Analysis of T Cell Populations in Clinically Stable ISHLT Grade A1 Lesions

Abstract

<h3>Purpose</h3> CD4+FOXP3+ Regulatory T cells (Tregs) may modulate acute cellular rejection (ACR)-associated immune responses and the progression of ACR to Chronic Lung Allograft Dysfunction (CLAD) following lung transplantation (LTx). Treg stability is enhanced by demethylation of the Treg-specific demethylated region (TSDR) in the Foxp3 locus. Grade A1 ACR is not treated in clinically stable patients at our centre, affording the opportunity to study its natural history in relation to its cellular composition. Our hypothesis was that a greater Treg content in first, clinically stable, grade A1 biopsies is associated with a lower risk of CLAD. <h3>Methods</h3> In this retrospective study, we selected clinically stable patients who received a bilateral LTx between 1 Jan 2010 and 18 Dec 2016 with a first episode of A1B0 ACR and who remained CLAD free for at least 5 years (n=16) or who developed CLAD within 2 years (n=9) following LTx. Immunofluorescence was used to quantify CD4+, CD8+ and FOXP3+ cells in transbronchial biopsies (Figure 1A). Separately, CD3+ cells were fluorescently labelled, micro-dissected (Figure 1B) and, using bisulfite conversion and pyrosequencing, the degree of TSDR methylation was determined. <h3>Results</h3> TSDR methylation was slightly lower in the CLAD group as compared to the CLAD-free group (Figure 1C), indicating increased Treg infiltration. Accordingly, the ratio of CD4+FOXP3+ cells to total CD4+ cells was slightly higher in the CLAD group (Figure 1D). These measures were weakly negatively correlated (r² = 0.250, p=0.0152) (Figure 1E). The ratio of CD4+:CD8+ cells was significantly lower in the CLAD group (p=0.0065) (Figure 1F). <h3>Conclusion</h3> Patients who developed CLAD within 2 years of LTx showed no significant difference in Treg, though greater CD8+ T cell, infiltration compared to stable patients. These results suggest that in asymptomatic patients with a first episode of A1 rejection, greater CD8+ T cell content may be indicative of a poorer long-term outlook. Validation in additional patients is now underway.