Cutting Edge: Self-Antigen Controls the Balance between Effector and Regulatory T Cells in Peripheral Tissues

Iris K. Gratz, Michael D. Rosenblum, Megan M. Maurano, Jonathan S. Paw, Hong-An Truong, Ann Marshak-Rothstein and Abul K. Abbas
J Immunol February 15, 2014, 192 (4) 1351-1355; DOI: https://doi.org/10.4049/jimmunol.1301777

Abstract

Immune homeostasis in peripheral tissues is achieved by maintaining a balance between pathogenic effector T cells (Teffs) and protective Foxp3+ regulatory T cells (Tregs). Using a mouse model of an inducible tissue Ag, we demonstrate that Ag persistence is a major determinant of the relative frequencies of Teffs and Tregs. Encounter of transferred naive CD4+ T cells with transiently expressed tissue Ag leads to generation of cytokine-producing Teffs and peripheral Tregs. Persistent expression of Ag, a mimic of self-antigen, leads to functional inactivation and loss of the Teffs with preservation of Tregs in the target tissue. The inactivation of Teffs by persistent Ag is associated with reduced ERK phosphorylation, whereas Tregs show less reduction in ERK phosphorylation and are relatively resistant to ERK inhibition. Our studies reveal a crucial role for Ag in maintaining appropriate ratios of Ag-specific Teffs to Tregs in tissues.

Introduction

The choice between tolerance and autoimmunity is determined, to a significant extent, by the relative generation and maintenance of effector T cells (Teffs) and regulatory T cells (Tregs) specific for self-antigens. An imbalance in this ratio is thought to underlie inflammatory and autoimmune disorders. Therefore, defining the variables that control these cell populations is critical for understanding the pathogenesis of autoimmune disease and for developing rational therapeutic approaches. Emerging treatments for both systemic and tissue-specific autoimmunity focus on inhibiting the activation of pathogenic cells and augmenting the pathways that suppress these cells and boost Treg function (1). It is also becoming increasingly clear that many of the dominant mechanisms of immune regulation occur in peripheral tissues and not in lymphoid organs, highlighting the importance of studying immune responses in tissues (2, 3).

To analyze the control of Teffs and Tregs in tissues, we have developed an experimental model in which a self-antigen is inducibly expressed in the skin (4). Studies with this model have shown that Ag recognition generates cytokine-producing Teffs that induce an inflammatory skin disease, followed by the activation of Foxp3+ Tregs that mediate disease resolution. A fraction of the Tregs survives in an IL-7–dependent manner (5) in the skin in the absence of continuous Ag expression, and these “memory Tregs” suppress subsequent inflammatory reactions in the tissue.

To further elucidate the mechanisms responsible for controlling the Teff/Treg balance, we use an adoptive transfer approach in which a single bolus of Ag-specific T cells is given to mice in which expression of tissue Ag can be turned on and off. An advantage of this system is the ability to follow Teffs and peripheral Tregs (pTregs) developing in the same animal from a single naive cell population. In addition, both the target tissue and lymphoid organs can be accessed and analyzed. For these reasons, our model is uniquely amenable to dissection of the mechanisms underlying the Teff/Treg balance in tissues. We have found that the duration of Ag exposure is a major determinant of the Teff/Treg balance in the Ag-expressing tissue, and that there are striking differences in the responses of these cells to transient or persistent Ag recognition. The implications of these results for self-tolerance and for therapeutic tolerance induction strategies are discussed.

Materials and Methods

Mice

All animal studies were performed in compliance with institutional guidelines in a specific pathogen-free facility. K5/rTA and TGO (TRE-OVA) mice were crossed and designated K5/TGO mice as described previously (4). The double-transgenic K5/TGO mouse line was crossed onto TCRα−/− mice on BALB/c background (6). DO11.10 TCR-transgenic mice (7) were crossed with Rag2−/−/CD90.1+ [wild type (WT)] or Rag2−/−/CD90.2+ mice carrying a mutation of Foxp3 (“scurfy”) (8).

