation of effector T cells, while depleting Tregs exerted the opposite effect, they strongly suggest that the immune repertoires of otherwise unmanipulated BALB/c mice contain Tregs that, like the Tregs from TS1xHA28 mice, can limit the recruitment of effector CD4+ and CD8+ T cells to the lungs of virus-infected mice.
IL-10 has been shown to be an important cytokine for the activity of Tregs at environmental interfaces such as the lung (26). Likewise, IL-10 has been shown to be crucial to counter an excessive pro-inflammatory response during influenza infection (27). Therefore, we examined the ability of the adoptively transferred 6.5+CD4+Foxp3+ cells, and of the endogenous 6.5−CD4+Foxp3+, CD4+Foxp3− and CD8+ cells to produce IL-10 and/or IFN-γ in PR8-infected mice. While very little or no cytokine production was detected in CD4+ or CD8+ T cells from the lungs of uninfected mice (data not shown), >25% of the CD4+Foxp3− cells, >35% of the CD4+Foxp3+ cells, and >20% of the CD8+ cells from lungs of PR8-infected BALB/c mice 8 d p.i. with PR8 virus could secrete either one or both of these cytokines (Fig. 4A, top panel). Notably, the majority of the 6.5+CD4+Foxp3+ cells accumulating in the lungs became IL-10-producers, and, in the presence of these IL-10-secreting virus-reactive Tregs, there were significant reductions in the numbers of CD4+Foxp3− and CD8+ effector T cells that produced either IFN-γ alone or both IFN-γ and IL-10 (Fig. 4A, bottom panel). Similarly, although the percentages of IL-10+ CD4+Foxp3− and IL-10+ CD4+Foxp3+ cells increased, their total cell number was reduced in the lungs of mice that had received 6.5+CD4+Foxp3+ cells. The 6.5−CD4+Foxp3+ cells in the lungs of PR8-infected mice that had not received Tregs from TS1xHA28 mice also secreted IL-10, and although the percentage that secreted IL-10 increased slightly in the presence of 6.5+CD4+Foxp3+ T cells, their number decreased, likely due to displacement by the transferred Tregs (Fig. 4A).
Despite the large increase in the frequency of IL-10-secreting 6.5+CD4+Foxp3+ cells in the lungs, there was little or no IL-10 secretion by 6.5+CD4+Foxp3+ cells isolated from the medLN of infected mice that had received Tregs. There was also very little IL-10 production by either endogenous 6.5−CD4+Foxp3+ Tregs, or by CD4+Foxp3− effector T cells in the medLN of infected mice, irrespective of whether they had or had not received Tregs from TS1xHA28 mice. Both CD4+Foxp3− and CD8+ effector cells from the medLN could, however, secrete IFN-γ, and the numbers of these cells were significantly lower in the medLN of mice that had received Tregs (Fig. 4B). The levels of a variety of serum cytokines, including IFN-γ, were also significantly lower, while levels of IL-10 were significantly higher in the serum of PR8-infected mice that had received Tregs from TS1xHA28 mice (Fig. 4C). Altogether, these data indicate that thymically-derived Tregs can become IL-10-competent by responding to a viral antigen in PR8-infected mice, and that the presence of a high frequency of these cells can lead to decreased frequencies of IFN-γ secreting CD4+ and CD8+ effector cells in the lungs and medLN, in addition to altering the systemic cytokine response to virus infection.
Tregs have been shown to be able to adopt transcriptional and functional programs that confer specificity to their ability to regulate effector T cells responses (12). Indeed, the ability to secrete IL-10 and suppress IFN-γ production is a hallmark of T-bet+ Tregs, and we therefore examined whether the virus-reactive Tregs in PR8-infected mice had acquired this transcriptional program. 6.5+CD4+Foxp3+ cells isolated from the lungs of infected mice expressed high levels of CXCR3 and T-bet, in addition to IL-10, indicative of their differentiation into T-bet+ Tregs (Fig. 5A). Interestingly, while the levels of IL-10 and T-bet in 6.5+CD4+Foxp3+ cells in the medLN of infected mice were significantly upregulated relative to those found in donor 6.5+CD4+Foxp3+ cells from TS1xHA28 mice they were in each case significantly lower than in 6.5+CD4+Foxp3+ cells isolated from the lungs of infected mice. By contrast, CXCR3 levels were highest in 6.5+CD4+Foxp3+ cells isolated from the medLN of infected mice, and while lower levels were found in 6.5+CD4+Foxp3+ cells isolated from the lungs, CXCR3 was nevertheless clearly upregulated in both lungs and medLN relative to cells obtained from donor TS1xHA28 mice (Fig. 5A).
