Journal: Journal for Immunotherapy of Cancer

Article Title: Combination of cancer vaccine with CD122-biased IL-2/anti-IL-2 Ab complex shapes the stem-like effector NK and CD8 + T cells against tumor

doi: 10.1136/jitc-2022-006409

Figure Lengend Snippet: Immunological response elicited by aAVC-WT1 treatment. (A) aAVC-WT1 treatment and assay protocol. WT1-expressing C1498 leukemic cell (C1498-WT1)-bearing mice were treated with aAVC-WT1 and monitored for immunological response and survival. (B) iNKT cell responses. The absolute number of iNKT cells (CD3 dim CD1d-tetramer/Gal + cells) was analyzed on days 10 and 14 using flow cytometry (mean±SEM, n=5; aAVC-WT1-treated day 10 vs others, ***p < 0.001; Tukey’s test). (C) NK cell responses. The absolute number of NK cells (CD3 − NK1.1 + cells) in the spleen and BM were analyzed (n=5/group; **p < 0.01, ***p < 0.001, Tukey’s test) (D) IFN-γ production by iNKT and NK cells in ELISpot assays (mean±SEM, n=5/group; ***p < 0.001; Tukey’s test). (E) CD8/Treg ratio in the BM at the indicated time points (n=5/group; ***p < 0.001; Tukey’s test). (F) WT1-specific CD8 + T-cell responses in IFN-γ ELISpot assays in C1498-WT1-bearing mice treated with or without aAVC-WT1. In some experiments, Irf8 cKO mice were used as recipients. (mean±SEM, n=5/group; ***p<0.001; Tukey’s test). aAVC, artificial adjuvant vector cell; BM, bone marrow; ELISpot, Enzyme-Linked Immunospot; IFN, interferon; iNKT, invariant natural killer T; i.v., intravenous; MNCs, mononuclear cells; NK, natural killer; SFC, spot-forming cell; WT1, Wilms’ tumor 1.

Article Snippet: For depletion experiments, CD8 + and NK cell depletion was performed using anti-CD8 Ab (clone 53.6.72; Bio X Cell) (200 μg/mouse) and anti-asialoGM1 Ab (WAKO Chemicals) (50 μL/mouse), respectively, twice a week from day 12 until the end of the experiments.

Techniques: Expressing, Flow Cytometry, Enzyme-linked Immunospot, Adjuvant, Plasmid Preparation, Wilms Tumor Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Combination of cancer vaccine with CD122-biased IL-2/anti-IL-2 Ab complex shapes the stem-like effector NK and CD8 + T cells against tumor

doi: 10.1136/jitc-2022-006409

Figure Lengend Snippet: Antitumor effects of aAVC and IL-2 or IL-2/anti-IL-2 complex. (A). The antitumor effects of aAVC-WT1 in a leukemia model. As described in , mice were challenged using 2×10 4 C1498-WT1 cells intravenously and then treated with aAVC-WT1 on day 7. Survival was monitored. (n=10/group; ***p<0.001; log-rank test). (B) Recall response to antitumor effects. Three to 6 months later, surviving mice from aAVC-WT1 traeted group were rechallenged using C1498-WT1 cells or irrelevant EL4 cells, and survival was assessed (n=4–5/group; ***p<0.001; log-rank test). (C) As in (A); however, high doses of C1498-WT1 cells (1×10 5 cells) were administered to mice. Mice were treated with or without aAVC-WT1 on day 7 and then injected i.p. with or without soluble human IL-2 (2.5×10 4 U/day) every day from days 14 to 20. (n=9–10/group, non-treated vs aAVC-WT1 or aAVC-WT1+IL-2, ***p<0.001; log-rank test). (D) As in (C), mice were challenged using high doses of C1498-WT1 (1×10 5 cells) and treated with aAVC-WT1 on day 7. With regard to the IL-2/anti-IL-2 complex (IL-2Cxs) combination, IL-2Cx(S4B6) or IL-2Cx(JES6) was injected into the mice every day from days 14 to 20. (n=7–9/group, non-treated vs others; ***p<0.001, aAVC-WT1 vs aAVC-WT1+IL-2Cx(S4B6) or IL-2Cx(JES6); ***p<0.001 and *p<0.05, respectively; aAVC-WT1+IL-2Cx(S4B6) vs aAVC-WT1+IL-2Cx(JES6); *p<0.05, log-rank test). (E) Depletion of CD8 + cell and NK cells on C1498-WT1 bearing mice treated with a combination of aAVC-WT1 and IL-2Cx(S4B6). Survival was monitored. (n=6/group; ns, not significant; log-rank test). aAVC, artificial adjuvant vector cell; IL, interleukin; i.p., intraperitoneal; i.v., intravenous; NK, natural killer; WT1, Wilms’ tumor 1.

Article Snippet: For depletion experiments, CD8 + and NK cell depletion was performed using anti-CD8 Ab (clone 53.6.72; Bio X Cell) (200 μg/mouse) and anti-asialoGM1 Ab (WAKO Chemicals) (50 μL/mouse), respectively, twice a week from day 12 until the end of the experiments.

