Fig. 2: CD61 transiently colocalizes with CD103.
a, Horizontal bar graph showing the average CD61 median fluorescence intensity (MFI) on CD103+ T cell lines following either activation by αCD3/CD28, or co-culture with antigenic cancer cells, or no activation by flow cytometry. ***P < 0.001, one-way ANOVA with Tukey’s multiple-comparison test. n = 7 patients, examined over three independent experiments. ***P values: (patient 1: 0.00098, patient 2: 0.00089, patient 3: 0.00001, patient 4: 0.000003, patient 5: 0.000, patient 6: 0.00002, patient 7: 0.00001). b, Representative synapse images of integrin β7, CD103, CD61 and merged, of a cancer-specific CD103+ TCR-T cell at 10 min after synaptic formation. Enlarged green box shows zoomed-in synaptic microcluster images of CD103 and CD61 colocalization, but not integrin β7. c,d, Representative dot plot showing CD103 and CD61 colocalization by PCC (P = 0.9435), or CD103 and integrin β7 negative PCC colocalization (P = 0.1973) at 5, 10 and 15 min after synaptic formation; each dot represents the average PCC per synapse. e, Representative synapse images showing internal reflection microscopy (IRM; denoting the area of synapse), integrin β7, CD49d and merged, of a cancer-specific CD103+ TCR-T cell at 5 min after synaptic formation. Enlarged green box shows zoomed-in images of colocalized integrin β7 and CD49d. f, Representative Co-IP immunoblot images of CD103 and CD61-flag on anti-flag IP pulldown lysate and whole-cell lysate, of CD103−CD61-flag+, CD103+CD61-flag− and CD103+CD61−flag+ T cell lines. Molecular weight (MW) of CD103: ~150 kDa, of CD61-flag: ~100 kDa. n = 3 lines examined over three independent experiments, with consistent results. g, Volcano plot showing enriched proteins on lysates of CD103+CD61+ T cells in comparison to CD103+CD61− T cells. n = 2 lines examined over one experiment. Dot represents one protein. One-way ANOVA with Tukey’s multiple-comparison test, converted to −log10 P values for each data point. Raw fold-change values were normalized using log2. h, Representative flow cytometry plots of intracellular and surface staining of CD61-flag with CD103-HA following initial transduction of primary T cells with CD103 (left), followed by secondary transduction with CD61 (right). n = 3 independent experiments, with consistent results. a,c,d. Data are presented as the median ± s.e.m., c,d. Two-way ANOVA with Tukey’s multiple-comparison test. b,e. n = 150 cells examined over three independent experiments with consistent observations per c and d; each dot represents the average PCC per synapse. c, time of 5 min: 72 synapses, time of 10 min: 102 synapses, time of 15 min: 141 synapses. d, time of 5 min: 53 synapses, time of 10 min: 81 synapses, time of 15 min: 40 synapses. b,e, Microscopy images: big scale bar, 5 μm; small scale bar, 1 μm. Ag, antigen. NS, not significant.
