Abstract
Personalized cancer vaccines are a promising approach for inducing T cell immunity to tumor neoantigens. Using a self-assembling nanoparticle vaccine that links neoantigen peptides to a Toll-like receptor 7/8 agonist (SNP-7/8a), we show how the route and dose alter the magnitude and quality of neoantigen-specific CD8+ T cells. Intravenous vaccination (SNP-IV) induced a higher proportion of TCF1+PD-1+CD8+ T cells as compared to subcutaneous immunization (SNP-SC). Single-cell RNA sequencing showed that SNP-IV induced stem-like genes (Tcf7, Slamf6, Xcl1) whereas SNP-SC enriched for effector genes (Gzmb, Klrg1, Cx3cr1). Stem-like cells generated by SNP-IV proliferated and differentiated into effector cells upon checkpoint blockade, leading to superior antitumor response as compared to SNP-SC in a therapeutic model. The duration of antigen presentation by dendritic cells controlled the magnitude and quality of CD8+ T cells. These data demonstrate how to optimize antitumor immunity by modulating vaccine parameters for specific generation of effector or stem-like CD8+ T cells.
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Data availability
The data that support the findings of this study are available from the corresponding author upon request. The scRNA-seq data have been uploaded to the Gene Expression Omnibus (accession number GSE158240).
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Acknowledgements
We thank members of the Seder lab for scientific discussions; S. Darko, A. Ransier and D. Douek for scRNA-seq advice; and M. Dillon, G. Salbador, S. Rush, L. Gilliam, O. Hernandez, C. Chiedi and D. Scorpio of the Translational Research Program (Vaccine Research Center) for their valuable support with the animal studies. This work was supported by the Intramural Research Program of the U.S. NIH (R.A.S., H.D.H. and J.S.T.), EMBO YIP and Singapore Immunology Network core funding (F.G.).
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F.B., A.S.I. and R.A.S. conceived and designed the experiments. F.B., R.A.R.-V., K.K.S.T. and H.Y. performed the experiments including animal work, flow cytometry and ELISA. R.A.R.-V., C-A.D. and A.K. performed the scRNA-seq data analysis with the support of M.P.M., A.J.M., J.S.T., X.M.Z. and F.G. F.B., G.V.R. and H.D.H. performed and analyzed the confocal microscopy data. J.A.H., J.P.F. and N.B. provided the mice and reagents for the experiments. G.M.L., V.L.C. and A.S.I. designed and prepared the vaccines for the experiments. F.B. and R.A.S. prepared the figures and wrote the manuscript with input from all authors.
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G.M.L., V.L.C., A.S.I. and R.A.S. are listed as inventors on patents describing polymer-based vaccines. G.M.L., V.L.C. and A.S.I. are employees of Avidea Technologies, which is commercializing polymer-based drug delivery technologies for immunotherapeutic applications.
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Extended data
Extended Data Fig. 1 Route and dose of SNP-7/8a immunization controls the magnitude and phenotype of antigen-specific CD8+ T cells.
a, Whole blood was collected on day 21 to measure the frequency of tetramer+ CD8+ T cells post boost. Bar graphs summarize the frequency of tetramer+ CD8+ T cells from blood (n = 10). b, Bar graphs summarize the frequency of IFNγ+ CD8+ T cells from blood (n = 10) on day 21. c, Bar graphs summarize the frequency of IFNγ+ CD4+ T cells from blood (n = 10). d, Bar graphs show proportions of MPEC/SLEC subpopulations in the blood (n = 10). e, Frequency of MPECs is negatively correlated to frequency of tetramer+ CD8+ T cells. f, g, Bar graphs show proportions of PD-1/Tim-3 subpopulations in the blood (n = 10) of tetramer+ cells (f) or IFNγ+ cells (g). Data are representative of two independent experiments. The bars represent the median (a–c) or mean ± s.e.m. (d, f, g). Statistics were assessed by Kruskal-Wallis with Dunn’s correction for multiple comparisons (a, b, d, f, g) and Spearman correlation (e).
