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. 2024 Oct 30;16(12):3033–3056. doi: 10.1038/s44321-024-00157-4

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© The Author(s) 2024

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Creative Commons Public Domain Dedication waiver http://creativecommons.org/publicdomain/zero/1.0/ applies to the data associated with this article, unless otherwise stated in a credit line to the data, but does not extend to the graphical or creative elements of illustrations, charts, or figures. This waiver removes legal barriers to the re-use and mining of research data. According to standard scholarly practice, it is recommended to provide appropriate citation and attribution whenever technically possible.

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(A) Quantitative analysis by flow cytometry of absolute count of CD4⁺ T cells (left) and CD8⁺ T cells (middle) in tumor samples from subcutaneous PDAC model treated with control vaccine (v-CTRL) and ADAM12 vaccine (v-A12), along with corresponding gating strategy (right). (n = 12 mice in v-CTRL and v-A12 respectively. Data were presented as mean ± SEM. Statistical test: unpaired two-tailed Student’s t-test.). (B) Quantitative analysis of CD4⁺ T cell population at the edge of PDAC tumor tissue (left, upper) and interior of PDAC tissue (left, bottom), along with corresponding representative images. (n = 5 mice in v-CTRL and n = 4 mice in v-A12. Data were presented as mean ± SEM. Statistical test: unpaired two-tailed Student’s t-test. Scale bar 50 μm). (C) Quantitative analysis of CD8⁺ T cell population at the edge of PDAC tumor tissue (left, upper) and interior of PDAC tissue (left, bottom), along with corresponding representative images. (n = 5 mice in v-CTRL and n = 6 mice in v-A12. Data were presented as mean ± SEM. Statistical test: unpaired two-tailed Student’s t-test. Scale bar 50 μm). (D) Representative flow cytometry gating (upper) of CD62L^- CD4⁺ effector T cells between control-vaccinated (v-CTRL) mice and ADAM12-vaccinated (v-A12) mice; corresponding quantitative analysis of naïve CD4⁺ T cells (bottom, left) and CD62L^- CD4⁺ effector T cells (bottom, right) within splenic CD4⁺ T cells from PDAC tumor-bearing mice treated with control vaccine (v-CTRL) and ADAM12 vaccine (v-A12). (n = 12 mice in v-CTRL and v-A12 respectively. Data were presented as mean ± SEM. Statistical test: unpaired two-tailed Student’s t-test.). (E) Representative flow cytometry gating of CD62L^- CD8⁺ effector T cells (upper) between control-vaccinated mice and ADAM12-vaccinated mice; corresponding quantitative result of naïve CD8⁺ T cells (bottom, left) and CD62L^- CD8⁺ effector T cells (bottom, right) within splenic CD8⁺ T cells from PDAC tumor-bearing mice treated with control vaccine (v-CTRL) and ADAM12 vaccine (v-A12). (n = 12 mice in v-CTRL and v-A12 respectively. Data were presented as mean ± SEM. Statistical test: unpaired two-tailed Student’s t-test.). (F) Spot-forming cells (SFC) in IFN-γ ELISpot of splenic CD4⁺ T cell and CD8⁺ T cell populations purified from tumor-bearing animals vaccinated with v-CTRL or v-A12 and restimulated with MutuDC1940 cells expressing ADAM12-derived epitopes (ADAM12 target) or with corresponding CTRL target (TurboGFP alone). (n = 6 independent samples in v-CTRL and v-A12 respectively. Data were presented as mean ± SEM. Statistical test: two-way ANOVA.). (G) Gene expression analysis of Adam12 in YAC-1, “KPPC”-derived KP2 PDAC cells (KP2) and ADAM12-expressing NIH 3T3 cells (NIH 3T3 A12). (H) Spontaneous baseline killing of YAC-1 cells by purified splenic CD8⁺ T cells from PDAC tumor-bearing mice vaccinated with v-CTRL or v-A12. (n = 3 independent samples in v-CTRL and v-A12, respectively. Data were presented as mean ± SEM. Statistical test: unpaired two-tailed Student’s t-test.). (I) Cytotoxic assay of purified splenic CD8⁺ T cells from PDAC tumor-bearing mice vaccinated with v-CTRL or v-A12 with target cells (KP2, NIH 3T3 A12). Data represented as fold change to v-CTRL. (n = 3 samples in v-CTRL and v-A12, respectively. Data were presented as mean ± SEM. Statistical test: unpaired two-tailed Student’s t-test.). (J) Spot-forming cells (SFC) in IFN-γ ELISpot of purified splenic CD8⁺ T cells from PDAC tumor-bearing mice vaccinated with control vaccine (v-CTRL) or ADAM12 vaccine (v-A12) co-cultured with target cells (KP2, NIH 3T3 A12). (n = 3 samples in v-CTRL and v-A12, respectively. Data were presented as mean ± SEM. Statistical test: unpaired two-tailed Student’s t-test). Source data are available online for this figure.