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. 2022 Sep 1;17(9):1976–1990. doi: 10.1016/j.stemcr.2022.08.002

Figure 3.

Figure 3

Immunogenicity of CXCL10 expressing SC-islets

(A) Transduced SC-islets with lentiviruses carrying Cas9 + gRNA (KO) or a given open reading frame (ORF) insert (overexpression [OE]), were co-cultured with allogeneic PBMCs.

(B) Flow cytometry for %TUNEL+ (apoptotic) SC-β cells (C-peptide+), following 48-h PBMC co-culture. Apoptosis was calculated by fraction from baseline (%TUNEL without PBMC). gRNA lentivirus transduced SC-islets were compared with non-targeting (NT) gRNA, and OE transduced SC-islets were compared with eGFP OE. n = 3 for ×5 PBMC donors (left; KO), n = 2–3 for ×2 donors (right; OE).

(C) Blocking antibodies prior to/with co-cultures: PBMCs with anti-CXCR3, or SC-islets with anti-TLR4, or anti-CXCL10 during co-culture.

(D) Flow cytometry analysis for apoptotic SC-β, following 48-h PBMC co-culture. n = 3 for ×2–6 donors.

(E) PBMCs were labeled with cell trace violet (CTV) prior to co-culture. Following a 48-h co-culture, PBMCs were separated and allowed to grow for 7 days. CD3+ were gated for the CTV-negative fraction of divided cells. n = 5 for ×3 donors. Error bars are mean ± SD. ns, not significant; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001, unpaired two-tailed t test.