In situ activation of helper T cells in the lung

Abstract

To better understand the lung and systemic responses of helper T cells mediating memory immunity to Mycobacterium tuberculosis, we used three- and four-color flow cytometry to study the surface phenotype of CD4(+) lymphocytes. Bronchoalveolar lavage (BAL) fluid and peripheral blood (PB) samples were obtained from a total of 25 subjects, including 10 tuberculosis (TB)-infected subjects, 8 purified-protein-derivative-negative subjects, and 7 purified-protein-derivative-positive subjects. In marked contrast to CD4(+) lymphocytes from PB (9% +/- 5% expressing CD45RA and CD29), the majority (55% +/- 16%) of CD4(+) lymphocytes in BAL (ALs) simultaneously expressed CD45RA, a naïve T-cell marker, and CD29, members of the very late activation family. Further evaluation revealed that CD4(+) ALs expressed both CD45RA and CD45RO, a memory T-cell marker. In addition, the proportion of CD4(+) lymphocytes expressing CD69, an early activation marker, was drastically increased in BAL fluid (83% +/- 9%) compared to PB (1% +/- 1%), whereas no significant difference was seen in the expression of CD25, the low-affinity interleukin 2 receptor (34% +/- 15% versus 40% +/- 16%). More importantly, we identified a minor population of CD69(bright) CD25(bright) CD4(+) lymphocytes in BAL (10% +/- 6%) that were consistently absent from PB (1% +/- 1%). Thus, CD4(+) lymphocytes in the lung paradoxically coexpress surface molecules characteristic of naïve and memory helper T cells as well as surface molecules commonly associated with early and late stages of activation. No difference was observed for ALs obtained from TB-infected and uninfected lung segments in this regard. It remains to be determined if these surface molecules are induced by the alveolar environment or if CD4(+) lymphocytes coexpressing this unusual combination of surface molecules are selectively recruited from the circulation. Our data suggest that ex vivo experiments on helper T-cell subsets that display distinctive phenotypes may be pivotal to studies on the human immune response to potential TB vaccines.

FIG. 1

Expression of CD29 and CD45RA and simultaneous expression of CD29 and CD45RA on helper T cells from BAL fluid and PB. Viable cells were labeled with FITC–anti-CD45RA, PE–anti-CD29, and CC–anti-CD4, fixed, and evaluated by FACS analysis. A minimum of 2,500 CD4⁺ lymphocyte-sized events were analyzed. The long bar indicates the median and the short bars indicate the first and third quartiles for each subject group. IN, involved lobe. BAL fluid values significantly different from PB values (P ≤ 0.05) are marked with an asterisk. All TB patients were nonanergic.

FIG. 2

Simultaneous expression of CD29, CD45RA, and CD45RO on CD4⁺ lymphocytes from the PB and BAL fluid obtained from the involved and uninvolved lobes of a TB patient. Viable cells were labeled with FITC–anti-CD45RA, PE–anti-CD29, APC–anti-CD45RO, and ECD–anti-CD4, fixed, and evaluated by FACS analysis. A minimum of 2,500 CD4⁺ lymphocyte-sized events were analyzed. The distribution of events in each quadrant is given as percent CD4⁺ lymphocytes. Quadrants located in the same position are used to analyze histograms within the same panel.

FIG. 3

Expression of CD25 and CD69 and enhanced expression of both markers on helper T cells from BAL fluid and PB. Viable cells were labeled with FITC–anti-CD69, PE–anti-CD25, and CC–anti-CD4, fixed, and analyzed with a FACS. A minimum of 2,500 CD4⁺ lymphocyte-sized events were analyzed. The long bar indicates the median and the short bars indicate the first and third quartiles for each subject group. IN, involved lobe. BAL fluid values significantly different from PB values (P ≤ 0.05) are marked with an asterisk. All TB patients were nonanergic.

FIG. 4

Expression of CD25 and CD69 on CD4⁺ lymphocytes from the PB and BAL fluid of a healthy PPD⁻ subject. Viable cells were labeled and analyzed as described for Fig. 3. The distribution of events in each quadrant is given as percent CD4⁺ lymphocytes. Quadrants located in the same position are used to analyze both histograms. A cluster of CD25^bright CD69^bright events in the right histogram is indicated by an arrowhead. This subset constituted 8.4% of the CD4⁺ lymphocytes in this BAL fluid specimen.