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. Author manuscript; available in PMC: 2020 Aug 15.

Published in final edited form as: Clin Cancer Res. 2019 May 20;25(16):4933–4944. doi: 10.1158/1078-0432.CCR-19-0183

Figure 3. — Magnitude and breadth of combined antigen-specific CD4⁺ and CD8⁺ T-cell responses post- (vs pre) BN-CV301 (MVA-BN-CV301/FPV-CV301) vaccine.

(A) Seven patients were tested and TAA responses compared both at 6 weeks (2 weeks after the 2^nd MVA-BN-CV301 prime, red) and 10 weeks (2 weeks after the 1^st FPV-CV301 boost, blue). The absolute number of CD4⁺ and/or CD8⁺ T-cells producing IFNγ, TNF, or IL-2, or positive for the degranulation marker CD107a per 1 × 10⁶ PBMC plated at the start of the stimulation assay was calculated. Any background signal (obtained with the HLA peptide pool) and any signal obtained prior to vaccination was subtracted ([post-TAA – post-HLA] – [pre-TAA – pre-HLA]). Each point indicates the magnitude of a cytokine/CD107a measure, with 8 measures assessed per patient (CD8⁺IFNγ⁺, CD8⁺TNF⁺, CD8⁺IL-2⁺, CD8⁺CD107a⁺, CD4⁺IFNγ⁺, CD4⁺TNF⁺, CD4⁺IL-2⁺, CD4⁺CD107a⁺). Frequency of positive measures (>250 CD4⁺ and CD8⁺ T-cells producing cytokine and/or positive for CD107a) is indicated. (B) Polyfunctional TAA responses (CD4⁺ and CD8⁺ T cells expressing 2 or more of the following: IFNγ, TNF, IL-2, or CD107a) were measured before and after any time point post-vaccination in all 12 patients. The frequency of patients developing a low, mid, or high magnitude of multifunctional TAA-specific T-cells after vaccination at any time point post- vs pre- is indicated. (C) TAA responses in 4 patients with known wild type KRAS (Black) and six patients with known/presumed KRAS mutations (Purple) were compared at 6 weeks (2 weeks after the 2^nd MVA Prime). The absolute number of CD4⁺ or CD8⁺ T-cells producing IFNg, TNF, or IL-2, or positive for the degranulation marker CD107a per 1×10⁶ PBMC plated at the start of the stimulation assay was calculated. Each point indicates the magnitude of a cytokine/CD107a measure, with 8 measures assessed per patient. Frequency of positive measures (>250 CD4⁺ and CD8⁺ T-cells producing cytokine and/or positive for CD107a) is indicated.

Figure 3. — Magnitude and breadth of combined antigen-specific CD4⁺ and CD8⁺ T-cell responses post- (vs pre) BN-CV301 (MVA-BN-CV301/FPV-CV301) vaccine.

(A) Seven patients were tested and TAA responses compared both at 6 weeks (2 weeks after the 2^nd MVA-BN-CV301 prime, red) and 10 weeks (2 weeks after the 1^st FPV-CV301 boost, blue). The absolute number of CD4⁺ and/or CD8⁺ T-cells producing IFNγ, TNF, or IL-2, or positive for the degranulation marker CD107a per 1 × 10⁶ PBMC plated at the start of the stimulation assay was calculated. Any background signal (obtained with the HLA peptide pool) and any signal obtained prior to vaccination was subtracted ([post-TAA – post-HLA] – [pre-TAA – pre-HLA]). Each point indicates the magnitude of a cytokine/CD107a measure, with 8 measures assessed per patient (CD8⁺IFNγ⁺, CD8⁺TNF⁺, CD8⁺IL-2⁺, CD8⁺CD107a⁺, CD4⁺IFNγ⁺, CD4⁺TNF⁺, CD4⁺IL-2⁺, CD4⁺CD107a⁺). Frequency of positive measures (>250 CD4⁺ and CD8⁺ T-cells producing cytokine and/or positive for CD107a) is indicated. (B) Polyfunctional TAA responses (CD4⁺ and CD8⁺ T cells expressing 2 or more of the following: IFNγ, TNF, IL-2, or CD107a) were measured before and after any time point post-vaccination in all 12 patients. The frequency of patients developing a low, mid, or high magnitude of multifunctional TAA-specific T-cells after vaccination at any time point post- vs pre- is indicated. (C) TAA responses in 4 patients with known wild type KRAS (Black) and six patients with known/presumed KRAS mutations (Purple) were compared at 6 weeks (2 weeks after the 2^nd MVA Prime). The absolute number of CD4⁺ or CD8⁺ T-cells producing IFNg, TNF, or IL-2, or positive for the degranulation marker CD107a per 1×10⁶ PBMC plated at the start of the stimulation assay was calculated. Each point indicates the magnitude of a cytokine/CD107a measure, with 8 measures assessed per patient. Frequency of positive measures (>250 CD4⁺ and CD8⁺ T-cells producing cytokine and/or positive for CD107a) is indicated.