Adoptive transfer of T cells

Single-cell suspensions from all lymph nodes (LNs) from DO11.10/Rag2−/−/CD90.1+ were prepared by mechanical disruption of LN. Three to 8 × 105 LN cells were adoptively transferred i.v. into sex-matched K5/TGO/TCRα−/− recipient mice.

Skin disease

To induce expression of the TGO transgene in the skin, K5/TGO/TCRα−/− mice were maintained on 1 g/kg doxycycline (dox) chow (Bio-Serv, Frenchtown, NJ). A 15-point clinical scoring scale was used to quantify skin disease. The clinical parameters of scaling, alopecia, erythema, level of activity, and periocular inflammation were each given a score of 0–3. Scores for individual clinical parameters were summed for each mouse.

Cell isolation from skin

Shaved dorsal and ventral trunk skin, and ears were harvested, minced, and digested for 40 min with 2.0 mg/ml collagenase from Clostridium histolyticum (Sigma), 0.5 mg/ml hyaluronidase (Sigma), and 0.1 mg/ml DNAse (Sigma), to isolate skin-infiltrating cells. Single cells were filtered, washed with tissue culture medium, and stained for flow cytometry or cultured for intracellular cytokine staining.

Restimulation for intracellular cytokine staining

Skin-draining LNs (sdLNs) or skin single-cell suspensions were cultured overnight with LPS/OVA-pulsed bone marrow–derived dendritic cells (4:1 skin or LN cells/dendritic cells) in supplemented RPMI 1640 tissue culture medium. Cultures were then treated with brefeldin A for 3 h and stained for FACS analysis.

In vitro culture of T cells

Single-cell suspensions of LNs and spleens of BALB/c mice were labeled with 1 μM CFSE and cultured for 6 d in 24-well plates coated with anti-CD3 and anti-CD28 (clones 2C11 and PV1, 1 μg/ml each). PD0325901 (Selleckchem), a selective and non–ATP-competitive MEK inhibitor that inhibits the phosphorylation of ERK1 and ERK2, was added at 0–10 nM.

Statistics

Statistical analysis was done using GraphPad Prism software (GraphPad). The p values were calculated using a two-tailed unpaired or paired (Fig. 4D, right panel) t test: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Results and Discussion

CD4+ T cells induce a spontaneously resolving inflammatory disease after encounter with cutaneous Ag

The model of inducible Ag we have developed (4) involves two transgenes. The “TGO” transgene expresses a membrane-associated form of OVA under the control of a tetracycline-response element, and the “K5” transgene expresses the tetracycline transactivator under the control of the keratin K5 promoter. In mice expressing both constructs (i.e., K5/TGO), expression of the Ag in the skin is dependent on exposure to the tetracycline analog, dox (4).

To define the consequences of Ag recognition on the generation and maintenance of Teffs and Tregs in a peripheral tissue, we transferred OVA-specific CD4+ T cells from DO11/Rag−/− donors (DO11 T cells expressing a single TCR) into K5/TGO recipients crossed onto a TCRα−/− background. Upon transfer of DO11 cells and induction of cutaneous Ag expression, K5/TGO/TCRα−/− mice developed a severe inflammatory dermatitis within 2 wk, associated with weight loss and death of ∼30% of the recipients (Fig. 1A–D). Disease was associated with expansion of the DO11 population in the sdLNs, production of the effector cytokine IFN-γ, and infiltration and persistence of these cells in the skin (Fig. 1E, 1F). Importantly, the production of IL-2 and IFN-γ by skin-infiltrating Teffs was transient, and appreciable levels of cytokines were detected only in the first 2 wk after transfer.

FIGURE 1.
FIGURE 1.

