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. 2004 Jul;114(1):28-38.
doi: 10.1172/JCI20509.

Tolerance induced by inhaled antigen involves CD4(+) T cells expressing membrane-bound TGF-beta and FOXP3

Marina Ostroukhova  1 , Carole Seguin-Devaux, Timothy B Oriss, Barbara Dixon-McCarthy, Liyan Yang, Bill T Ameredes, Timothy E Corcoran, Anuradha Ray
Affiliations

Tolerance induced by inhaled antigen involves CD4(+) T cells expressing membrane-bound TGF-beta and FOXP3

Marina Ostroukhova et al. J Clin Invest. 2004 Jul.

Abstract

Under normal circumstances, the respiratory tract maintains immune tolerance in the face of constant antigen provocation. Using a murine model of tolerance induced by repeated exposure to a low dose of aerosolized antigen, we show an important contribution by CD4(+) T cells in the establishment and maintenance of tolerance. The CD4(+) T cells expressed both cell surface and soluble TGF-beta and inhibited the development of an allergic phenotype when adoptively transferred to naive recipient mice. While cells expressing cell surface TGF-beta were detectable in mice with inflammation, albeit at a lower frequency compared with that in tolerized mice, only those from tolerized mice expressed FOXP3. Blockade of TGF-beta in vitro and in vivo interfered with immunosuppression. Although cells that expressed TGF-beta on the cell surface (TGF-beta(+)), as well as the ones that did not (TGF-beta(-)), secreted equivalent levels of soluble TGF-beta, only the former were able to blunt the development of an allergic phenotype in mice. Strikingly, separation of the TGF-beta(+) cells from the rest of the cells allowed the TGF-beta(-) cells to proliferate in response to antigen. We propose a model of antigen-induced tolerance that involves cell-cell contact with regulatory CD4(+) T cells that coexpress membrane-bound TGF-beta and FOXP3.

