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. 2020 Jun 1;9(1):1771142. doi: 10.1080/2162402X.2020.1771142

Figure 5.

Figure 5.

ARG2-specific T-cells recognize ARG2-expressing malignant myeloid cells. (a) To identify HLA-matched malignant T-cells, the ARG2-specific T-cells were examined in IFNγ ELISPOT response toward different relevant cancer cell lines pre-pulsed with ARG2-1 peptide. The same cancer cell lines without peptide stimulation were examined as control. Effector-to-target ratio 1:1 with 1 × 104 effector cells plated per well. * p ≤ 0.05 or ** p ≤ 0.01 according to the distribution free resampling rule. TNTC, too numerous to count (>500). (b) FNγ ELISPOT response of the ARG2-specific T-cells toward THP-1 cells pulsed with ARG2-1 peptide and class II blockers. (c) ARG2 expression in THP-1 evaluated by RT-qPCR following 48-h incubation of THP-1 cells with different cytokines. Data are represented as fold change vs unstimulated THP-1 cells; mean+SD, n = 4. (d) FNγ ELISPOT response of the ARG2-specific T-cells toward THP-1 cells stimulated with the cytokine cocktail. Effector-to-target ratio 5:1 with 1.5 × 105 effector cells plated per well. ** p ≤ 0.01 and ns = not significant according to the distribution free resampling rule. (e) Intracellular staining of TFNα and IFNγ production from CD4 + T-cells in the ARG2-specific T-cell culture when incubated with unstimulated THP-1 cells or THP-1 cells pre-stimulated with cytokine cocktail for 48 h. Effector-to-target ratio 2:1 with 500,000 effector cells used per condition. (f) RG1 and ARG2 expression in THP-1 cells evaluated by RT-qPCR following 48-h incubation with cytokine cocktail. Unstimulated THP-1 cells served as control. Data are represented as relative expression to the housekeeping gene ACTB; mean+SD, n = 4. (g) IFNγ ELISPOT response of the ARG2-specific T-cells toward THP-1 cells stimulated with the cytokine cocktail (THP-1 + cyto) and the class II blocker, aHLA-DR. Effector-to-target ratio 5:1 with 1.5 × 105 effector cells plated per well. ** p ≤ 0.01 and ns = not significant according to the distribution free resampling rule. (h) ARG2 expression in THP-1 cells evaluated by RT-qPCR following 48-h stimulation with cytokine cocktail (Th2 cocktail) or IFNγ. Unstimulated THP-1 cells were included as control. Data are represented as relative expression to the housekeeping gene RPO; mean+SD, n = 4. (i) IFNγ ELISPOT response of the ARG2-specific T-cells toward THP-1 cells pre-stimulated with the cytokine cocktail (THP-1 + cytokines) or IFNγ (THP-1 + IFNγ). Effector-to-target ratio 2.5:1 with 5 × 104 effector cells plated per well. ** p ≤ 0.01 and ns = not significant according to the distribution free resampling rule.