T cells induce a spontaneously resolving inflammatory disease after encounter with cutaneous Ag. DO11/Rag−/− cells were adoptively transferred i.v. into K5/TGO/TCRα−/− recipients. Mice were fed with dox chow from the time of T cell transfer (day 0). (A) Representative pictures of clinical appearance of mice on day 19. (B) Course of clinical skin disease. Error bars show SEM. (C) Weight loss during the disease course. (D) Survival of K5/TGO/TCRα−/− recipients. Single-transgenic TCRα−/− recipients served as controls (n = 19). (E and F) Skin or sdLN cells were stimulated overnight with OVA-pulsed bone marrow–derived dendritic cells and stained for expression of IL-2, IFN-γ, and IL-17A. FACS data are gated on live donor CD4+ KJ-126+ Foxp3− CD90.1+ DO11 cells. Cytokine production within the Foxp3− DO11 population in (E) skin and (F) sdLNs over the course of disease is shown. Cumulative data from four experiments, n = 3–5 mice/time point. Error bars show SEM.

Frequency of Tregs increases progressively and Foxp3+ cells are required for resolution of disease

One of the striking features of this acutely induced inflammatory reaction was that it resolved spontaneously, despite the fact that mice were maintained on dox and Ag was continuously expressed, and Ag-specific T cells remained in the skin (Fig. 2A). Resolution was associated with the generation and progressive accumulation of Foxp3+ Tregs. Because the starting population of DO11/Rag−/− T cells does not contain Foxp3-expressing Tregs (9), all Tregs observed in this system are pTregs. Approximately 40 d after Ag induction there was a reversal of the predominant cell population in the skin from Foxp3− effector cells to Foxp3+ Tregs (Fig. 2B). To test whether T cell lymphopenia affected the generation and maintenance of pTregs in our model, we adoptively transferred about an 8-fold excess of WT polyclonal T cells into K5/TGO/TCRα−/− recipients before DO11 T cell transfer and found that the generation of Ag-specific Foxp3+ T cells was unaffected by the presence of irrelevant T cells (data not shown).

FIGURE 2.
FIGURE 2.

The frequency of Foxp3+ Tregs increases progressively. DO11/Rag−/−/CD90.1+ LN cells were adoptively transferred into K5/TGO/TCRα−/−. (A) Skin (left panel) and sdLN (right panel) single-cell suspensions from recipients over the course of disease were stained for CD90.1, CD4, KJ1-26 (specific for the DO11 TCR), and analyzed by flow cytometry, excluding dead cells. Error bars show SEM; n = 2–8/time point. (B) Percentage of Foxp3− Teffs and Foxp3+ Tregs within the skin-infiltrating LNs (left panel) and sdLNs (right panel) DO11.10 population over the course of disease. Error bars show SEM; cumulative data from 10 independent experiments, n = 2–8/time point. (C and D) A total of 0.45 × 106 DO11/Rag−/− (WT) or DO11/scurfy/Rag−/− (scurfy) LN cells were adoptively transferred. (C) Survival of K5/TGO/TCRα−/− recipients. (D) Course of clinical skin disease. scurfy: n = 7; WT: n = 5.

To determine whether Tregs were required for disease resolution, we used CD4+ T cells that lack functional Foxp3+ Tregs because of the scurfy mutation in Foxp3 (8). These cells induced nonresolving disease that led to death in 100% of the animals (Fig. 2C, 2D), which indicated that potential Teff intrinsic tolerance mechanisms (e.g., exhaustion or deletion) were not sufficient for disease resolution, but that pTregs were required to induce resolution.

Persistent exposure to self-antigen leads to loss of Teffs and relative preservation of Tregs

To test the hypothesis that the duration of Ag exposure may influence the Teff/Treg balance, we transferred DO11 T cells into K5/TGO/TCRα−/− recipients, and dox was administered either for 7 d only (transient Ag expression) or for 60–80 d (persistent Ag expression), and all mice were examined on days 60–80 after cell transfer. The initial skin inflammation was comparable in both groups. The proportion of Foxp3+ and Foxp3− T cells, however, was markedly different, especially in the skin. When Ag expression was persistent, ∼80% of the DO11 cells that accumulated in the skin were Foxp3+ Tregs. In contrast, after transient (7-d) Ag expression, most of the DO11 cells in the skin were Foxp3− cells (Fig. 3A). Differences in the ratio of Foxp3+ and Foxp3− T cells were due to the loss of Foxp3− T cells with persistent Ag expression, as the absolute number of Foxp3+ T cells was virtually unchanged (Fig. 3B). The reduction in Foxp3− cells was more pronounced in the skin than in the sdLNs. Initially, both populations underwent a rapid burst of proliferation upon induction of Ag expression (as measured by expression of Ki67; Fig. 3C, 3D). After transient Ag exposure, Foxp3− cells continued to proliferate in the absence of Ag, which is expected for Teff memory cells (10, 11), whereas persistent Ag expression led to a marked reduction in the proliferation of Foxp3− cells (Fig. 3C). In contrast, with either persistent Ag or transient Ag, 30% of Ag-specific Foxp3+ Tregs within the sdLNs were proliferating at days 66–82 (Fig. 3D).

