Airway hyperresponsiveness through synergy of gammadelta} T cells and NKT cells

Abstract

Mice sensitized and challenged with OVA were used to investigate the role of innate T cells in the development of allergic airway hyperresponsiveness (AHR). AHR, but not eosinophilic airway inflammation, was induced in T cell-deficient mice by small numbers of cotransferred gammadelta T cells and invariant NKT cells, whereas either cell type alone was not effective. Only Vgamma1+Vdelta5+ gammadelta T cells enhanced AHR. Surprisingly, OVA-specific alphabeta T cells were not required, revealing a pathway of AHR development mediated entirely by innate T cells. The data suggest that lymphocytic synergism, which is key to the Ag-specific adaptive immune response, is also intrinsic to T cell-dependent innate responses.

FIGURE 1

Vγ1⁺ γδ T cells from naive donors enhance AHR when αβ T cells are present. AHR was monitored by measuring R_L (a, c, and e) and C_dyn (b, d, and f). a and b, Reconstitution of γδ T cell-deficient mice with Vγ1⁺ cells restores AHR. OVA-sensitized B6.TCR-δ^−/− mice received 1 × 10⁴ splenic Vγ1⁺ γδ T cells from untreated (NT) or sensitized (2ip) C57BL/6 donors, before airway challenge. Untreated recipients (NT) and recipients that were sensitized and challenged, but did not receive cells (2ip3N), are also shown. Results for each group are presented as means ± SEM (n = 8). Significant differences between 2ip3N and 2ip3N + Vγ1 groups are indicated: **, p < 0.01; ***, p < 0.001. c and d, Vγ1⁺ cells from αβ T cell-deficient mice are still capable of restoring AHR. OVA-sensitized B6.TCR-δ^−/− mice received 1 × 10⁴ splenic Vγ1⁺ γδ T cells from untreated (NT) or sensitized (2ip) B6.TCR-β^−/− donors, before airway challenge. Recipients that were sensitized and challenged, but did not receive cells (2ip3N), are also shown. Results for each group are presented as means ± SEM (n = 12). Significant differences between 2ip3N and 2ip3N + Vγ1 groups are indicated as follows: *, p < 0.05; **, p < 0.01; ***, p < 0.001. e and f, Vγ1⁺ cells together with αβ T cells restore AHR in T cell-deficient mice. OVA-sensitized B6.TCR-β^−/−δ^−/− mice received 1 × 10⁴ splenic Vγ1⁺ γδ T cells from sensitized B6.TCR-β^−/− donors and 2 × 10⁶ αβ T cells from sensitized B6.TCR-δ^−/− donors (Vγ1 + αβ), before airway challenge. Recipients that were sensitized and challenged, but did not receive cells (2ip3N), or received only Vγ1⁺ cells (Vγ1) or only αβ T cells (αβ), are also shown. Results for each group are presented as means ± SEM (n = 4–9). Significant differences between sensitized and challenged mice, which received no cells, or Vγ1⁺ cells plus αβ T cells, are indicated as follows: *, p < 0.05; **, p < 0.01; ***, p < 0.001.

FIGURE 2

Vγ1⁺ γδ T cells fail to mediate AHR when NK1.1⁺ cells are absent. AHR was monitored by measuring R_L (a and e) and C_dyn (b and f). a and b, Vγ1⁺ cells fail to restore AHR in γδ T cell-deficient mice pretreated with anti-NK1.1 mAb. OVA-sensitized B6.TCR-δ^−/− mice were treated with mAb PK136 (200 μg i.v.), received 1 × 10⁴ splenic Vγ1⁺ γδ T cells 3 days later, and were then challenged via the airways (NK1.1-depleted, 2ip3N + Vγ1). Recipients that were only sensitized and challenged (2ip3N) and those that were sensitized and challenged and treated with the Ab (NK1.1-depleted, 2ip3N) are also shown. Results for each group are presented as means ± SEM (n = 4). Significant differences between 2ip3N and 2ip3N, NK1.1-depleted groups are indicated as follows: *, p < 0.05. c and d, Depletion of NK1.1⁺ cells within the Vγ1⁺ population. C57BL/6 mice were treated with mAb PK136 (200 μg i.v.) and 3 days later, NAD splenocytes were stained for TCR-δ, Vγ1, and NK1.1. Cytofluorimetric analysis shows that ~20% of gated splenic Vγ1⁺ γδ T cells express NK1.1 (c) and that the treatment with mAb PK136 removes most of these cells (d). e and f, NK1.1⁻ Vγ1⁺ γδ T cells enhance AHR. OVA-sensitized B6.TCR-δ^−/− mice received 1 × 10⁴ splenic Vγ1⁺ γδ T cells from C57BL/6 donors before airway challenge. The cell donors were either untreated or received mAb PK136 i.v., 3 days before cell transfer (Vγ1 from B6 and Vγ1 from NK1.1-depleted B6). Recipients that were sensitized and challenged, but did not receive cells, are also shown (no cells transferred). Results for each group are presented as means ± SEM (n = 7–8). Significant differences between mice that received no cells or Vγ1 cells are indicated as follows: *, p < 0.05; **, p < 0.01; ***, p < 0.001. Significant difference between mice that had received Vγ1⁺ cells from B6 vs from NK1.1-depleted B6 (f) is as follows: #, p < 0.05.

