Cutting edge: contribution of lung-resident T cell proliferation to the overall magnitude of the antigen-specific CD8 T cell response in the lungs following murine influenza virus infection

Abstract

Following influenza virus infection, CD8 T cells encounter mature, Ag-bearing dendritic cells within the draining lymph nodes and undergo activation, programmed proliferation, and differentiation to effector cells before migrating to the lungs to mediate viral clearance. However, it remains unclear whether CD8 T cells continue their proliferation after arriving in the lungs. To address this question, we developed a novel, in vivo, dual-label system using intranasal CFSE and BrdU administration to identify virus-specific CD8 T cells that are actively undergoing cell division while in the lungs. With this technique we demonstrate that a high frequency of virus-specific CD8 T cells incorporate BrdU while in the lungs and that this lung-resident proliferation contributes significantly to the magnitude of the Ag-specific CD8 T cell response following influenza virus infection.

FIGURE 1

Proliferation within the lungs by pulmonary antigen-specific CD8 T cells following influenza virus infection. On days 0–10 p.i., influenza-infected mice were treated i.n. with CFSE followed by BrdU as shown in supplemental Figure 2A. 4 h later, mice were sacrificed and their lungs examined by flow cytometry for total numbers of CFSE⁺BrdU⁺ (black bars), CFSE⁺BrdU^neg (grey bars) and CFSE^neg NP₁₄₇-specific tetramer⁺(A) and HA₅₃₃-specific tetramer⁺(B) CD8 T cells. The lungs were also analyzed for the frequency (C) of CFSE⁺ NP₁₄₇-specific tetramer⁺(squares) and HA₅₃₃-specific tetramer⁺(triangles) CD8 T cells that incorporated BrdU during the 4-hour window of analysis. Data are representative of 2 separate experiments. n=3–5 mice/group.

FIGURE 2

Lung-resident CD8 T cell proliferation contributes to the overall magnitude of the virus-specific CD8 T cell response in the lungs. Influenza-infected mice were administered FTY720 i.p. daily starting on days 4 (grey bars), 5 (black bars), 6 (diagonal bars), 7 (horizontal bars) or 8 (hatched bars) p.i‥ Mice that did not receive FTY720 were included as controls (Nil, white bars). Groups were treated with CFSE-BrdU on days 5, 6, 7, 8 and 10 p.i. as shown in supplemental Figure 2A. 4 h later, the mice were sacrificed and their lungs examined by flow cytometry for: (A) cell numbers of influenza virus-specific tetramer⁺ CD8 T cells; and (B) frequencies of influenza virus-specific tetramer⁺ CD8 T cells that incorporated BrdU during the analysis. Data are representative of 2 separate experiments. n=3–5 mice/group.