American Society of Hematology

Figure 3.

Antigen-specific CD8 T-cell killing by armed 225Ac-tetramers. (A) 225Ac-tetramers (LMP1) were added to LMP1-CD8 (•) or Flu58-66-CD8 T cells (○) and then incubated at 37° C for 72 hours. The 225Ac-LMP1 tetramers (5 nCi/μg) efficiently killed the targeted LMP1-specific CD8 T cells with great potency (•, ED50 = 5-8 nCi/mL or 1-1.6 μg/mL). About 15- to 40-fold–higher doses (ED50 = 110-200 nCi) of 225Ac-LMP1 tetramers (○) or equivalent 225Ac DOTA alone (▪) were required for nonspecific killing (P < .001). Much higher levels (100-140 μg/mL) of unlabeled specific LMP1 tetramer alone (▵) were required to induce mild cytotoxicity in targeted LMP1 CD8+ T cells. (B) 225Ac-LLO91-99 tetramers (•) specifically killed mouse LLO91-99-specific CD8+ T cells (ED50 = 5-7 nCi/mL or 1-1.4 μg/mL). More than 15-fold–larger amounts of 225Ac (▴) were required to kill control p60217-225-specific CD8+ T cells. Addition of excess (50-fold) unlabeled LLO91 tetramers (○) partially blocked cell killing of 225Ac-LLO91-99 tetramers. (C) 225Ac-LLO91-99 tetramers at 1 to 30 nCi/mL (0.2-6 μg/mL) were added to mixed cell (50:50) cultures of target LLO91-99-specific CD8 T cells and p60217-225-specific CD8 T cells. 225Ac-LLO91 tetramers (▴) selectively killed LLO91-CD8 T cells in a mixed cell culture, and were as effective as killing in cultures of purified LLO91-CD8 T cells alone (○). 225Ac-LLO91 tetramers produced minimal cytotoxicity in control p60217-225-specific CD8 T cells in a mixed cell culture (▪; P < .0001). Data represent the mean plus or minus the standard error (SE) of 3 tests in a single representive experiment done 2 times. (D) 225Ac-LLO91-99 tetramers (5 nCi/mL or 1 μg/mL) reduce IFN-γ–secreting clones in targeted LLO91-99-specific CD8+ T cells. The level of IFN-γ–secreting cells in suicide tetramer–treated LLO91-99-specific CD8+ T cells (▪) decreased significantly (83 ± 14 colonies) when compared with the nontreated control T cells (□; 219 ± 21 colonies). The control p60217-specific CD8 T cells showed only modest nonspecific reduction in IFN-γ–secreting cells (133 ± 7 vs 105 ± 5). Negative control stimulations with irrelevant peptides produce few spots (□). Bars represent normalized percent of spots from each CD8 cell line either treated with 225Ac-LLO91 tetramers or controls.

Antigen-specific CD8 T-cell killing by armed 225Ac-tetramers. (A) ²²⁵Ac-tetramers (LMP₁) were added to LMP₁-CD8 (•) or Flu_58-66-CD8 T cells (○) and then incubated at 37° C for 72 hours. The ²²⁵Ac-LMP₁ tetramers (5 nCi/μg) efficiently killed the targeted LMP₁-specific CD8 T cells with great potency (•, ED₅₀ = 5-8 nCi/mL or 1-1.6 μg/mL). About 15- to 40-fold–higher doses (ED₅₀ = 110-200 nCi) of ²²⁵Ac-LMP₁ tetramers (○) or equivalent ²²⁵Ac DOTA alone (▪) were required for nonspecific killing (P < .001). Much higher levels (100-140 μg/mL) of unlabeled specific LMP₁ tetramer alone (▵) were required to induce mild cytotoxicity in targeted LMP₁ CD8⁺ T cells. (B) ²²⁵Ac-LLO_91-99 tetramers (•) specifically killed mouse LLO_91-99-specific CD8⁺ T cells (ED₅₀ = 5-7 nCi/mL or 1-1.4 μg/mL). More than 15-fold–larger amounts of ²²⁵Ac (▴) were required to kill control p60_217-225-specific CD8⁺ T cells. Addition of excess (50-fold) unlabeled LLO₉₁ tetramers (○) partially blocked cell killing of ²²⁵Ac-LLO_91-99 tetramers. (C) ²²⁵Ac-LLO_91-99 tetramers at 1 to 30 nCi/mL (0.2-6 μg/mL) were added to mixed cell (50:50) cultures of target LLO_91-99-specific CD8 T cells and p60_217-225-specific CD8 T cells. ²²⁵Ac-LLO₉₁ tetramers (▴) selectively killed LLO₉₁-CD8 T cells in a mixed cell culture, and were as effective as killing in cultures of purified LLO₉₁-CD8 T cells alone (○). ²²⁵Ac-LLO₉₁ tetramers produced minimal cytotoxicity in control p60_217-225-specific CD8 T cells in a mixed cell culture (▪; P < .0001). Data represent the mean plus or minus the standard error (SE) of 3 tests in a single representive experiment done 2 times. (D) ²²⁵Ac-LLO_91-99 tetramers (5 nCi/mL or 1 μg/mL) reduce IFN-γ–secreting clones in targeted LLO_91-99-specific CD8⁺ T cells. The level of IFN-γ–secreting cells in suicide tetramer–treated LLO_91-99-specific CD8⁺ T cells (▪) decreased significantly (83 ± 14 colonies) when compared with the nontreated control T cells (□; 219 ± 21 colonies). The control p60₂₁₇-specific CD8 T cells showed only modest nonspecific reduction in IFN-γ–secreting cells (133 ± 7 vs 105 ± 5). Negative control stimulations with irrelevant peptides produce few spots (□). Bars represent normalized percent of spots from each CD8 cell line either treated with ²²⁵Ac-LLO₉₁ tetramers or controls.

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