Cutting edge: Pseudomonas aeruginosa abolishes established lung transplant tolerance by stimulating B7 expression on neutrophils

Abstract

The mechanisms that link bacterial infection to solid organ rejection remain unclear. In this study, we show that following the establishment of lung allograft acceptance in mice, Pseudomonas aeruginosa airway infection induces a G-CSF-dependent neutrophilia that stimulates acute rejection. Graft-infiltrating neutrophils sharply upregulate the B7 molecules CD80 and CD86, but they do not express CD40 or MHC class II in response to P. aeruginosa infection. Neutrophil B7 promotes naive CD4(+) T cell activation and intragraft IL-2(+), IFN-γ(+), and IL-17(+) T lymphocyte accumulation. Intravital two-photon microscopy reveals direct interactions between neutrophils and CD4(+) T cells within pulmonary allografts. Importantly, lung rejection in P. aeruginosa-infected recipients is triggered by CD80/86 on neutrophils and can be prevented by B7 blockade without affecting clearance of this pathogen. These data show that neutrophils enhance T cell activation through B7 trans-costimulation and suggest that inhibiting neutrophil-mediated alloimmunity can be accomplished without compromising bacterial immune surveillance.

Figure 1

PA-induced neutrophilia prevents established lung allograft tolerance. Balb/c → B6 lung allografts analyzed by (a) H & E graft histology (representative of N ≥ 4) or for (b) the accumulation of indicated intragraft T lymphocytes (N ≥ 4) up to 3-weeks after either saline or PA airway instillation. (c) Intragraft CD4⁺ T cells isolated 3-weeks after either saline or PA airway instillation into Balb/c → B6 recipients were stimulated with Balb/c splenocytes and analyzed for IL-17 or IFN-γ expression (N ≥ 3). (d) Balb/c → B6 lung allograft BAL neutrophil numbers after either saline or PA airway instillation (N ≥ 4) or (e) CFU after PA infection (N=3). (f) BAL neutrophils quantified 1-day after hkPA or saline airway instillation into Balb/c → B6 recipients (N=4) treated with indicated Abs or 1-day after hkPA airway instillation into Balb/c → CD11b^−/− recipients (N=3). (g) Allograft tissue isolated 3-weeks after PA infection from lung recipients treated as in (f) and analyzed by H & E histology (N ≥ 3) or for (h) indicated intragraft T lymphocyte accumulation (N ≥ 3).

Figure 2

Neutrophil B7 directly enhances naïve CD4⁺ T cell activation. (a) BAL neutrophils from uninfected (black line) or PA-infected (red line) Balb/c → B6 recipients were stained with indicated Abs or isotype (shaded histogram) Abs (N=4). (b) IL-2 levels from naïve CD4⁺ T cells cultured with neutrophils from PA-infected mice (B6(PA)) or stimulated by CD3 beads in the absence (no PMN) or presence of neutrophils from uninfected mice (B6), B6(PA), PA-infected CD80^−/−86^−/− mice (CD80^−/− 86^−/− (PA)) or B6(PA) and 15 μg/ml CTLA4Ig. (c) CFSE-labeled naïve CD4⁺ T cells cultured as in (b) for 72 hrs and analyzed for responder frequency where mitotic divisions are shown below the x ordinate (N=4).

Figure 3

Lung allograft rejection in PA-infected recipients is neutrophil B7 dependent. (a) Percent abundance of indicated intragraft CD4⁺ and CD8⁺ T cells one day after saline or PA inoculation into Balb/c → B6 lung recipients that either received control Ig or G-CSF Abs with B6 or CD80^−/−86^−/− neutrophils (N ≥ 3). (b) H & E graft histology (N=4) and indicated intragraft T lymphocyte accumulation (N=4) of Balb/c → B6 lung recipients 3-weeks after that receiving PA, G-CSF Abs and either B6 or CD80^−/−86^−/− neutrophils. (c) Intravital 2-photon imaging within allografts of Balb/c → B6 CD11c-EYFP LysM-GFP lung recipients that received CMTPX-labeled CD4⁺ T cells one day after PA-infection. A zoomed view of a typical interaction between a CD4⁺ T cell (red-yellow arrows), DC (green-hollow arrow) and neutrophil (blue-white arrows) in the y-x (left) and z-x (right) plane (N=2). (d) IL-2 produced by intragraft CD4⁺ T cells from uninfected Balb/c → B6 lungs cultured alone (No DC) or co-cultured with allogeneic Balb/c bone-marrow derived DC (DC) or co-cultured with DC in combination with B6 neutrophils from PA-infected B6 mice (DC + B6 PMN) or DC in combination with PMN from PA-infected CD80^−/−86^−/− mice (DC + CD80^−/−86^−/− PMN) or co-cultured with DC separated from B6 neutrophils by transwells (DC II B6 PMN) (N ≥ 3) Lung recipients on POD 7 received PA along with 200μg of Control Ig, CD154 or CTLA4Ig and were either analyzed 3-weeks later for (e) H & E graft histology (N ≥ 3) and (f) intragraft T lymphocyte accumulation (N ≥ 3) or assessed for (g) BAL PMN numbers (N ≥ 3) one day after infection or (f) BAL CFU (N ≥ 3) for up to 3-days after infection.