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Figures

Figure 1
Figure 1
Repeated antigen exposure induces tolerance in the respiratory tract. Mice were exposed to aerosolized PBS alone (airway inflammation [inf.]; black bars) or 1% OVA in PBS (tolerance, [tol.]; white bars) daily for 20 minutes each day for 10 consecutive days (days 0–10). Both groups were then immunized intraperitoneally with 10 ∝g OVA and 1 mg alum on days 21 and 27. Treated mice were challenged by exposure to aerosolized 1% OVA in PBS for 20 minutes each day for 7 days from day 34 through day 42 (A–D, G) or used for spleen CD4 T cell isolation on day 34 (E and F). In the case of mice challenged by inhaled OVA, on day 43 BAL was performed, serum was collected, and lungs were removed for histological evaluation. Shown is a representative experiment of five, with three to six animals per group in each experiment. (A) OVA-specific serum IgE concentration was measured by ELISA. Shown is the mean plus or minus SD; *P < 0.05 versus mice with airway inflammation. (B) Cytokine profile in the BAL fluid of tolerized mice compared with mice with airway inflammation as measured by ELISA. Shown is mean plus or minus SD with three mice per group (*P < 0.05). (C) Mean plus or minus SD of total and differential cell (eosinophils, Eos; neutrophils, Neu; lymphocytes, Lymph; and macrophages, Mac) counts in the airways of tolerized mice and mice with airway inflammation as recovered by BAL (*P < 0.05). (D) H&E staining demonstrating the absence of airway inflammation in the lungs of tolerized mice. Magnification ∞10. (E) Six animals were used per group and subjected to the inflammation or tolerance protocol, immunized with OVA and alum, and spleens isolated. Spleens from two animals were pooled in each group (three pools per group), and CD4+ T cells were isolated and stimulated with OVA and APCs in vitro for 5 days. Shown are the cytokine profiles in the supernatants as determined by ELISA (*P < 0.05). (F) Nuclear factor expression in nuclear fractions of the CD4+ T cells after 5-day culture as determined by Western blot analysis using specific Ab’s. CREB-1 expression is shown as a marker for protein loading. (G) Lung-draining lymph nodes (LNs) were harvested from mice on day 37 after initial exposure to OVA or PBS, immunization with OVA/alum, and 3 days of challenge with inhaled OVA. LNs were pooled from three mice, cells recovered from the LNs were cultured with OVA and APCs for 3 days, and cytokines were measured in the culture supernatants by ELISA.
Figure 2
Figure 2
Suppressive function of CD4+ T cells from tolerized mice. (A) Mice were exposed to PBS (airway inflammation) or 1% OVA (tolerance) and subsequently immunized with OVA and alum on days 21 and 27 after the initiation of OVA/PBS exposure. Splenic CD4+ T cells isolated from mice on day 34 were stimulated in vitro with different concentrations of OVA (0.01–100 ∝g/ml) and mitomycin C–treated, T cell–depleted APCs at equivalent cell numbers (105 cells each per well). After 72 hours of incubation, small samples of culture supernatants were removed for cytokine determination, and the remaining cells were pulsed for measurement of [3H]-thymidine incorporation. *P < 0.025 compared with proliferation of cells from mice immunized for inflammation. The proliferative response of CD4+ T cells from naive mice (open diamonds) is shown as a negative control. An additional control used was a mixture of cells from the inflammation group and from naive mice used in a 1:2 ratio. Each data point represents the mean plus or minus SEM of triplicate wells. Shown is a representative experiment of three. (B) As described above, CD4+ T cells isolated after day 34 were subjected to two rounds of stimulation with OVA and APCs in vitro, and nuclear extracts were prepared. Expression of GATA-3 and FOXP3 was assessed in the nuclear extracts (7–10 ∝g of total protein) by Western blotting techniques. CREB-1 expression is shown as a marker for protein loading. (C) Shown is an average densitometric reading of FOXP3 and GATA-3 expression relative to that of CREB-1 from two independent experiments.
Figure 3
Figure 3
Inhibition of development of the allergic phenotype in mice that received CD4+ T cells from tolerized mice. On day 21 after the initial 10-day exposure to OVA/PBS, CD4+ T cells were purified from spleens of mice from both groups and 5 ∞ 105 cells were adoptively transferred into naive BALB/c mice that were then immunized with 10 ∝g of OVA and 1 mg of alum intraperitoneally (day 0). Recipients were boosted with OVA/alum 7 days after transfer (day 7) and were challenged by exposure to an aerosol of 1% OVA for 7 days. Twenty-four hours after the last exposure, mice were evaluated for (A) blood IgE levels, (B) cytokine levels, and (C) cell differentials in the BAL fluid. *P < 0.05 in all panels compared with data from inflammation group. (D) Cytospin preparations of cells in the BAL fluid are shown in the upper panels and lung tissue histology is shown in the lower panels. Results are representative of three independent experiments with three to five mice per group.
Figure 4
Figure 4
Cell contact and TGF-β–dependent inhibition of proliferation by CD4+ T cells from tolerized mice. (A) Mice were first exposed to PBS (inflammation group) or 1% OVA (tolerance group) daily for 10 days and then were immunized with OVA/alum on days 21 and 27. Splenic CD4+ T cells isolated on day 34 were stimulated in vitro with different concentrations of OVA (10–200 ∝g/ml) and APCs at equivalent cell numbers (105 cells per well). Cells were mixed as described in the legend to Figure 2 or separated by transwell. In the transwell experiments, cells from the inflammation group were plated in the wells, and cells from tolerized mice on the insert and thymidine incorporation in the former group was measured. *P < 0.05 versus proliferation of cells in the inflammation group. (B) Chicken IgY anti–TGF-β1 (100 ng/ml) or isotype control (chicken IgY) was added to mixed cultures. **P < 0.05 of mixed cultures incubated with anti–TGF-β1 compared with mixed cultures incubated without Ab. (C) Anti–IL-10 (1 mg/ml) or isotype control was added to mixed cultures. All assays were incubated for 72 hours, after which the cells were pulsed for measurement of [3H]-thymidine incorporation. Each data point represents the mean plus or minus SEM of triplicate wells. Shown is a representative experiment of three experiments.
Figure 5
Figure 5