Techniques: Injection, Adjuvant, Plasmid Preparation, Wilms Tumor Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Combination of cancer vaccine with CD122-biased IL-2/anti-IL-2 Ab complex shapes the stem-like effector NK and CD8 + T cells against tumor

doi: 10.1136/jitc-2022-006409

Figure Lengend Snippet: aAVC/IL-2Cx(S4B6) combination therapy induces CD8 + T cells with stem-like features. As shown in ; however, splenic CD4 + and CD8 + T cells were analyzed on day 14. (A) Gating strategy of natural killer cells (left) and absolute number of total CD8 + T cells in the spleen (right panel) was analyzed using flow cytometry (mean±SEM, n=4–5/group; ***p<0.001; Tukey’s test). (B) Representative histogram of expression of the transcription factors TCF-1, T-bet, Eomes, and Blimp-1 in total CD8 + T cells in the spleen. (C) Representative dot plots showing phenotypic profiles of splenic CD8 + T cells. (D) The frequencies of central memory T (CD44 + CD62L + ) and effector memory T (CD44 + CD62L − ) subsets are summarized (mean±SEM, n=4/group; *p<0.05, **p<0.001, ***p<0.001; Tukey’s test). (I) Gating strategy of OVA-SIINFEKL-tetramer + (OVA-tet + ) CD8 + T cells in the spleen (left) and summary of absolute number of OVA-tet + CD8 + T cells (right) (mean±SEM, n=4–5/group; ***p<0.001; Tukey’s test). (F) Representative histogram of expression of the transcription factors TCF-1, T-bet, Eomes, and Blimp-1 in OVA-tet + CD8 + T cells in the spleen. (G) Kinetics of the frequency of OVA-tet + CD8 + T cells in the blood (lower panel). Representative flow cytometry data of OVA-tet + CD8 + T cells in the blood at day 14 are shown (upper panel) (mean±SEM, n=6–7/group; **p<0.001, ***p<0.001; Tukey’s test). (H) Gating strategy of Foxp3 + CD4 + T cells in splenic CD4 + T cells (left) and summary of absolute number of Foxp3 + CD4 + T cells (right panel) (mean±SEM, n=4–5/group; **p<0.001, ***p<0.001; Tukey’s test). (I) CD8/Treg ratio in spleen (mean±SEM, n=4–5/group; ***p<0.001; Tukey’s test). (J) Mass cytometry results for splenic CD8 + T-cell compartment. As shown in , spleen cells were harvested on day 21. t-SNE plots showing annotated clusters (upper panel), heatmap (upper right panel), and frequency (lower) of each cluster in splenic CD8 + T cells (mean±SEM, n=4–7/group; *p<0.05, **p<0.001, ***p<0.001; Tukey’s test). aAVC, artificial adjuvant vector cell; IL-2Cx, interleukin 2 complex; Treg, regulatory T cell; WT1, Wilms’ tumor 1; t-SNE, t-distributed Stochastic Neighbor Embedding; TCR, T-cell receptor.

Article Snippet: For depletion experiments, CD8 + and NK cell depletion was performed using anti-CD8 Ab (clone 53.6.72; Bio X Cell) (200 μg/mouse) and anti-asialoGM1 Ab (WAKO Chemicals) (50 μL/mouse), respectively, twice a week from day 12 until the end of the experiments.

Techniques: Flow Cytometry, Expressing, Mass Cytometry, Adjuvant, Plasmid Preparation, Wilms Tumor Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Combination of cancer vaccine with CD122-biased IL-2/anti-IL-2 Ab complex shapes the stem-like effector NK and CD8 + T cells against tumor

doi: 10.1136/jitc-2022-006409

Figure Lengend Snippet: Tumor-site-infiltrating CD8 + T and NK cells induced by the combination therapy. As shown in , the mice were challenged using C1498-WT1 and then treated with aAVC-WT1 or aAVC-WT1+IL-2Cx(S4B6). On day 21, the bone marrow cells from these mice were analyzed using mass cytometry. (A) Mass cytometry results of the BM CD8 + T-cell compartment. t-SNE plots showing annotated clusters (upper), heatmap (right upper), and frequency (lower) of each cluster in BM CD8 + T cells (mean±SEM, n=4–7/group; *p<0.05, **p<0.001, ***p<0.001; Tukey’s test). (B) Mass cytometry results for the BM NK cell compartment. T-SNE plots showing annotated clusters (upper panel), heatmap (right panel), and frequency (lower panel) of each cluster in the splenic NK cells. (mean±SEM, n=3–6/group; *p<0.05, **p<0.001, ***p<0.001; Tukey’s test). (C) PCA and clustering using the KMeans of BM CD8 + T cells and NK cells. Left: colored dots indicate the samples from naïve (red), D21 C1498-WT1 bearing (light green), D21 C1498-WT1 bearing, aAVC-WT1 treated (green) and D21 C1498-WT1 bearing, aAVC-WT1 and IL-2Cx(S4B6) treated (blue). Middle: colored dots indicate the samples of CD8 + T cells (red) and NK cells (blue). Right: Data shows clustering into three clusters using the KMeans. Cluster 1 (green), Cluster 2 (yellow) and Cluster 3 (violet). aAVC, artificial adjuvant vector cell; BM, bone marrow; IL-2Cx, interleukin 2 complex; NK, natural killer; PCA, principal component analysis; WT1, Wilms’ tumor 1; t-SNE, t-distributed Stochastic Neighbor Embedding.

Article Snippet: For depletion experiments, CD8 + and NK cell depletion was performed using anti-CD8 Ab (clone 53.6.72; Bio X Cell) (200 μg/mouse) and anti-asialoGM1 Ab (WAKO Chemicals) (50 μL/mouse), respectively, twice a week from day 12 until the end of the experiments.

Techniques: Mass Cytometry, Adjuvant, Plasmid Preparation, Wilms Tumor Assay