FIGURE 3.
FIGURE 3.

Persistent Ag expression leads to a specific loss of Teffs in the periphery and relative preservation of Tregs. (A) DO11/Rag−/− cells were adoptively transferred to K5/TGO/TCRα−/−, and recipient mice were either fed with dox for 7 d and then switched back to regular feed (transient expression) or kept on dox feed for the entire time of the experiment (persistent expression). Single-cell suspensions of skin (left panel) and sdLNs (right panel) were prepared between days 60 and 82 posttransfer and analyzed by FACS. Percentage of Foxp3+ and Foxp3− T cells within the skin-infiltrating DO11 T cell population. Data pooled from three independent experiments. Total number of mice analyzed, n = 3–5/condition; error bars show SD. (B) As in (A), showing the absolute number of Foxp3+ and Foxp3− DO11 T cells/105 live cells in the skin (left panel) or sdLN (right panel). (C) sdLN cells were analyzed for expression of Foxp3 and Ki67 on indicated days after DO11 transfer. Data are gated on live donor CD4+, CD90.1+ cells. (D) The percentage of Ki67-expressing LN DO11 cells within the Fopx3+ and Foxp3− population, respectively, was calculated. Error bars show SD; n = 2–3/condition and time point. *p < 0.05, ***p < 0.001.

In addition to reduced proliferation of Foxp3− T cells, persistent Ag expression resulted in a diminution of both IL-2 and IFN-γ expression in Foxp3− cells upon restimulation with OVA when compared with the same population transiently exposed to Ag (Supplemental Fig. 1). Again, this effect was most pronounced in the skin compared with sdLNs. Foxp3+ Tregs did not produce IL-2, IFN-γ, or IL17 under any condition.

Signaling pathways influenced by duration of Ag exposure

Numerous studies have suggested differences in signaling pathways between Teffs and Tregs (12–14). Using phosphoflow analyses after ex vivo restimulation, we found that both populations in sdLNs showed reduced p-ERK induction upon ex vivo restimulation after prolonged Ag exposure in vivo (Supplemental Fig. 2A). Similar results were seen in skin-infiltrating cells, although the percentage of cells in which p-ERK was induced was less than in sdLNs (Supplemental Fig. 2B). In vivo stimulation with Ag also resulted in reduced p-ERK in both populations after prolonged Ag exposure (Fig. 4A). However, the proportion of p-ERK+ cells was lower in the Foxp3− subset with the persistence of Ag than in the Foxp3+ subset when restimulated with i.v. OVA (Fig. 4B, right panel). To further ask whether ERK phosphorylation differentially impacts the proliferation of Tregs and Teffs, we inhibited ERK in vitro using the MEK inhibitor PD0325901. This treatment inhibited the proliferation of Foxp3− cells at a 10-fold lower concentration than that of Foxp3+ cells, which were only mildly affected at the highest dose (Fig. 4C, 4D). Increasing doses of inhibitor thus resulted in a relative enrichment of Foxp3+ cells in the culture (Fig. 4E). These data indicate that the proliferation of Foxp3+ cells is relatively independent of ERK phosphorylation compared with Foxp3− cells, and downmodulation of ERK signals in response to chronic TCR stimuli might be a means to control the balance of self-reactive Foxp3+ and Foxp3− cells.

FIGURE 4.
FIGURE 4.

