Fig 1.
Transient Treg-cell depletion without adjuvants induces neutralizing antibodies to authentic SARS-CoV-2.
(A) A schematic diagram showing the experimental workflow. Wild type (WT) and Foxp3-DTR (Foxp3DTR) mice were injected with diphtheria toxin (DT) (50 μg/kg) intraperitoneally (i.p.) and SARS-CoV-2 spike protein 1 (S1) (10 μg) subcutaneously (s.c.) into four footpads on day 0. At the indicated days, peripheral blood mononuclear cells (PBMCs), draining lymph nodes (axillary and popliteal), spleen and sera were analyzed. (B) PBMCs were analyzed for Foxp3 at the indicated time points. Representative FACS plots from two independent experiments, gated on CD45+ CD4+ cells, are shown (S1A Fig, for gating strategy). Representative cell frequency data from two separate experiments are shown (n = 3/group). Data are presented as the mean ± SEM, two-way ANOVA followed by Bonferroni’s multiple comparisons test. (C) The levels of whole S1 or receptor-binding domain (RBD)-specific IgM and IgG in sera on day 10 post-injection were determined by ELISA. Viral antibody endpoint titers against S1 and RBD were expressed as the reciprocal of the highest dilution with an optical density at 490 nm (OD490) cutoff value > 0.1. Sera obtained from three independent experiments were analyzed. Data represents the mean ± SEM (DT -injected WT mice, n = 4; DT -injected Foxp3DTR mice, n = 3; DT+S1-injected WT mice, n = 6; DT+S1-injected Foxp3DTR mice, n = 6). Data were analyzed using the Kruskal-Wallis test with Dunn’s multiple comparisons test. (D) Levels of neutralizing antibodies to authentic SARS-CoV-2 in sera on day 10 post-injection were analyzed by plaque reduction neutralization tests (PRNT). The highest dilution reducing plaque numbers by 50% (PRNT50) titers were determined. Data represent the mean ± SEM from three independent experiments (DT-injected WT mice, n = 4; DT-injected Foxp3DTR mice, n = 3; DT+S1-injected WT mice, n = 6; DT+S1-injected Foxp3DTR mice, n = 6). Data were analyzed using the Kruskal-Wallis test with Dunn’s multiple comparisons test. The horizontal broken line indicates the detection limit. (E) Levels of anti-dsDNA IgG and total IgG in sera on day 10 post-injection were measured by ELISA. Data represent the mean ± SEM from three independent experiments (DT+S1-injected WT mice, n = 6; DT+S1-injected Foxp3DTR mice, n = 6). The sera from 10-week female MRL/lpr (MRLlpr) mice were used as a positive control (n = 4). The horizontal broken lines in the right panel indicate the detection limits. Kruskal–Wallis test with Dunn’s multiple comparisons test.
Fig 2.
Transient Treg-cell depletion induces the formation of follicular helper T cells and antigen-specific B cells in S1-injected mice.
(A) A schematic diagram showing the experimental workflow. As in Fig 1A, but follicular helper T(Tfh), follicular regulatory T (Tfr) and B cells from draining (axillary and popliteal) lymph nodes and spleens were analyzed on day 10 post-injection. (B) Representative gating strategy used to identify Tfr and Tfh cells (S1B Fig, gating strategy). Representative FACS plots of two independent experiments are shown. Representative graphs of two independent experiments are shown as the mean ± SEM (n = 3/group). Data were analyzed using unpaired Student’s t-test. (C) Representative FACS plots of GL7+CD38- germinal center (GC) B cells gated on live B220+ CD4- cells (S1C Fig, gating strategy). IgM- IgD- class-switched GC B cells were gated on GL7+ CD38- GC B cells. RBD-binding class-switched GC B cells were gated on IgM- IgD- class-switched GC B cells. Influenza virus HA protein was used as the negative control for RBD-binding. Representative FACS plots of two independent experiments are shown. Representative frequencies of GC B, class-switched GC B, and RBD-binding GC B cells from two independent experiments are plotted as the mean ± SEM (n = 3/group). Data were analyzed using unpaired Student’s t-test.
Fig 3.
DCs undergo maturation without adjuvants in transient Treg cell-depleted mice.