FIGURE 3

Vγ1⁺ γδ T cells synergize with NKT cells in mediating AHR. AHR was monitored by measuring R_L (a, c, and e) and C_dyn (b, d, and f). a and b, Vγ1⁺ cells together with NK1.1⁺ αβ T cells restore AHR in T cell-deficient mice. OVA-sensitized B6.TCR-β^−/−δ^−/− mice received 1 × 10⁴ splenic Vγ1⁺ γδ T cells from sensitized B6.TCR-β^−/− donors and 2 × 10⁴ NK1.1⁺ αβ T cells from sensitized B6.TCR-δ^−/− donors (Vγ1 + NK1.1⁺αβ), before airway challenge. Recipients that were sensitized and challenged, but did not receive cells (no cell transferred), or that received 2 × 10⁴ NK1.1⁺ αβ T cells (NK1.1⁺αβ) alone, Vγ1⁺ cells plus 2 × 10⁶ NK1.1⁻ αβ T cells (Vγ1 + NK1.1⁻αβ), or Vγ1⁺ cells plus 9 × 10⁴ NK1.1⁺ non-T cells (Vγ1 + NK), are also shown. Results for each group are presented as means ± SEM (n = 6–7). Significant differences between mice that received no cells or Vγ1⁺ cells plus NK1.1⁺ αβ T cells are indicated as follows: **, p < 0.01; ***, p < 0.001. c and d, Vγ1⁺ cells together with CD1d tetramer⁺ αβ T cells restore AHR in T cell-deficient mice. OVA-sensitized B6.TCR-β^−/−δ^−/− mice received 1 × 10⁴ splenic Vγ1⁺ γδ T cells from sensitized B6.TCR-β^−/− donors and 2 × 10⁴ CD1d tetramer⁺ αβ T cells from sensitized B6.TCR-δ^−/− donors (Vγ1 + Tet⁺ αβ), before airway challenge. Recipients that were sensitized and challenged, but did not receive cells (no cells), or that received 2 × 10⁴ Tet⁺ αβ T cells (Tet⁺ αβ) alone, or Vγ1⁺ cells plus 2 × 10⁴ Tet- αβ T cells (Vγ1 + Tet⁻ αβ), are also shown. Results for each group are presented as means ± SEM (n = 4–5). Significant differences between mice that received no cells or Vγ1⁺ plus NK1.1⁺ αβ T cells are indicated as follows: ***, p < 0.001. e and f, Only NK1.1⁺ αβ T cells that are also CD1d tetramer⁺ synergize with Vγ1⁺ γδ T cells in mediating AHR. OVA-sensitized B6.TCR-β^−/−δ^−/− mice received 1 × 10⁴ splenic Vγ1⁺ γδ T cells from sensitized B6.TCR-β^−/− mice and 2 × 10⁴ NK1.1⁺ CD1d tetramer⁺ αβ T cells from sensitized B6.TCR-δ^−/− donors (Vγ1 + NK1.1⁺Tet⁺ αβ), before airway challenge. Recipients that were sensitized and challenged, but did not receive cells (no cell transferred), or that received Vγ1⁺ cells and 2 × 10⁴ NK1.1⁺Tet⁻ αβ T cells (Vγ1 + NK1.1⁺Tet⁻ αβ), or Vγ1⁺ cells plus 2 × 10⁴ NK1.1⁻Tet⁻ αβ T cells (Vγ1 + NK1.1⁻Tet⁻ αβ), are also shown. Results for each group are presented as means ± SEM (n = 5–6). Significant differences between mice that received no cells or Vγ1⁺ cells plus NK1.1⁺ αβ T cells are indicated as follows: *, p < 0.05; **, p < 0.01; ***, p < 0.001.

FIGURE 4

Comparison of Vγ1⁺ γδ T cells expressing different Vδs for their ability to mediate AHR. a, Vδ expression among Vγ1⁺ γδ T cells in B6.TCR-β^−/− spleen. NAD splenocytes of adult B6.TCR-β^−/− mice were stained with Abs against Vγ1, TCR-δ, and several Vδs, and analyzed cytofluorimetrically. Results for each group are presented as means ± SEM (n = 5–11). b and c, Reconstitution of γδ T cell-deficient mice with Vδ⁺ fractions of Vγ1⁺ cells. OVA-sensitized B6.TCR-δ^−/− mice received 1 × 10⁴ sorted Vγ1⁺ γδ T cells from untreated B6.TCR-β^−/− spleen, expressing the indicated Vδs, before airway challenge (2ip3N + Vγ1). Recipients that were sensitized and challenged, but did not receive cells (no cell transferred), are also shown. AHR was monitored by measuring R_L (b) and C_dyn (c). Results for each group are presented as means ± SEM (n = 5–8). Significant differences between mice that received no cells or Vγ1⁺ cells are indicated as follows: **, p < 0.01; ***, p < 0.001.