Additional file 1 of Nanocages engineered from Bacillus Calmette-Guerin facilitate protective Vγ2Vδ2 T cell immunity against Mycobacterium tuberculosis infection
Creators: name: Pi, Jiang
name: Zhang, Zhiyi
name: Yang, Enzhuo
name: Chen, Lingming
name: Zeng, Lingchan
name: Chen, Yiwei
name: Wang, Richard
name: Huang, Dan
name: Fan, Shuhao
name: Lin, Wensen
name: Shen, Hongbo
name: Xu, Jun-Fa
name: Zeng, Gucheng
name: Shen, Ling
Publisher: name: figshare
Year: 2022
Subjects: subject: Immunology
subject: FOS: Clinical medicine

description: Additional file 1: Fig. S1. Size of BCG-Nanocage upon -80℃storage. Size distribution of BCG-Nanocage before and after 70 days storage at −80℃, samples were thawed at room temperature for size measurements. Fig. S2. BCG preferentially target macrophages (MΦ), but not T cells, B cells or endothelium ex vivo. (A) Confocal imaging for cellular uptake of GFP-BCG by macrophages (MΦ) from the freshly isolated bronchoalveolar lavage (BAL) fluid of rhesus macaques after 1 h treatment of GFP-BCG, followed with anti-human CD11b-APC antibody and anti-human CD3-PE antibody staining, scale bar: 20 μm. (B) Flow cytometry analysis for cellular uptake of GFP-BCG by macrophages, T cells, B cells and endothelium from the freshly isolated intraepithelium (IEL) of rhesus macaques after 1 h treatment. (C) Cellular uptake of GFP-BCG by macrophages, T cells, B cells and endothelium from the freshly isolated intraepithelial lymphocytes of rhesus macaques after 1 h treatment, n = 4, ***p &lt; 0.001. Fig. S3. Comparison of BCG and BCG-Nanocage uptake by macrophages (MΦ), T cells, B cells and endothelium ex vivo. Cellular uptake of GFP-BCG or GFP-BCG-Nanocage by macrophages, T cells, B cells and endothelium from the freshly isolated intraepithelial lymphocytes of rhesus macaques after 1 h treatment, n = 4. Cellular uptake of GFP-BCG or GFP-BCG-Nanocage by macrophages, T cells, B cells and endothelium from the freshly isolated intraepithelial lymphocytes of rhesus macaques after 3 h treatment, n = 4, **p &lt; 0.01. Fig. S4. Effects of BCG and BCG-Nanocage on the viability of CD14+ macrophages, CD3+ T cells, CD20+ B cells and CD56+ NK cells in PBMC from rhesus after 6 day treatment. All cells were gated in lymphocytes and macrophages population of PBMC. (A) Effects of BCG and BCG-Nanocage on the viability of CD14 + macrophages, n = 3, Mean ± S.D, *p &lt; 0.05. (B) Effects of BCG and BCG-Nanocage on the viability of CD3+ T cells, n = 3, Mean ± S.D, ***p &lt; 0.001. (C) Effects of BCG and BCG-Nanocage on the viability of CD20+ B cells, n = 3, Mean ± S.D, *p &lt; 0.05. (D) Effects of BCG and BCG-Nanocage on the viability of CD56 + NK cells, n = 3, Mean ± S.D, *p &lt; 0.05. Fig. S5. Ex vivo activation and expansion of CD4+ CD161+ T cells in PBMC from rhesus macaques induced by co-stimulation with BCG-Nanocage and IL2. (A) Typical flow cytometry panel for CD4+ CD161+ cells gated in T cells from PBMC with or without indicated (co-)stimulations of BCG/IL2, BCG-Nanocage/IL2 or IL2 for 6 days. (B) Effects of indicated (co-)stimulations of BCG/IL2, BCG-Nanocage/IL2 or IL2 on CD4+ CD161+ cells in T cells after 6 day treatment, n = 3, *p &lt; 0.05. (C) Typical flow cytometry panel for Granzyme B+ Perforin + cells gated in CD4+ CD161+ T cells from PBMC with or without indicated (co-)stimulations of BCG/IL2, BCG-Nanocage/IL2 or IL2 for 6 days. (D) Effects indicated (co-)stimulations of BCG/IL2, BCG-Nanocage/IL2 or IL2 on CD4+ CD161+ Granzyme B+ Perforin+ cells in T cells after 6 day treatment, n = 3, *p &lt; 0.05. (E) Typical flow cytometry panel for Granulysin + IFN-γ + cells gated in CD4+ CD161+ T cells from PBMC with or without indicated (co-)stimulations of BCG/IL2, BCG-Nanocage/IL2 or IL2 for 6 days. (F) Effects of indicated (co-)stimulations of BCG/IL2, BCG-Nanocage/IL2 co- or IL2 on CD4+ CD161+ Granulysin + IFN-γ+ cells in T cells after 6 day treatment, n = 3, *p &lt; 0.05. Fig. S6. Ex vivo activation and expansion of CD8+ cytotoxic T cells in PBMC from rhesus macaques induced by BCG-Nanocage and IL2 co-stimulation. (A) Typical flow cytometry panel for CD8 + NKG2C + cells gated in T cells from PBMC with or without (co-)stimulations using