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. Author manuscript; available in PMC: 2019 Aug 13.
Published in final edited form as: Nature. 2019 Feb 13;567(7746):43–48. doi: 10.1038/s41586-019-0942-8

Extended Data Fig. 10. Evaluation of the specificity and cytotoxic properties of CTL clones.

Extended Data Fig. 10.

(a) Design of cytotoxic T lymphocyte (CTL) killing assays. As target cells, monotypic pseudoislets (αGFP, αPM, or βGFP) after 1–2 week culture were dissociated and labelled with chromium (51Cr). In some control conditions, islet cells were loaded with either DRiP or PPI peptide epitopes. Then, target cells were co-cultured with effector cells (CTLs), which were either CMV-directed (CMV: negative control clones), DRiP-directed (DRiP: targeting stressed β-cells), PPI-directed (PPI: recognizing preproinsulin), or alloreactive (HLA-A2: positive control) CTL clones at 3 different effector/target (E/T) ratios. DRiP and PPI CTLs are autoreactive T-cell clones derived from T1D patients. After 4h-coculture, the release of 51Cr from islet-cells was measured with γ-counter to calculate the specific cell-lysis. (See Extended Data Fig. 10 to validate the specific killing capability of CTL clones). (b) Schema of validation for CTLs. To evaluate the specificity and function of CTL clones, JY cells, Epstein-Barr virus (EBV)-immortalised B lymphoblastoid cell line (HLA class-I A2+), were used as target cells. As positive control groups, JY cells were loaded with either INS-DRiP1–9 (DRiP) or preproinsulin (PPI) peptide epitope and labelled with chromium (51Cr). Then they were co-cultured with effector cells (CTLs), which is either CMV-directed (CMV), DRiP-directed (DRiP), PPI-directed (PPI), or alloreactive (HLA-A2) CTL clone. (b) CTL killing assay against JY cells. JY cells were killed by the alloreactive HLA-A2 CTLs, but not by CMV-directed CTL, β-cell-specific CTLs anti-PPI or anti-DRiP CTLs. When target cells were loaded with the PPI or DRiP peptide epitope, those JY cells were killed by the respective CTLs, confirming that the specific CTLs function and kill when they recognize their epitope. Each dot represents independent measurement from 3 independent experiments. ****p < 0.0001, one-way ANOVA with Holm-Sidak’s multiple comparisons test.