To more closely evaluate the differential expression of IL-10 and T-bet in the lungs versus the medLN of infected mice, we purified 6.5+CD4+eGFP+ cells from these sites by FACS and determined the levels of IL-10 and T-bet (Tbx21) mRNA relative to the input 6.5+CD4+eGFP+ cells from TS1xHA28 mice. Notably, the levels of IL-10 and T-bet transcripts that were found in 6.5+CD4+eGFP+ cells analyzed directly ex vivo closely paralleled the amounts detected by intracellular cytokine staining following stimulation with PMA and ionomycin: IL-10 and T-bet mRNA levels were highest in 6.5+ Tregs isolated from the lungs of infected mice, and although lower than in the lungs, the levels found in Tregs from the medLN were higher than were found in the input 6.5+ Tregs from TS1xHA28 mice (Fig. 5B). Interestingly, mRNA levels for the transcription factor Blimp-1 (Prdm1) showed a similar pattern of expression, with highest levels in 6.5+ Tregs obtained from the lungs and a slight increase relative to input in cells from the medLN (Fig. 5B). This is noteworthy since Blimp-1 has previously been implicated in the acquisition of IL-10 secretion by both Tregs and CD8+ T cells (28, 29); by contrast, mRNAs encoding other molecules that have previously been implicated in processes associated with either Th1- (IFN-γ, IL-27, IL-27R-α), Th2- (IRF4 and GATA3), Th17- (ROR-γt) and follicular helper T-cell conversion (Bcl-6) of Tregs were not found to be differentially expressed in these different 6.5+ Treg populations (16, 17, 30, 31) (Fig. 5B).
To establish whether T-bet+ Treg specialization can occur in the Tregs of mice that had not received an extraneous source of Tregs, we examined CD4+Foxp3+ cells from the lungs and medLN of mice that had been infected with PR8 virus but had not received Tregs from TS1xHA28 mice. In this case, CD4+Foxp3+ cells in both the lungs and the medLN showed significant upregulation of CXCR3 (Fig. 5C). The levels of IL-10 and T-bet were also significantly higher than isotype controls in CD4+Foxp3+ cells from the lungs, but not from the medLN (Fig. 5C).
Finally, to determine whether TCR specificity contributes to T-bet+ Treg specialization, we compared 6.5+CD4+Foxp3+ cells with endogenous 6.5−CD4+Foxp3+ cells in the lungs of mice that had received Tregs from TS1xHA28 mice. The 6.5+CD4+Foxp3+ cells expressed significantly higher levels of CXCR3, T-bet and IL-10 than were found in endogenous 6.5−CD4+Foxp3+ cells (Fig. 5D). We also purified 6.5+CD4+eGFP+ and 6.5−CD4+eGFP+ cells from the lungs of infected BALB/c.Foxp3eGFP mice that had been seeded with Tregs from TS1xHA28.Foxp3eGFP mice, and found significantly higher levels of both T-bet and Blimp-1 mRNAs in the GFP+ cells expressing the clonotypic 6.5 TCR than in endogenous Tregs (Fig. 5E). Altogether, these data indicate that virus-reactive Tregs undergo differentiation to acquire distinct T-bet+ Treg phenotypes in the lungs and medLN of infected mice. In addition, within cells that were present in the lungs of infected mice, the levels of IL-10, T-bet and Blimp-1 were highest in Tregs expressing a TCR that confers strong reactivity toward the viral antigen.
To examine how T-bet+ Tregs were leading to decreased numbers of effector T cells in the lungs of infected mice we analyzed cell division using the proliferation marker Ki-67. Sizable fractions (from 60% to 80%) of the CD8+ and CD4+Foxp3− effector cells in the lungs of infected mice that had not received Tregs were Ki-67+, consistent with previous studies showing that effector cells undergo active proliferation in the lungs of influenza virus-infected mice (27, 32) (Fig. 6A). Notably, the percentages of CD8+ and CD4+Foxp3− cells that were Ki-67+ were significantly reduced in the lungs of mice that had received Tregs from TS1xHA28 mice. It was also noteworthy that most of the Foxp3+ Tregs (both the 6.5+CD4+Foxp3+ cells derived from TS1xHA28 mice and the endogenous 6.5−CD4+Foxp3+ cells) in the lungs were Ki-67+, and that the presence of Tregs from TS1xHA28 mice did not lead to decreases in Ki-67 expression by the endogenous Tregs, in contrast to their effects on CD8+ and CD4+Foxp3− effector cells (Fig. 6A).
Contrary to the abundant Ki-67 expression that was evident in the lungs of infected mice, smaller percentages (~10%) of the CD8+ and CD4+ T cells in the medLN of infected mice were Ki-67+ (Fig. 6B). Moreover, there was no difference in the percentage of Ki-67+ cells in the medLN of mice that had or had not received virus-reactive Tregs. There were also no differences in the expression of CXCR3 by CD4+ and CD8+ T cells in the medLN; since CXCR3 plays a major role in directing the migration of T cells from the medLN to the lungs during respiratory infection (33), this argues against the possibility that Tregs affect the accumulation of effector cells in the lungs by blocking their migration from the medLN (34) (Fig. 6C). Instead, virus-reactive T-bet+ Tregs prevent the accumulation of effector T cells primarily by suppressing their proliferation at the site of infection.