Extended Data Fig. 2 Intravenous administration of SNP-7/8a generates TCF1+ CD8+ T cells with anti-tumor capacity upon anti-PD-L1 treatment.
a, Tumor growth curves of mice unvaccinated (black) or vaccinated with SNP-SC (blue) or SNP-IV (red) with (dotted line) or without α-PD-L1 (solid line) (n = 10). b, Flow cytometric analysis of single cells from spleen (concatenated, n = 6) after SNP-SC (top panel) or SNP-IV (bottom panel). Cells were stained with Reps1 tetramer and other antibodies. Numbers indicate percentage of cell population within the gate. c, Mice were vaccinated with SNP-7/8a containing Reps1, E7, OVA or Trp1 antigens (n = 5). Spleens were collected 7 days post prime. d, Splenocytes were stained with tetramers specific for the respective antigens. Bar graph summarizes the frequencies of antigen-specific CD8+ T cells following SNP-SC (blue) or SNP-IV (red) (n = 5). e, Bar graph summarizes the frequencies of TCF1 subpopulations in the spleen (n = 5) after SNP-SC or SNP-IV. f, Frequency of TCF1+PD-1+ cells is negatively correlated to frequency of tetramer+ CD8+ T cells. g, Bar graph summarizes the frequencies of early effector cells (EEC, gray), memory precursor effector cells (MPEC, tan), double positive effector cells (DPEC, lilac) and short lived effector cells (SLEC, crimson) in the spleen (n = 5) after SNP-SC or SNP-IV. h, Frequency of MPEC is negatively correlated to frequency of tetramer+ CD8+ T cells. The bars represent mean ± s.e.m. (d,e,g). Statistics were assessed by Mann Whitney test (d, e, g) and Spearman correlation (f, h).
Extended Data Fig. 3 Single-cell analysis of neoAg+ CD8+ T cells by RNA sequencing identifies stem-like gene signature in SNP-IV and effector gene signature in SNP-SC cells.
a, C57BL/6 mice (n = 5) were vaccinated subcutaneously or intravenously at 8 nmol on day 0 and day 14 with SNP-7/8a containing Reps1. Spleens were collected on day 28 and tetramer+ CD8+ T cells were sorted by flow cytometry. Flow plots show gating strategy for cell sorting. b, Mice were individually labeled with distinct hashtag oligo-tagged antibodies and pooled for 10x and RNA sequencing. Individual UMAPs show gene expression of each mouse vaccinated SC (left panel) or IV (right panel). c, Bar graph summarizes the frequency of the twelve Monocle 3 clusters that are represented by each vaccination route (n = 5). d, Density plots to identify stability states corresponding to higher density areas on UMAP, based on 2D kernel density estimation. e, Expression of top differentially expressed genes (DEG) of naïve cells are presented in meaning plots. f, Heatmap summarizes the number of cells that share a clonotype based on paired alpha and beta complementarity-determining region 3 (CDR3) sequences in each individual animal. g, Bar graph shows numbers of stem-like cells (clusters 2 and 4) and effector cells (clusters 1, 3, 5, 7 and 8) in each clonotype from SC or IV vaccinated mice. Only clonotypes expressed by more than 100 cells are represented in the graphs. h, Heatmap of DEG expressed in each cluster organized along the pseudotime trajectory.
Extended Data Fig. 4 Therapeutic vaccination with SNP-IV generates neoAg-specific CD8+ T cells with superior anti-tumor capacity.
a, Tumor growth of mice treated with SNP-7/8a with (red) or without agonist (gray) (n = 10). b, Average tumor growth of SNP-IV (red), SNP-SC given twice (blue), once on day 7 (dotted blue) or twice at a lower dose (light blue) (n = 10). c, Total numbers of CD8+ T cells, CD4+ T cells and NK cells and d, frequency of tetramer+ CD8+ T cells from blood in mice treated with isotype control antibody (red) or blocking antibodies against CD8β (black), CD4 (blue) or NK1.1 (purple) as assessed by flow cytometry (n = 10). e–h, Spleens and tumors were harvested on day 14 (n = 10) and day 21 (spleen, n = 5; tumor, n = 3). e, Stem-like cells (TCF1+PD-1+; dark blue), f, effector cells (Granzyme B+TCF1–; orange) or g, exhausted cells (PD-1+Tim-3+) of tetramer+ cells were identified by flow cytometry. Bar graphs summarize the frequency of cells in the spleen and tumors on day 14 (filled bar) or day 21 (checked bar). h, Bar graphs summarize the frequency of Ki-67+ cells in different tissues on day 14 (red bar) or day 21 (checked bar) post SNP-IV. Data are representative of four independent experiments. Mean ± s.e.m. Statistics were assessed by two-way ANOVA with Bonferroni correction (a, b) and Mann Whitney test (e–h).