CD4+ T cells in tolerized mice express both soluble and membrane-bound TGF-β. On day 34, splenic CD4+ T cells from the two groups of mice (tolerance and airway inflammation), as well as cells from naive animals, were isolated and stimulated with OVA/APCs in vitro for 48 hours. The T cells were then examined for both (A) intracellular and (B) cell-surface expression of TGF-β by flow cytometry. Appropriate isotype controls are shown, and the percentages presented for the stained cells have had the indicated background isotype control levels subtracted. (A) T cells were stained for cell-surface CD4 expression, then were fixed, permeabilized, and stained for intracellular expression of TGF-β. The isotype for CD4 is shown for the naive animals. For the airway inflammation and tolerance conditions, the control is stained for CD4 and for the isotype for TGF-β. (B) T cells were double-stained for cell-surface expression of CD25 and TGF-β.
Figure 6
Figure 6
Administration of anti–TGF-β1 interferes with tolerance development in vivo. Chicken IgY anti–TGF-β (50 ∝g/mouse) or matching isotype control was administered intraperitoneally into naive BALB/c mice at three time points: 1 hour prior to primary exposure to OVA, on day 5 of exposure, and 1 hour prior to first OVA/ alum immunization on day 21. On day 43, BAL was performed, serum was collected, and lungs were removed for histological evaluation. Shown is a representative experiment of two experiments, with three animals per group in each experiment. (A) IgE levels in blood and (B) cytokine (IL-13) levels in BAL fluid of animals in each group. Shown are mean plus or minus SD with three mice per group (*P < 0.05 versus levels in inflammation group; **P < 0.05 versus animals treated with isotype control). (C) Differential cell counts in BAL fluid. *P < 0.05 compared with inflammation group; **P < 0.05 compared with tolerized group. (D) Lung tissue histology. The grade of inflammation was +5 in all animals immunized for airway inflammation, less than +1 in tolerized animals and in animals that received the isotype control, and between +2 and +4 in mice that received anti–TGF-β1 Ab.
Figure 7
Figure 7
Stable cell surface TGF-β expression is enhanced on CD4+ T cells from tolerized mice relative to those with airway inflammation. Splenic CD4+ T cells were isolated from mice on day 34 after initial exposure and immunization with OVA/alum, subjected to two rounds of in vitro stimulation with OVA/APCs, and then examined for cell surface expression of TGF-β and CD25 by flow cytometry. Before restimulation in vitro, dead cells were removed by density gradient centrifugation using lymphocyte separation medium. Quadrant locations on dot plots were determined using appropriate isotype control Ab’s as shown in the left-hand panel. The indicated level of background for the isotype control staining was subtracted from positively staining samples to arrive at the percentages shown in the upper-right quadrants of the right-hand panels.
Figure 8
Figure 8
Expression of membrane-bound TGF-β on freshly isolated cells from both groups of mice and their similar cytokine secretion profile. (A) Cell surface TGF-β and CD25 expression on freshly isolated CD4+ T cells from inflammation and tolerance groups. The boxed area denotes cells expressing high levels of CD25. (B) CD4+ T cells from the two groups’ cells were cultured with OVA/APCs for two rounds of stimulation (maintaining equal numbers of cells during restimulation). Cells expressing cell surface TGF-β were isolated using PE-labeled anti–TGF-β Ab, anti-PE microbeads, and separation on magnetic columns. Equal numbers of positively selected cells were restimulated with OVA/APCs for 72 hours, and the indicated cytokines in the culture supernatants were measured by ELISA. Cells expressing IL-10 were isolated using the MACS IL-10 secretion assay, stimulated, and assessed for cytokine production as described above. All data are representative of two independent experiments.
Figure 9
Figure 9
Selective FOXP3 expression in cells expressing membrane-bound TGF-β from tolerized mice. TGF-β+ and TGF-β– cells were isolated from both groups of mice, and 4 ∞ 106 cells of each type from each group were maintained in culture for 5 days with Ag and APCs. FOXP3 expression was investigated in the nuclear extracts of the cells (20 mg total protein) and was readily detectable only in the TGF-β+ cells from tolerized mice. CREB-1 expression was used as a loading control.
Figure 10
Figure 10
Cell surface TGF-β is key to the immunosuppressive properties of CD4+ T cells from tolerized mice. Mice were exposed to 1% OVA (tolerance) for 10 days, and spleens were harvested on day 21. CD4+ T cells were prepared by negative selection, and cells expressing TGF-β on the cell surface (TGF-β+) were separated from those devoid of cell surface TGF-β (TGF-β–). The purity of the two populations was assessed by FACS analysis (approximately 75% enrichment of TGF-β+ cells; not shown). (A) Proliferative potential of TGF-β– cells in the absence of TGF-β+ cells. Equal numbers of the two populations were tested for cell proliferation, and culture supernatants were assayed for soluble TGF-β. (B) Production of soluble TGF-β by the two populations. For each group, an average of the concentrations detected with the different doses of OVA is shown.
Figure 11
Figure 11
Adoptive transfer of TGF-β–expressing cells from tolerized mice into naive mice significantly attenuates the development of airway inflammation in the recipient mice. Cells (105) from each group were adoptively transferred into naive BALB/c mice, which were immunized intraperitoneally at the same time with OVA/alum (day 0). The recipients were boosted with OVA/alum 7 days after transfer (day 7) and were challenged by exposure to aerosol of 1% OVA for 7 days, from day 14 to 21. Control mice were immunized with OVA/alum and challenged with aerosolized OVA (not shown). Twenty-four hours after the last OVA challenge, mice were processed for (A) IgE levels in blood, (B) cytokine (IL-13) levels in BAL fluid, and (C and D) pulmonary inflammation. Lung infiltrates were graded as +5 in all TGF-β− cell transfers or in control OVA/OVA immunized mice (not shown) and were between +1 and +2 in mice that received TGF-β+ cells. There were three mice per group, and the results are representative of two independent experiments. *P < 0.05 versus animals that received TGF-β− cells. (E) In separate experiments, after adoptive transfer of TGF-β+ or TGF-β− cells and immunization with OVA/alum, splenic CD4+ T cells were isolated by positive selection from two recipients in each group on day 14. Cells were stimulated with OVA/APCs for 5 days in vitro. Nuclear extracts were prepared and subjected to Western blot analysis for GATA-3 and STAT-6.
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