Local and Systemic CD4⁺ T Cell Exhaustion Reverses with Clinical Resolution of Pulmonary Sarcoidosis

Figure 6

Immune dysfunction in sarcoidosis BAL versus PBMC. BAL cells and matching PBMC isolated from sarcoidosis subjects were analyzed according to immune parameters associated with T cell exhaustion. (a, b, c, and d) Intracellular staining for cytokine production: cells were incubated in the absence (a and b) or presence (c and d) of TCR stimulation for 6 hours with Golgi Stop. Cells were then stained with anti-CD3, anti-CD4, and anti-CD8 before permeabilization and staining with PE-conjugated anti-IL-2. Flow cytometry was conducted and live, singlet cells were gated to assess the percentage of CD4⁺CD3⁺ cells that had produced IL-2 or IFN-γ. Unstimulated cells (baseline) were used to help determine the IL-2 production in TCR-stimulated cells. (a and b) Baseline IL-2 and IFN-γ production. (c and d) TCR-stimulated IL-2 and IFN-γ production. (e) The percentage of apoptosing CD4⁺ T cells in matching BAL and PBMC was assessed using 7AAD and Annexin V staining. (f) PD-1 expression in the population of CD4⁺ T cells was determined in matching BAL and PBMC. Statistical analysis was performed using the Wilcoxon matched-pairs signed-rank test. . NS: not significant.

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