We have shown here that Tregs that were generated intrathymically based on specificity for a self-peptide can undergo functional specialization during the course of an immune response to influenza virus infection. Virus-reactive T-bet+ Tregs isolated from the medLN and lungs of PR8-infected mice in each case expressed elevated levels of CXCR3, along with additional molecules associated with Treg cell function and/or activation, such as CTLA4, CD44 and ICOS. The levels of T-bet and IL-10 expressed by 6.5+CD4+Foxp3+ cells were also elevated in cells isolated from both the lungs and the medLN than were those found in cells analyzed directly after isolation from these tissues in TS1xHA28 mice, consistent with differentiation into T-bet+ Tregs. Interestingly, however, the 6.5+ T-bet+ Tregs cells isolated from the lungs of infected mice expressed significantly higher levels of T-bet, Blimp-1 and IL-10 than did cells from the medLN, providing evidence that Treg cells with identical antigenic specificity can differentiate into phenotypically distinct subsets of T-bet+ Tregs at different anatomical locations in an infected host.
The signals that induce functional specialization of Tregs in different settings are still being defined. T-bet+ Tregs were first identified based on the ability of Foxp3+ T cells to up-regulate T-bet in response to IFN-γ, which in turn promoted CXCR3 expression and was accompanied by increased IL-10 production (15). More recently, Foxp3+ T cells expressing Blimp-1 were found to be highly represented in the lungs of Blimp1-reporter mice (29). T-bet was also found to be expressed in these cells but was dispensable for IL-10 production; instead, Blimp-1 acting in concert with IRF-4, was required for the ability of these cells to produce IL-10. The studies here provide evidence that TCR signaling can also be important in determining the amount of IL-10 that is expressed, because T-bet, Blimp-1 and IL-10 levels were all significantly higher in Tregs expressing the clonotypic 6.5 TCR than in the endogenous (6.5−) Tregs in the lungs of infected mice. This disparity must be due to differences in TCR signaling, because the 6.5+ and 6.5− Tregs had been isolated from infected lungs simultaneously and thus had been exposed to the same cytokine environment. In support of this conclusion, Blimp-1 mRNA has previously been shown to increase substantially in Tregs in response to TCR stimulation (35), and T-bet expression by effector CD4+ T cells is also sensitive to TCR stimulation (14). Since the levels of Blimp-1 and T-bet in lung Tregs also correlated with the amount of IL-10 that is produced, the data here further suggest that the differing levels of Blimp-1 and/or T-bet that are induced in response to TCR stimulation can determine the amount of IL-10 that differentiated Tregs produce. By contrast, IRF4 levels did not differ between the input Foxp3+ Tregs and those isolated from infected mice, suggesting that its expression levels do not play a role in modulating the extent of IL-10 production by Tregs.
There are several mechanisms that could potentially account for the significant differences in Blimp-1, T-bet and IL-10 expression that were found between T-bet+ Tregs isolated from the lungs and the medLN of infected mice. One possibility is that these phenotypes arise progressively, for example if virus-reactive Tregs are initially activated in the medLN where they upregulate T-bet, Blimp-1 and CXCR3, they produce low levels of IL-10, and they then migrate to the lungs and undergo further differentiation. Consistent with this possibility, CD4+Foxp3+ T cells accumulated most rapidly in the medLN at 5 d p.i. (increasing ~100-fold in the medLN relative to uninfected mice, versus 4-fold in the lungs), whereas they did not reach peak numbers in the lungs until 8 d p.i.. Another mechanism, that is not mutually exclusive, is that differing levels of cytokines and/or specialized cell types support Treg differentiation into cells that express high versus low amounts of IL10 in the lungs versus the medLN. For example, neutrophil-derived IL-27 acting in concert with IL-2 has previously been shown to induce IL-10 production in CD8+ T cells in the lungs of influenza virus-infected mice by a Blimp 1-dependent mechanism (28), and differences in the local availability of these or other cytokines might contribute to the different levels of Blimp-1, T-bet and IL-10 that we found among 6.5+ T-bet+ Tregs isolated from these two sites. Lastly, the extent of differentiation might be linked to the degree of proliferation of the Tregs. Previous studies have indicated that proliferation by lung-resident CD8+ T cells makes a substantial contribution to the overall immune response during influenza virus infection (32). It is possible then that proliferation of Tregs in the lungs induces more extensive Treg differentiation, leading to the accumulation of virus-reactive T-bet+ Tregs with significantly different phenotypes in the lungs and the medLN under steady state conditions during influenza virus infection.
