Donor leukocyte trafficking during human ex vivo lung perfusion

Abstract

Background: Ex vivo lung perfusion (EVLP) is used to assess and preserve lungs prior to transplantation. However, its inherent immunomodulatory effects are not completely understood. We examine perfusate and tissue compartments to determine the change in immune cell composition in human lungs maintained on EVLP.

Methods: Six human lungs unsuitable for transplantation underwent EVLP. Tissue and perfusate samples were obtained during cold storage and at 1-, 3- and 6-h during perfusion. Flow cytometry, immunohistochemistry, and bead-based immunoassays were used to measure leukocyte composition and cytokines. Mean values between baseline and time points were compared by Student's t test.

Results: During the 1st hour of perfusion, perfusate neutrophils increased (+22.2 ± 13.5%, p < 0.05), monocytes decreased (-77.5 ± 8.6%, p < 0.01) and NK cells decreased (-61.5 ± 22.6%, p < 0.01) compared to cold storage. In contrast, tissue neutrophils decreased (-22.1 ± 12.2%, p < 0.05) with no change in monocytes and NK cells. By 6 h, perfusate neutrophils, NK cells, and tissue neutrophils were similar to baseline. Perfusate monocytes remained decreased, while tissue monocytes remained unchanged. There was no significant change in B cells or T cell subsets. Pro-inflammatory cytokines (IL-1b, G-CSF, IFN-gamma, CXCL2, CXCL1 granzyme A, and granzyme B) and lymphocyte activating cytokines (IL-2, IL-4, IL-6, IL-8) increased during perfusion.

Conclusions: Early mobilization of innate immune cells occurs in both perfusate and tissue compartments during EVLP, with neutrophils and NK cells returning to baseline and monocytes remaining depleted after 6 h. The immunomodulatory effect of EVLP may provide a therapeutic window to decrease the immunogenicity of lungs prior to transplantation.

Keywords: donor leukocyte; ex vivo lung perfusion; immune modulation.

FIGURE 1

Physiologic data of ex vivo lung perfusion of six human lungs. (A) Pulmonary artery pressure. (B) Post organ perfusate pH. (C) P/F ratio – ratio of partial pressure of oxygen (PaO₂) of perfusate downstream of organ to fraction of inspired oxygen (FiO₂). (D) Ratio wet:dry. * – Lung 11 (red) demonstrated persistently elevated pulmonary artery perfusion pressure in addition to histology suggestive of chronic intra‐alveolar hemorrhage

FIGURE 2

Representative histology for donor lung demonstrated structural integrity after 6 h of perfusion. H&E staining was used to identify airway (2.5×), alveoli (2.5×) and interstitial compartment (10×), while CD31 immunohistochemistry was used to identify pulmonary capillaries (20×). (All slides obtained from lung 8)

FIGURE 3

Percentage change of each myeloid immune cell frequency during perfusion. A percentage change of 0% suggests there is no change in the innate immune cell frequency from time point 0th hour. Average values of percentage changes (orange line) as well as standard deviations were calculated (vertical bar). A two‐sided, paired t test was performed to compare percentages of each time point to those at time 0 h. (*: p < 0.05; **: p < 0.01). (A) Neutrophil percentage change in perfusate (ANOVA p value = 0.14) and lung tissue (ANOVA p value = 0.21). (B) Eosinophil percentage change in perfusate (ANOVA p value = 0.05) and lung tissue (ANOVA p value < 0.01). (C) Classocal monocyte percentage change in perfusate (ANOVA p value < 0.01) and lung tissue (ANOVA p value = 0.10). (D) Intermediate monocyte percentage change in perfusate (ANOVA p value < 0.01) and lung tissue (ANOVA p value = 0.29). (E) Non‐classical monocyte percentage change in perfusate (ANOVA p value = 0.51) and lung tissue (ANOVA p value = 0.48)

FIGURE 4

Percentage change of each lymphoid immune cell frequency during perfusion. A percentage change of 0% suggests there is no change in the innate immune cell frequency from time point 0th hour. Average values of percentage changes (orange line) as well as standard deviations were calculated (vertical bar). A two‐sided, paired t test was performed to compare percentages of each time point to those at time 0 h. (*: p < 0.05; **: p < 0.01). (A) NK cell percentage change in perfusate (ANOVA p value < 0.01) and lung tissue (ANOVA p value = 0.70). (B) NK T cell percentage change in perfusate (ANOVA p value = 0.30) and lung tissue (ANOVA p value = 0.58). (C) CD4 T cell percentage change in perfusate (ANOVA p value = 0.47) and lung tissue (ANOVA p value = 0.63). (D) CD8 T cell percentage change in perfusate (ANOVA p value = 0.10) and lung tissue (ANOVA p value = 0.81). (E) B cell percentage change in perfusate (ANOVA p value = 0.44) and lung tissue (ANOVA p value = 0.51)

FIGURE 5

Identification of Treg cells using immunohistochemistry (ROI: 931 um × 698 um). (A) Representative immunohistochemistry histology. DAPI‐ Blue; CD3‐ Cyan; CD4‐Yellow; FoxP3‐Red. (Taken from Lung 8). (B) Tissue regulatory T cell percentages prior to (0 h) and following EVLP (6 h). *Lung 11 was excluded from calculation of average values