Extended Data Fig. 5 Transient vaccine distribution to spleen and activation of migratory cDC1 and moDC in after SNP-IV.
a, Whole body images of mice following SNP-SC or SNP-IV with labeled vaccines. b, Confocal images of LN or spleen sections of an unvaccinated mouse. c, Confocal image of popliteal LN section post SNP-SC. Detailed overlay of additional markers. White, vaccine; red, ERTR7 (stroma); orange, CD11b (monocytes, macrophages or cDC2); CD11c (moDC or cDC). Scale bar, 200 µm or 50 µm (inset). Arrows show co-localization of vaccine and CD11b+CD11c+ cells. d, Gating strategy to identify various populations from popliteal LN and spleen after SNP-SC and SNP-IV: MoDC (red), monocytes (pink), subcapsular sinus macrophages, SCS (gray), red pulp macrophages, RPM (dark gray), cDC1 (maroon), cDC2 (coral). Kinetics of MNPs that are vaccine+ in e, popliteal LNs or f, spleens after SNP-SC or SNP-IV respectively (n = 3). g, Histograms show MFI of CD80, CD86, CCR7 and labeled vaccine in migratory or resident cDC1 or moDC in popliteal LN of naïve (gray) or SNP-SC mice after vaccination (concatenated, n = 3). h, i, Flow cytometric analysis of single cells stained with XCR1 and CD86 after gating on cDC1s in spleens or popliteal LNs of mice post SNP-IV or SNP-SC respectively (n = 3). Mean ± s.e.m. (i). Data are representative of two independent experiments.
Extended Data Fig. 6 Prolonged antigen presentation by DC drives CD8+ T cell responses after SNP-SC.
a, WT, Batf3–/– or Ccr2–/– mice (n = 10) were vaccinated SC or IV at 8 nmol on day 0 and day 14 with SNP-7/8a (Reps1). b, Total number of cDC1 in spleen and popliteal LN, or monocytes in popliteal LN (right panel) of WT, Batf3–/– or Ccr2–/– were measured (n = 3). c, Bone marrow (BM) chimeras were performed by irradiating WT CD45.1 mice and transferring BM from Ccr2DTR or WT CD45.2 mice. After 8 weeks of reconstitution, mice were treated with DT (n = 3). d, Total number of monocytes, cDC1 and cDC2 in spleen of Ccr2DTR mice 24 h after DT treatment was measured (n = 3). e, Kinetics of neoAg-specific CD8+ T cell responses after SNP-IV in blood of Ccr2DTR without DT treatment, or WT CD45.2 BM chimera with or without prior DT treatment showed similar responses (n = 5). f, Sera were collected after SNP-SC (blue) or SNP-IV (red). IL-12 (left panel) or IFN-α (right panel) were measured by ELISA (n = 3). g, Total number of monocytes and cDC1 in popliteal LN of WT, Ifnar–/– or Tlr7–/– were measured by flow cytometry. h, Histograms of EOMES gated on tetramer+ cells post SNP-SC in WT or IL12b–/– mice (n = 4). Mean ± s.e.m. (e–g). Data are representative of two independent experiments. Statistics were assessed by Mann Whitney test (d) or Kruskal-Wallis with Dunn’s correction for multiple comparisons (g).
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Baharom, F., Ramirez-Valdez, R.A., Tobin, K.K.S. et al. Intravenous nanoparticle vaccination generates stem-like TCF1+ neoantigen-specific CD8+ T cells. Nat Immunol 22, 41–52 (2021). https://doi.org/10.1038/s41590-020-00810-3
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DOI: https://doi.org/10.1038/s41590-020-00810-3
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