Immunology and Inflammation

Vaccination with mycobacterial lipid loaded nanoparticle leads to lipid antigen persistence and memory differentiation of antigen-specific T cells

Eva Morgun
Jennifer Zhu
Sultan Almunif
Sharan Bobbala
Melissa S. Aguilar
Junzhong Wang
Kathleen Conner
Yongyong Cui
Liang Cao
Chetan Seshadri
Evan A. Scott author has email address
Chyung-Ru Wang author has email address

Department of Microbiology and Immunology, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA
Department of Biomedical Engineering, Northwestern University, Evanston, IL, USA
Department of Medicine, University of Washington School of Medicine, Seattle, WA, USA

https://doi.org/10.7554/eLife.87431.1

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Figures and data

Physicochemical characterization of BCN and PLGA-NP.
(A) Dynamic light scattering (DLS) analysis of blank BCN and MA-BCN. (B) CryoTEM of MA-BCN (scale = 500 nm). (C) Negative staining TEM images of MA-BCN. (D) SAXS of blank BCN and MA-BCN. (E) DLS analysis of blank PLGA and MA PLGA. (F) MA encapsulation efficiency for MA-BCN and MA PLGA. N = 3 per condition. Data represented as mean ± SD.

MA-BCN effectively activated MA-specific T cells in vitro and in vivo.
(A, B) BMDCs were pulsed for 18 hrs. with selected nanoparticles at various concentrations, co-cultured for 48 hrs. with DN1 T cells, and T cell activation was measured. (A) Percentage of CD69 and CD25-expressing DN1 T cells. (B) IFN-g production measured by ELISA. (N=3). (C, D) ELISPOT of IFN-g from MA-specific human M11 T cells cultured for 16 hours with monocyte-derived dendritic cells with selected nanoparticles at high (C) and low (D) concentrations. Data are representative of two independent experiments and displayed as mean ± SEM. Statistical analysis: 2-way ANOVA, significance designated for MA-BCN vs free MA. (E-G) hCD1Tg mice were IN vaccinated with selected nanoparticles and CellTrace Violet-labeled DN1 T cells were adoptively transferred the next day. After 1 week, DN1 T cell activation and proliferation was measured. (E) Representative FACS plots of DN1 T cells in the LN. Percentage of proliferating (F) and CD44-expressing (G) DN1 T cells in the LN and lung. Data represented as mean ± SEM. Statistical analysis: 2-way ANOVA. *p<0.05, **p<0.01, ***p<0.001, ****p< 0.0001.

Vaccination with MA-BCN leads to antigen persistence 6 weeks post-vaccination.
hCD1Tg mice were IT vaccinated with MA-BCN or BCN. 6 weeks later, CellTrace-labeled p25 and DN1 T cells were adoptively transferred, and T cell activation was measured after 1 week. (A) Experimental design. (B) Representative FACS plots of p25 and DN1 T cells in the LN. Percentage of proliferating (C) and CD44-expressing (D) p25 and DN1 T cells in the LN, lung, and spleen. N = 3 or 6 per condition. Outliers were identified through Grubbs method with alpha=0.05 with one sample removed. Data represented as mean ± SEM. Statistical analysis: 2-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Ag85B and MA dual loaded BCNs activate antigen-specific T cells without Ag85B persistence.
(A) DLS analysis of blank BCN and Ag85B-MA-BCN. (B-D) hCD1Tg mice were IT vaccinated with Ag85B-MA-BCN or BCN and CellTrace-labeled p25 and DN1 T cells were adoptively transferred the next day. After 1 week, T cell activation and proliferation was measured. (B) Representative FACS plots of p25 and DN1 T cells in the LN. Percentage of proliferating (C) and CD44-expressing (D) p25 and DN1 T cells in the LN, lung, and spleen (N = 4 or 5). (E-G) hCD1Tg mice were IT vaccinated with blank BCN or Ag85B-MA-BCN. 6 weeks later, CellTrace-labeled p25 and DN1 T cells were adoptively transferred, and T cell activation was measured after 1 week. (E) Representative FACS plots of p25 and DN1 T cells in the LN. (F) Percentage of proliferating p25 and DN1 T cells. (G) Percentage of CD44-expressing p25 and DN1 T cells in the LN and lung. (N = 4). Data represented as mean ± SEM. Statistical analysis: 2-way ANOVA. ns = not significant, *p<0.05, **p<0.01, ***p<0.001, ****p< 0.0001.