Persistent Ag expression results in reduced phospho-ERK induction. DO11/Rag−/− cells were adoptively transferred into K5/TGO/TCRα−/− recipients. Recipient mice were treated as in Fig. 3 (i.e., transient or persistent Ag expression). (A and B) Five to 70 d posttransfer, mice were i.v. injected with 86 μg OVA peptide, and 17 min later sdLN cells were fixed with 1.6% paraformaldehyde (Electron Microscopy Sciences), permeabilized with ice-cold methanol, and stained with p-p44/42 MAPK (Erk1/2; Thr202/Tyr204, 197G2; Cell Signaling Technologies) for analysis by flow cytometry. (A) Representative histograms showing phospho-ERK in sdLNs. (B) Geometric mean fluorescent intensity (MFI) of phospho-ERK signal of DO11 T cells in sdLNs gated on either the Foxp3+ or Foxp3− population (left panel). Same data as left panel, but Foxp3+ or Foxp3− population within individual mice paired (right panel). Data pooled from two independent experiments. (C–E) CFSE-labeled cells were continuously stimulated in vitro with plate-bound anti-CD3/CD28 for 6 d in the presence of increasing concentrations of the MEK inhibitor PD0325901. (C) Representative FACS plots of CFSE signals of T cells gated on CD4+Foxp3+ and CD4+Foxp3−. (D) MFI of CFSE of Foxp3+CD4+ and Foxp3−CD4+ cells are summarized. (E) Relative enrichment of Foxp3+ cells with increasing concentrations of PD0325901 is quantified. Each data point measured in triplicates; one representative experiment of two. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

These studies highlight the essential role of peripherally generated Tregs in regulating pathologic immune responses directed at tissue Ag, and the importance of chronicity of Ag exposure in regulating the Teff/Treg balance in the target tissue. Persistent Ag expression resulted in tolerance, manifested by loss and functional inactivation of Foxp3− effector cells and relative preservation of suppressive Foxp3+ Tregs. Tregs continued to proliferate even in the face of reduced p-ERK, likely because Treg proliferation is less dependent on p-ERK than Teff proliferation. In fact, other studies have shown that Tregs do not require many signals thought to be essential for the responses of Teffs, such as Akt and mTOR (15). These results suggest that Tregs are more resistant to tolerance than Teffs, which allows Tregs to function in the face of constant encounter with a self-antigen. Elucidating the mechanisms that determine the strikingly different reactions of Teffs and Tregs to tissue Ag in vivo may reveal the critical pathways that control the Teff/Treg balance and provide insight into the pathogenesis of tissue-specific autoimmunity.

Disclosures

The authors have no financial conflicts of interest.

Acknowledgments

We thank Carlos Benitez (University of California San Francisco [UCSF]) for assistance in animal husbandry, Sara Isakson (UCSF) for genotyping, Dr. Katya Ravid and Greg Martin (Transgenic/Knockout Core, Boston University School of Medicine, Boston, MA) for derivation of TRE-TGO transgenic mice, and Haopeng Wang (UCSF) and Bill O’Gorman (Stanford University, Stanford, CA) for advice with TCR signaling experiments. We thank Dr. Steven F. Ziegler (Benaroya Research Institute, Seattle, WA) for K5-rTA transgenic mice.

Footnotes

  • This work was supported by the National Institutes of Health (Grants R01 AI73656 and U19 AI56388 to A.K.A.; Grant 1R03AR064554-01 to I.K.G.; Grant 1K08AR062064-01 to M.D.R.), an Erwin Schroedinger Fellowship from the Austrian Science Fund (Fonds zur Förderung der Wissenschaftlichen Forschung [FWF], J2997-B13 to I.K.G.), a Burroughs Wellcome Career Award for Medical Scientists (to M.D.R.), the Scleroderma Research Foundation (to M.D.R.), and the Department of Dermatology, University of California San Francisco (to M.D.R.).

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article:

    dox
    doxycycline
    LN
    lymph node
    pTreg
    peripheral regulatory T cell
    sdLN
    skin-draining LN
    Teff
    effector T cell
    Treg
    regulatory T cell
    UCSF
    University of California San Francisco
    WT
    wild type.

  • Received July 10, 2013.
  • Accepted December 11, 2013.

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