(a) Representative IF-FISH and (b, top) pooled data showing telomere elongation in nonsenescent CD4 + T cells and concomitant telomere shortening in APCs after forming the synapse. APCs from human donors were pre-loaded with cytomegalovirus lysates and allowed to interact with autologous nonsenescent CD4+ T cells in 3:1 ratio for the indicated time points. Conjugates were fixed and analysed by IF-FISH. Data are from n = 3 donors (three independent experiments). Scale bar, 10 µm. T = 0, initial time at which conjugates are observed (20 min). Images were z-stacked, and the raw telomere integrated fluorescence signals (AU, arbitrary units) are shown. One-hundred two conjugates were analysed. (b, bottom) Analysis of telomere length by qPCR in APCs and nonsenescent CD4 + T cells after forming conjugates. (c-d) Primary human nonsenescent CD4+ T cells were transfected by nucleofection with sgCtrl or sgTERT CRISPR constructs, activated with anti-CD3 plus anti-CD28 and elimination of telomerase was confirmed by (c) qPCR and (d) TRAP assay. (e) Telomerase positive (transfected with sgCTRL) and negative (transfected with sgTERT) nonsenescent T cells were exposed to APCs in the presence of antigen pool, and telomere content was quantified by Flow-FISH using TelC telomere probe. Absolute telomere length by Flow-FISH was determined from Mean Fluorescence Intensity (MFI) values using a standard curve formed by cryopreserved samples with known telomere length as determined by TRF. (f) Telomere content was measured by flow-FISH using either TelC or TelG telomere PNA specific probes coupled to anti-BrdU detection (to monitor telomere elongation vs DNA synthesis in T cells) in telomerase negative nonsenescent CD4+ T cells (CRISPR KO sgTERT) and control T cells (sgCtrl) stimulated with antigen pool for 48 h. (g) Telomere length by flow-FISH demonstrating telomere elongation in primary human nonsenescent CD4+ T cells treated with DNA polymerase inhibitors aphidicolin and thymidine prior to exposure to APCs for 48 h. Data are from n = 3 donors throughout. Statistical Tests are provided in the Supplementary Table 1. Error bars indicate S.E.M. throughout.
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