The distinct phenotypes displayed by 6.5+CD4+Foxp3+ cells in the lungs and medLN are also notable in light of differences in the effects of Treg activity on effector CD4+ and CD8+ cells at these different sites. There were significant reductions in the numbers of IFN-γ-secreting CD4+Foxp3− and CD8+ T cells in both the lungs and the medLN of PR8-infected mice that had received 6.5+ Tregs from TS1xHA28 mice. However, whereas the presence of 6.5+ Tregs inhibited proliferation and accumulation of IFN-γ-secreting CD4+ and CD8+ cells in the lungs, proliferation and accumulation appeared to be little affected in the medLN. Since a prominent difference between the Foxp3+ Tregs that accumulate at these sites is the amount of IL-10 that they can produce, the suppression of effector CD4+ and CD8+ T cell proliferation in the lungs may be mediated by high local concentrations of IL-10 that are produced by Blimp-1hi T-bethi IL-10hi Tregs and that may act either directly on T cells themselves or by modifying local APC activity. In support of this possibility, IL-10 expression by Tregs was previously shown to be required to prevent spontaneous inflammatory responses at mucosal sites (including the lungs), but did not appear to be required to prevent systemic immune activation in sites such as LN (26). In addition, IL-10R blockade resulted in lethal pulmonary inflammation during influenza infection, consistent with a role for IL-10 in controlling inflammatory responses in the lungs of infected mice (27). By contrast, the Tregs in the medLN produced little or no IL-10, and it is possible that these Tregs could inhibit cytokine production, but not the proliferation of effector T cells, by an alternative mechanism. For example, CTLA-4 expression by the Tregs may be more important in regulating the immune responses that develop in the medLN versus the lung, consistent with studies indicating that mice whose Tregs lack CTLA-4 expression develop excessive effector T cell responses systemically (i.e. in sites such as LN), but not in the tissues (36). Whatever the basis, the studies here are significant because they contrast and extend previous studies indicating that Tregs modify the immune response to pathogens primarily by controlling the homing of effector T cells from the draining LN to the infected site (37), since we have shown that Tregs can exert regulatory effects primarily through their ability to suppress the differentiation and proliferation of CD4+ and CD8+ effector T cells in the infected tissues themselves.
The findings here point to a prominent role for thymically-derived Tregs in modulating the immune response to an infectious agent, while conventional CD4+ T cells with identical specificity for the pathogen underwent little or no conversion to become “adaptive” Tregs in influenza virus-infected mice. CD4+Foxp3+ T cells expressing a pathogen-specific transgenic TCR were similarly found to expand in response to Mycobacterium tuberculosis, while conventional CD4+ T cells with the same specificity underwent little or no conversion to become Foxp3+ Tregs (11). It was notable that depletion of Tregs using anti-CD25 treatment induced reciprocal effects; this implies that the immune repertoire of otherwise unmanipulated mice contains Tregs that exert similar effects on the immune response as we observe in mice with increased Treg cell frequencies. In addition, the finding that anti-CD25 treatment led to enhanced, rather than diminished effector T cell responses in the lungs, implies that this treatment was primarily removing a regulatory component (since a direct effect of anti-CD25 treatment on effector cells would most likely have caused a reduced, rather than increased effector T cell response). Lastly, it is interesting to consider whether the high frequencies of virus-reactive Tregs that are present in the adoptive transfer setting we employed here might also arise in the setting of natural influenza virus infections. It is currently unclear whether the T-bet+ Tregs generated during an infection can adopt long-lived “memory” phenotypes; if so, then virus-reactive Tregs would be present at increased frequencies when an individual is re-infected with a pathogen bearing a crossreactive antigen, creating a similar situation to the one we have generated experimentally here using an adoptive transfer approach. It will also be important in future experiments to determine whether self-peptide-selected Tregs can become activated by viral antigens with which they are only weakly crossreactive, as we previously demonstrated based on in vitro suppression assays (38). Anti-viral CD8+ T cell responses have previously been shown to be modified by prior infections with crossreactive antigen due to perturbations of the available immune repertoire (39). For influenza infection, it is possible that virus-reactive T-bet+ Tregs can be expanded by repeated exposure to crossreactive viral epitopes over an individual’s lifetime, and contribute to the decreased immune responsiveness that is found in aged individuals (40).
We thank Krystyna Mozdzanowska, the Wistar Flow Cytometry Core and the UPenn Human Immunology Core for technical expertise.
This work was supported by NIH AI083022, NIH AI51966, NCI P30 CA10815, and the Commonwealth of Pennsylvania. FB was supported by NCI T32 CA09171 and G-S.C. by NIH T32 HL07748.
The authors declare no financial conflicts of interest.