Oligoclonal CD4+ T cells in the lungs of patients with severe emphysema

Abstract

Rationale: Within the lungs of patients with severe emphysema, inflammation continues despite smoking cessation. Foci of T lymphocytes in the small airways of patients with emphysema have been associated with disease severity. Whether these T cells play an important role in this continued inflammatory response is unknown.

Objective: The aim of this study was to determine if T cells recruited to the lungs of subjects with severe emphysema contain oligoclonal T-cell populations, suggesting their accumulation in response to antigenic stimuli.

Methods: Lung T-cell receptor (TCR) Vbeta repertoire from eight patients with severe emphysema and six control subjects was evaluated at the time of tissue procurement (ex vivo) and after 2 weeks of culture with interleukin 2 (in vitro). Junctional region nucleotide sequencing of expanded TCR-Vbeta subsets was performed.

Results: No significantly expanded TCR-Vbeta subsets were identified in ex vivo samples. However, T cells grew from all emphysema (n = 8) but from only one of the control lung samples (n = 6) when exposed to interleukin 2 (p = 0.0013). Within the cultured cells, seven major CD4-expressing TCR-Vbeta subset expansions were identified from five of the patients with emphysema. These expansions were composed of oligoclonal populations of T cells that had already been expanded in vivo.

Conclusion: Severe emphysema is associated with inflammation involving T lymphocytes that are composed of oligoclonal CD4+ T cells. These T cells are accumulating in the lung secondary to conventional antigenic stimulation and are likely involved in the persistent pulmonary inflammation characteristic of severe emphysema.

Figure 1.

Surface markers expressed by T cells isolated from lungs and blood of patients with severe emphysema (n = 6). The upper panel demonstrates the percentage of CD4⁺ T cells that express CD28 (a costimulatory receptor), CD45RO (a marker of previous activation), CD62L (a lymph node homing molecule), and CCR7 (a homing receptor specific for the lymph node). The lower panel demonstrates CD8⁺ T-cell expression of these same markers. Data are presented as mean ± SEM. White bars = peripheral blood mononuclear cells; gray bars = upper lung lymphocytes; black bars = lower lung lymphocytes.

Figure 2.

T-cell receptor (TCR) Vβ region expression on T cells from the blood and lower and upper lung of patients with severe emphysema (n = 7). The upper panel demonstrates TCR-Vβ expression on CD4⁺ T cells, and the lower panel demonstrates expression on CD8⁺ cells. Data are expressed as mean ± SEM.

Figure 3.

Blasting of lung T cells from subjects with emphysema and normal subjects after 2 weeks in culture with interleukin (IL) 2. (A) Light microscopy of lung tissue culture reveals large clusters of proliferating lymphocytes in a representative emphysema culture (left panel), which are distinctly absent in a representative control culture (right panel). (Original magnification: 100×) (B) Forward- versus side-scatter density plots are shown for representative emphysema (left panel) and control (right panel) lung tissue culture. Blasting lymphocytes are found within the upper gate with the resting lymphocytes in the lower gate. The percentage of blasting or resting lymphocytes per total cells collected is shown. (C) A density plot of CD4 versus CD8 expression on CD3⁺ T cells from a representative subject with emphysema after in vitro culture in the presence of IL-2 is shown.

Figure 4.

Density plots for CD4 versus TCR-Vβ6.7 and TCR-Vβ17 from a representative emphysematous lung tissue culture with IL-2. Representative density plot from Patient 1 demonstrating increased expression of TCR-Vβ6.7 and TCR-Vβ17 on cultured CD4⁺ T-cell blasts derived from the upper and lower lobes is shown.

Figure 5.

TCR-Vβ repertoire in cultured CD4⁺ T cells. TCR-Vβ repertoire of cultured CD4⁺ T cells compared with ex vivo expression in four subjects with emphysema who demonstrated an expansion (> twofold) in the lung after IL-2 exposure in culture compared with ex vivo expression. Data are expressed as the mean percentage of CD4⁺ T cells expressing a particular Vβ. Asterisks denote the expanded TCR-Vβ region in the individual patients.

Figure 6.

Analysis of deduced TCRB junctional region amino acid sequences expressed in ex vivo T cells and cultured CD4⁺ T cells from the lungs of four patients. TCRB sequences found three times or more in any sample or present in both ex vivo and cultured T-cell populations are shown. For each T-cell clone, the entire TCRB junctional region is shown, extending from the 5′ end of the selected TCRBV family gene, including the highly rearranged NBDN gene segment, and ending at the selected BJ gene segment. The column TCRBJ notes for each clone which BJ gene family member was selected during genetic rearrangement. For more detail on the gene rearrangement of TCRB junctional region, see the online supplement. The NBDN region of sequences found in both ex vivo and cultured T-cell populations are shown in bold. The number of identical sequences (defined at the nucleotide level) is shown over the total number of sequences analyzed for a given anatomic site or culture sample. These sequence data are available from GenBank under accession numbers AY726734 to AY726754.