BCG/IL2, BCG-Nanocage/IL2 or IL2 for 6 days. (B) Typical flow cytometry panel for Granzyme B + Perforin + cells gated in CD8+ NKG2C+ T cells from PBMC with or without (co-)stimulations using BCG/IL2, BCG-Nanocage/IL2 or IL2 for 6 days. (C) Typical flow cytometry panel for Granulysin + IFN-γ + cells gated in CD8 + NKG2C + Granzyme B + Perforin + T cells from PBMC with or without (co-)stimulations using BCG/IL2, BCG-Nanocage/IL2 or IL2 for 6 days. (D) Effects of (co-)stimulations using BCG/IL2, BCG-Nanocage/IL2 or IL2 on CD8 + NKG2C + Granzyme B + Perforin + Granulysin + IFN-γ + cells in T cells after 6 day treatment, n = 3, **p &lt; 0.01. Fig. S7. BCG and BCG-Nanocage immunization in macaques promote IFN-γ production in CD4+ and CD8+ T cells against stimulation co-stimulations with anti-CD28+ anti-CD49d and BCG-Nanocage. Typical flow images for intracellular IFN-γ levels in CD4+ T cells from PBMC of rhesus macaques before and after BCG immunization or BCG-Nanocage immunization with (A) anti-CD28/anti-CD49d stimulation or (B) anti-CD28/anti-CD49d and BCG-Nanocage stimulation. (C) Statistical results for anti-CD28/anti-CD49d and BCG-Nanocage stimulation induced intracellular IFN-γ levels in CD4+ T cells from PBMC of rhesus macaques before and after BCG immunization or BCG-Nanocage immunization, data were expressed as percentages of control, n = 4. Typical flow images for intracellular IFN-γ level in CD8+ T cells from PBMC of rhesus macaques before and after BCG immunization or BCG-Nanocage immunization with (D) anti-CD28/anti-CD49d stimulation or (E) anti-CD28/anti-CD49d and BCG-Nanocage co-stimulation. (F) Statistical results for anti-CD28/anti-CD49d and BCG-Nanocage stimulation induced changes of intracellular IFN-γ levels in CD8+ T cells from PBMC of rhesus macaques before and after BCG immunization or BCG-Nanocage immunization, data were expressed as percentages of control, n = 4. Fig. S8. BCG-Nanocage immunization in macaques promote IFN-γ production in CD4+ and CD8+ T cells against co-stimulation with anti-CD28+ anti-CD49d and BCG-Nanocage. Typical flow images for intracellular Granzyme B levels in CD4+ T cells from PBMC of rhesus macaques before and after BCG immunization or BCG-Nanocage immunization with (A) anti-CD28/anti-CD49d stimulation or (B) co-stimulation with anti-CD28/anti-CD49d and BCG-Nanocage. (C) Statistical results for co-stimulation anti-CD28/anti-CD49d and BCG-Nanocage induced changes of intracellular Granzyme B levels in CD4+ T cells from PBMC of rhesus macaques before and after BCG immunization or BCG-Nanocage immunization, data were expressed as percentages of control, n = 4. Typical flow images for intracellular Granzyme B levels in CD8+ T cells from PBMC of rhesus macaques before and after BCG immunization or BCG-Nanocage immunization with (D) anti-CD28/anti-CD49d stimulation or (E) co-stimulation with anti-CD28/anti-CD49d and BCG-Nanocage. (F) Statistical results for anti-CD28/anti-CD49d and BCG-Nanocage stimulation induced changes of intracellular Granzyme B levels in CD8+ T cells from PBMC of rhesus macaques before and after BCG immunization or BCG-Nanocage immunization, data were expressed as percentages of control, n = 4. Fig. S9. BCG-Nanocage boost immunization induce rapid peripheral Th1/Th1-like responses to Mtb challenge. Typical flow images for IFN-γ + CD4 + Th1 effector cells in the T cells of PBMC from (A) saline, (B) BCG-immunized and (C) BCG-Nanocage-immunized Rhesus before and after Mtb infection. Typical flow images for IFN-γ + CD8+ Th1-like effector cells in T cells in PBMC from (D) saline, (E) BCG-immunized and (F) BCG-Nanocage-immunized Rhesus before and after Mtb infection. (G) Changes of IFN-γ + CD4 + Th1 effector cells in the T cells of PBMC from saline, BCG-immunized and BCG-Nanocage-immunized Rhesus before and after Mtb infection. (H) Changes of IFN-γ + CD8 + Th1-like effector cells in T cells in PBMC from saline, BCG-immunized and BCG-Nanocage- immunized Rhesus before and after Mtb infection. Fig. S10. The swelling of hilar lymph nodes (HLN) in saline-treated but not in BCG- immunized or BCG-Nanocage-immunized rhesus macaques after Mtb challenge. Blue arrow indicated the swelling structures of HLN.