Route of vaccination or vector type do not affect antigen persistence.
hCD1Tg mice were IT vaccinated with MA or MA-BCN pulsed BMDCs at 6 weeks or 1 week prior to the adoptive transfer of CellTrace-labeled DN1 T cells. T cell activation and proliferation was measured 1 week after adoptive transfer. (A) Experimental design. (B, C) Percentage of proliferating (B) and CD44-expressing (C) DN1 T cells in the LN, lung, and spleen of vaccinated mice. N = 4 per condition. N = 4 per condition. hCD1Tg mice were vaccinated SC (subcutaneously) or IV (intravenously) with blank BCN, MA-BCN, or attenuated Mtb strain and 6 weeks later CellTrace-labeled DN1 T cells were adoptively transferred. (D) Representative FACS plots of DN1 T cells in the LN of mice vaccinated with indicated conditions. (E) Percentage of proliferating DN1 T cells and (F) Percentage of CD44-expressing DN1 T cells in the LN and spleen of mice vaccinated with Blank-BCN (SC), MA-BCN (IV and SC), and attenuated Mtb. N = 3-5 per condition. Data represented as mean ± SEM. Statistical analysis: 2-way ANOVA. ns = not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Persistent MA remains in part due to encapsulation inside alveolar macrophages and activates T cells through DCs.
B6 or hCD1Tg mice were IT vaccinated with BCN loaded with a hydrophobic fluorescent dye (DiD) at 6 weeks, 6 days, 48 hours, 24 hours, or 4 hours prior to the experiment. LN, lung, and spleen were analyzed using (A) In Vivo Imaging System (IVIS). Single cell suspension was then obtained and presence of DiD BCN was quantified in (B) total lung, and (C) CD45⁻ cells, neutrophils (Ly6G⁺), alveolar macrophages (CD11c⁺SiglecF⁺), DCs (CD11c⁺), monocytes (CD11b⁺CD11c⁻), B cells (CD19⁺), T cells (CD3⁺), NK cells (NK1.1⁺), eosinophils (CD11c⁻SiglecF⁺) by flow cytometry. Gating strategy exemplified in Suppl Fig 4. hCD1Tg mice were IT vaccinated with either BCN or MA-BCN. After 6 weeks, alveolar macrophages were enriched from lungs using anti-SiglecF. Enriched or flow through cells were co-cultured with DN1 T cells in the presence or absence of hCD1Tg-expressing BMDCs for 48 hours, and DN1 T cells activation was measured. (D) Experimental design. (E) Percentage of CD25-expressing DN1 T cells. Data represented as mean ± SEM. Statistical analysis: 2-way ANOVA. ns = not significant, *p<0.05, **p<0.01, ***p<0.001.

MA-BCN vaccination leads to DN1 T cell differentiation into T follicular helper-like T cells.
Mixed bone marrow chimeric mice were created by adoptive transfer of DN1 bone marrow and congenic CD45.1 hCD1Tg bone marrow to irradiated hCD1Tg mice. After 5 weeks, mice were vaccinated with either MA-BCN or PBS, and DN1 surface marker expression was monitored in the blood and in organs at 6 wks. (A) Experimental design. (B) Percent CD44⁺ DN1 T cells in the blood at various time points post vaccination. (C) Percent CD44⁺CD62L⁺ DN1 T cells in indicated organs at 6 weeks post vaccination. Memory (CD44⁺CD62L⁺) and naïve (CD44⁻CD62L⁺) DN1 T cells were sorted from MA-BCN vaccinated hCD1Tg-DN1 BM chimeras at 6 weeks post vaccination and subjected to RNAseq analysis. N=3 per condition (D, E) PCA of log fold change of gene expression for each T cell subset was performed relative to internal naïve T cell control. (F) Relative expression of key T_FH cell and DEGs in memory subset. (G) Gene enrichment analysis of upregulated DEGs was performed using Metascape. (H) Representative FACS plots of DN1 T cells in the LN of mice. Percentage of (I) CXCR5⁺PD1⁺ or (J) KI67⁺ DN1 T cells DN1 T cells in LN, lung, and spleen. N = 3-5 per condition. Data represented as mean ± SEM. Statistical analysis: 2-way ANOVA. *p<0.05, **p<0.01, ****p < 0.0001.

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