Segregated autologous CD4⁺CD28⁺ and CD4⁺CD28^null cells from peripheral blood of IPF patients (n = 9) had similar proliferations (determined by BrdU incorporation) in 5-day control (unstimulated) cultures, as well as after stimulation with plate-bound anti-CD3 monoclonal antibody.

<p>Segregated autologous CD4⁺CD28⁺ and CD4⁺CD28^null cells from peripheral blood of IPF patients (n = 9) had similar proliferations (determined by BrdU incorporation) in 5-day control (unstimulated) cultures, as well as after stimulation with plate-bound anti-CD3 monoclonal antibody.</p

Subject Characteristics.

<p>CD28% High denotes those subjects in whom ≥82% of their circulating CD4 T-cells co-express CD28. CD28% Low denotes those subjects in whom <82% of their circulating CD4 T-cells express CD28. CD28% is defined here as the proportion of circulating CD4 T-cells that also express CD28. FVC; forced vital capacity, FVC%p percentage of normal predicted FVC, FEV1/FVC; forced expiratory volume in 1 second, DLCO; diffusing capacity for carbon monoxide, DLCO%p; percentage of normal predicted DLCO. One CD28% High biopsy showed end-stage, honeycombed fibrotic lung, whereas all other histologic patterns were usual, interstitial pneumonia. *p<0.0001, **p = 0.007; †p<0.045.</p

Cytokine elaborations by autologous CD4 subpopulations of IPF patients.

<p>Initial (left) data point in each series represents control unstimulated (basal) condition, while second (right) data point delineates productions of cells after stimulation with plate bound anti-CD3 antibody. These paired specimens (control and stimulated) are also connected by lines. CD4⁺CD28^null cells from IPF patients (open circles with paired specimens connected by dashed lines) tend to elaborate greater amounts of pro-inflammatory and T_H1 cytokines (top two rows), whereas CD4⁺CD28⁺ cells (open squares with paired specimens connected by solid lines) have an apparent T_H2 bias, with the exception of IL-4 production (bottom row) (n = 6 randomly-selected specimens in each measure).</p

Associations of CD28% espression with clinical outcome.

<p><i>A.)</i> Survival curves show cumulative freedom from major adverse events (lung transplantation or death) of IPF patients. Those subjects with the most extreme CD28 down-regulation, with CD28 expressed on <82% of their circulating CD4 T-cells (CD28% Low), had much worse outcomes than the cohort with greater proportions of CD4-Tcells that expressed CD28 (CD28% High). Numbers in parenthesis at the ends of survival curves denote remaining, unafflicted subjects that were censored at 12 months of observation. <i>B.)</i> Survival curves showing that cummulative freedom from major adverse events of IPF patients who have either significant CD28 downregulation (CD28% Low) or diffusing capacities for carbon monoxide, as percentages of predicted normal values (DLCO%p) <38, had worse outcomes than the IPF cohort who were both CD28% High and had more normal DLCO%p.</p

Characteristics of CD4 T-cell subpopulations in IPF patients.

<p><i>A:</i> The proportions of circulating CD4 T-cells that also expressed CD28 (CD28%) were reduced in many IPF patients. The horizontal line denotes the population means. <i>B:</i> In contrast to autologous CD4⁺CD28⁺ cells, the CD4⁺CD28^null T-cells of IPF patients more often express major histocompatibility antigen (MHC) Class II (DR), but less frequently express CD25. CD4⁺CD28^null T-cells of IPF patients less frequently produce transcription factor FoxP3 (a putative marker of regulatory T-cells), but much more frequently produce cytotoxic mediators granzyme B (GB) and perforin (Perf). For each measure n = 24, and p values for all intergroup comparisons (CD4⁺CD28⁺ vrs. CD4⁺CD28^null cells) are <0.0001.</p

Flow cytometry methodology (see also references 20, 52).

<p>Aliquots of fresh, live peripheral blood mononuclear cells (PBMNC) were stained with anti-CD4-allophycocyanin (APC), anti-CD28-fluorescein isothiocyanate (FITC), and phycoerythrin (PE)-conjugated antibodies against other cell epitopes. <i>A.)</i> Ten thousand (10,000) or more live cells were selected for further study, based their side scatter (SSC) and forward scatter (FSC) characteristics (G1). <i>B.)</i> The brightly staining CD4 cells among these also expressed CD3 (Cy-Chrome) and, thus, are T-cells <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008959#pone.0008959-Zhu1" target="_blank">[52]</a>. <i>C.)</i> These CD4 T-cells were further characterized based on their expression of CD28. The proportions of CD4⁺CD28⁺ T-cells among the total CD4⁺ T-cell population (upper left and upper right quadrants) defines the CD28%. The respective proportions of CD4⁺CD28⁺ and CD4⁺CD28^null cells that co-expressed other cell determinants of interest (in this case MHC Class II [DR]) were quantitated. Numbers within the delineated region/quadrants denote the proportions of cells with these respective characteristics.</p