Bronchoalveolar immunologic profile of acute human lung transplant allograft rejection

Abstract

Bronchoalveolar lavage fluid (BALF) offers a potential means to diagnose acute rejection and could provide insight into the immune mechanisms responsible for lung allograft rejection. Transbronchial biopsies from 29 bronchoscopic procedures were assessed for rejection. Concurrent BALF lymphocyte subsets were examined by flow cytometry, including CD4 and CD8 T cells and their activation status by CD38 expression, natural killer (NK), NK-like T (NT), B, regulatory T, and invariant receptor NK-T cells. Percentages of CD4 were reduced, and CD8 and activation of CD4 T cells correlated with rejection. There were trends for increased NT, reduced NK, and increased B cell percentages with rejection, suggesting potential roles of these cells. Among regulatory cells, the percentages of regulatory T cells decreased and CD4/CD8 invariant NK-T cells increased during rejection, suggesting a proinflammatory profile. A unique BALF lymphocyte profile was associated with rejection and may provide insight into the pathogenesis of allograft rejection.

Figure 1. Flow cytometric definition of lymphocyte subsets

Representative assessment of BALF lymphocytes is shown. Top row: Total lymphocytes were gated using CD45/log side scatter (SSClog), followed by exclusion of dead cells by forward scatter (FSC)/SSClog gating. The CD3⁺/CD14⁻ cells defined by these gates were analyzed as CD4⁺ and CD8⁺ T cell subsets. Middle Row: B cells (CD3⁻/CD19⁺), NK (CD3⁻/CD16⁺/CD56⁺) and NT (CD3⁺/CD16⁺/CD56⁺) cells were defined within a gate of total CD45⁺ lymphocytes with FSC/SSClog exclusion of dead cells. Bottom Row: T cells were defined by CD3/SSClog gating, followed by a FSC/SSClog gating to exclude dead cells. Treg cells (CD4⁺/CD25⁺/Foxp3⁺), activated T cells (CD4⁺/CD38⁺ or CD8⁺/CD38⁺) and iNKT (6B11⁺/Vα24⁺ and CD4^±/CD8^±) were defined from this total T cell population.

Figure 2. Comparisons of lymphocyte subsets in BALF of non-rejectors versus rejectors

Lymphocyte subsets were defined as described in Figure 1. Statistically significant differences between group medians are indicated by asterisks placed above the groups to indicate association with non-rejection or rejection. The number of samples in each group is given above the x-axis labels. The rugs plot each point value and are given along the y-axis. Outliers, if present, are indicated with a ‘q’ located beyond the boxplot whiskers. A) BALF effector and helper T cells and their activation status, B) BALF NK, NT and B cells, C) BALF regulatory cells.

Figure 2. Comparisons of lymphocyte subsets in BALF of non-rejectors versus rejectors

Lymphocyte subsets were defined as described in Figure 1. Statistically significant differences between group medians are indicated by asterisks placed above the groups to indicate association with non-rejection or rejection. The number of samples in each group is given above the x-axis labels. The rugs plot each point value and are given along the y-axis. Outliers, if present, are indicated with a ‘q’ located beyond the boxplot whiskers. A) BALF effector and helper T cells and their activation status, B) BALF NK, NT and B cells, C) BALF regulatory cells.

Figure 2. Comparisons of lymphocyte subsets in BALF of non-rejectors versus rejectors

Lymphocyte subsets were defined as described in Figure 1. Statistically significant differences between group medians are indicated by asterisks placed above the groups to indicate association with non-rejection or rejection. The number of samples in each group is given above the x-axis labels. The rugs plot each point value and are given along the y-axis. Outliers, if present, are indicated with a ‘q’ located beyond the boxplot whiskers. A) BALF effector and helper T cells and their activation status, B) BALF NK, NT and B cells, C) BALF regulatory cells.