ADAM17-Mediated Processing of TNF-α Expressed by Antiviral Effector CD8+ T Cells Is Required for Severe T-Cell-Mediated Lung Injury

Influenza infection in humans evokes a potent CD8+ T-cell response, which is important for clearance of the virus but may also exacerbate pulmonary pathology. We have previously shown in mice that CD8+ T-cell expression of TNF-a is required for severe and lethal lung injury following recognition of an influenza antigen expressed by alveolar epithelial cells. Since TNF-a is first expressed as a transmembrane protein that is then proteolytically processed to release a soluble form, we sought to characterize the role of TNF-a processing in CD8+ T-cell-mediated injury. In this study we observed that inhibition of ADAM17-mediated processing of TNF-a by CD8+ T cells significantly attenuated the diffuse alveolar damage that occurs after T-cell transfer, resulting in enhanced survival. This was due in part to diminished chemokine expression, as TNF-aprocessing was required for lung epithelial cell expression of CXCL2 and the subsequent inflammatory infiltration. We confirmed the importance of CXCL2 expression in acute lung injury by transferring influenza-specific CD8+ T cells into transgenic mice lacking CXCR2. These mice exhibited reduced airway infiltration, attenuated lung injury, and enhanced survival. Theses studies describe a critical role for TNF-a processing by CD8+ T cells in the initiation and severity of acute lung injury, which may have important implications for limiting immunopathology during influenza infection and other human infectious or inflammatory diseases

ADAM17 expression on transferred CD8⁺ T cells is required for enhanced airway inflammation.

<p>SPC-HA transgenic mice received 10⁷ WT or ADAM17^−/− HA-specific CD8⁺ T cells via tail vein injection. Twenty-four hours after T-cell transfer, cell-free BAL fluid was prepared and the levels of (A) CXCL2, (B) CCL2, (C) IP-10, (D) CCL11, (E) CXCL5, and (F) G-CSF were determined by Luminex assay. Data represent mean ± standard deviation. Data are representative of two mice from two independent experiments with a total of 4 mice per group. *<i>P</i><0.05, **<i>P</i><0.01.</p

CD8⁺ T-cell-mediated acute lung injury depends in part on CXCR2 signaling.

<p>WT and CXCR2^−/− SPC-HA transgenic mice received 5×10⁶ WT HA-specific CD8⁺ T cells via tail vein injection. (A) Survival of mice after T-cell transfer was monitored daily and a striking difference in survival (<i>P</i><0.001) was observed. Representative H&E stained lung sections from (B) WT-HA or (C) CXCR2^−/−-HA mice harvested 5 days after transfer of WT HA-specific CD8⁺ T cells shown at 10x magnification with 40x inset. Data are representative of at least two independent experiments with 4-5 mice per group.</p

CD8⁺ T-cell processing of TNF-α is required for lung epithelial cell expression of CXCL2.

<p>MLE-K^d cells were pulsed with HA_210−219 peptide and co-cultured with HA-specific CD8⁺ T cells for 5 hours. In some experiments, recombinant mouse TNF-α was added to the culture supernatant. Cell-free supernatant was analyzed for expression of (A) TNF-α, (C) CCL2, and (D) CXCL2 by ELISA. (B) Alternatively, total TNF-α production by HA-specific CD8⁺ T cells after PMA/ionomycin stimulation with protease inhibitor and detergent solubilization was measured by ELISA. Data represents mean ± standard deviation. Data are representative of three independent experiments with each condition conducted in triplicate. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001.</p

ADAM17 expression on transferred CD8⁺ T cells is required for acute lung injury.

<p>SPC-HA transgenic mice received WT or ADAM17^−/− HA-specific CD8⁺ T cells via tail vein injection. (A) Survival of mice after transfer of 10⁷ T cells was monitored and a difference in survival (<i>P</i><0.05) was observed. Representative H&E stained lung sections from SPC-HA transgenic mice harvested 5 days after transfer of 5×10⁶ (B) WT or (C) ADAM17^−/− CD8⁺ T cells shown at 10x magnification with 40x inset. (D) ELISA was used to assess the levels of albumin in cell-free BAL fluid after transfer of 10⁷ T cells. (E) Peripheral oxygen saturation was measured in mice before and after transfer of 10⁷ T cells using the MouseOx System. Data represent mean ± standard deviation. Data are representative of at least three independent experiments with 3-4 mice per group. *<i>P</i><0.05, **<i>P</i><0.01.</p

Processing of TNF-α by CD8⁺ T cells is required for severe and lethal lung injury.

<p>SPC-HA transgenic mice received WT or tmTNF HA-specific CD8⁺ T cells via tail vein injection. (A) Survival of mice after transfer of 10⁷ T cells was monitored and a significant difference in survival (<i>P</i><0.05) was observed. Representative H&E stained lung sections from SPC-HA transgenic mice harvested 5 days after transfer of 5×10⁶ (B) WT or (C) tmTNF CD8⁺ T cells shown at 10x magnification with 40x inset. (D) Carbon monoxide uptake was measured to assess lung function. (E) Percentage of neutrophils in whole lung homogenates was determined by morphological analysis of cells after staining cytospin preparations. (F) ELISA was used to assay CXCL2 expression in cell-free whole lung homogenates. Data represent mean ± standard deviation. Data are representative of at least two independent experiments with 3-4 mice per group. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001.</p

CD8⁺ T-cell expression of ADAM17 is required for TNF-α processing following antigen recognition <i>in vivo</i>.

<p>SPC-HA transgenic mice received 10⁷ WT or ADAM17^−/− HA-specific CD8⁺ T cells via tail vein injection. ELISA was used to assess (A) TNF-α and (B) IFN-γ expression in cell-free BAL fluid. Alternatively, HA-specific CD8⁺ T cells were labeled with CFSE prior to transfer. Twenty-four hours after transfer, cells were recovered from (C) BAL and (D) lung homogenates and assessed by flow cytometric analysis to determine the total number of transferred HA-specific CD8⁺ T cells present. Data represent mean ± standard deviation. Data are representative of two or more independent experiments with 4 mice per group. **<i>P</i><0.01.</p