Figure 3

E. coli and Zoledronate-expanded γδT cells phagocytose E. coli with similar dynamics in a TCR-dependent manner. Freshly-isolated PBMC (n = 5) were expanded with UV-irradiated E. coli or zoledronate for 14 days, and examined for E. coli uptake on day 14. To determine uptake, PBMC were incubated with IgG-opsonized E. coli for 60 min. PBMC were pre-cultured with normal media (control), anti-γδTCR mAb (clone: B1) or isotype-matched control. The effect of pre-culture with anti-γδTCR mAb on phagocytosis was examined; data is shown for (A) E. coli-expanded or (B) zoledronate-expanded γδT cells, incubated with FITC-E. coli or pHrodo-E. coli. (C) PBMC were incubated with opsonized green fluorescent beads and quenched post-culture with Trypan Blue. Shown are representative stains, gated on γδT cells: i) non-quenched co-culture indicating total bead fluorescence (black, solid, unshaded), ii) quenched co-culture indicating intracellular bead fluorescence (red, dotted, unshaded), iii) cells alone (gray, shaded). (D) The effect of anti-γδTCR mAb was examined on γδT cell uptake of opsonized bead in quenched and non-quenched samples (n = 3). (E) TCR sequencing was carried out on E. coli or zoledronate-expanded and fresh non-expanded γδT cells. Representative heat maps of one donor E. coli and zoledronate-expanded γδT cell Vγ (VG), Vδ (VD) and Vj (VJ) chain reads are shown. (F) The number of shared TCR CDR3 sequences was tallied and compared in E. coli or zoledronate-expanded and fresh unexpanded γδT cells. A comparison of the number of sequencing reads shared as well as the number of unique sequences in each category is shown for both Vγ and Vδ TCR chains. For all samples, total depth of sequencing was equivalent. Only sequences with more than 5000 reads were tallied to select for the commonest clones. (G) TCR CDR3 spectratyping was carried out for E. coli or zoledronate-expanded Vγ9Vδ2 T cells. Representative spectratypes of one donor are shown.