(A) A schematic diagram showing the experimental workflow. As in Fig 1A, but dendritic cells (DCs) from draining lymph nodes (axillary and popliteal) were analyzed on day 2. (B) A representative FACS plot from two independent experiments is shown for MHCIIhigh CD11cint migratory DCs, MHCIIint CD11chigh resident DCs, and MHCII+ CD11c- non-DCs gated on CD45+ cells (S1D Fig, gating strategy). Frequency of resident DCs, migratory DCs and MHCII+ CD11c- non-DCs from draining lymph nodes of DT+S1-injected WT and Foxp3DTR mice. The data are represented summaries from two independent experiments as the mean ± SEM (DT+S1-injected WT mice, n = 4; DT+S1-injected Foxp3DTR mice, n = 3). Data were analyzed using unpaired Student’s t-test. (C) Representative histograms from two independent experiments measuring CD80 (top) and CD86 (bottom) in resident DCs, migratory DCs, and non-DCs are shown. The graphic data showing the mean fluorescence intensity (MFI) are summarized from two independent experiments (DT+S1-injected WT mice, n = 4; DT+S1-injected Foxp3DTR mice, n = 3). Data were analyzed using unpaired Student’s t-test.
Fig 4.
DCs capture S1 protein and undergo maturation in transient Treg cell-depleted mice.
(A) A schematic diagram showing the experimental workflow. WT and Foxp3DTR mice were injected with DT (50 μg/kg) i.p. and Alexa Fluor 647 labeled S1(S1AF467) (5 μg) s.c. into two fore footpads on day 0. Cells from draining (axillary) lymph nodes and distal (inguinal) lymph nodes were analyzed on day 2. (B) Representative FACS plots of S1AF647+ cells in the distal and draining lymph nodes from WT mice on day 2. Cells were gated on live cells. Percentage of S1AF647+ CD45+ cells / live cells in distal and draining lymph nodes from DT+S1 AF647-injected WT and Foxp3DTR mice. Representative data from two independent experiments were plotted as the mean ± SEM (n = 3 mice /group). Data were analyzed using two-way ANOVA with Tukey’s multiple comparisons test. (C) Percentage of S1AF647+ cells in resident DCs, migratory DCs or MHCII+ CD11c- non-DCs in draining lymph nodes from DT+S1 AF647-injected WT and Foxp3DTR mice. Representative data from two independent experiments is plotted as the mean ± SEM (n = 3 mice /group). Data were analyzed using two-way ANOVA with Tukey’s multiple comparisons test. (D) A representative FACS plot to identify migratory DCs, resident DCs, and MHCII+ CD11c- non-DCs, gated on live CD45+ S1AF647+ cells from draining lymph nodes of DT+S1AF647-injected Foxp3DTR mice are shown (top). Representative overlaid contour plots of CD80 and CD86 expression gated on S1AF647+ resident DCs, S1AF647+ migratory DCs or S1AF647+ MHCII+ CD11c- non-DCs from draining lymph nodes of DT+S1 AF647-injected WT or Foxp3DTR mice are shown (WT, green; Foxp3DTR, red-purple). Representative of two independent experiments for FACS plots and graphics are shown. The graphic data are presented as the mean ± SEM of the MFI (n = 3 / group). Data were analyzed using unpaired Student’s t-test.
Fig 5.
SARS-CoV-2 antigen-specific IFN-γ producing CD4+ and CD8+ T cells are successfully induced in transient Treg cell-depleted mice.
(A) A schematic diagram showing the experimental workflow. As in Fig 1A, but IFN-γ-secreting cells in draining lymph nodes (axillary and popliteal) and spleen on day 10 were analyzed using ELISpot and intracellular cytokine staining. (B) Representative pictures of IFN-γ ELISpot assays from draining lymph nodes and spleens on day 10 following S1 and DT injection are shown (top). The plates of the ELISpot Kit are from R&D Systems Inc.. IFN-γ-secreting spots were quantified after 36 h of stimulation with 1 μg/mL S1 peptide pool. The graphic data are a summary of two independent experiments. The data are represented as the mean ± SEM (DT-injected WT mice, n = 3; DT-injected Foxp3DTR mice, n = 2; DT+S1-injected WT mice, n = 5; DT+S1-injected Foxp3DTR mice, n = 5). Data were analyzed using two-way ANOVA with Tukey’s multiple comparisons test. (C) IFN-γ-secreting cells from draining lymph nodes on day 10 were analyzed by flow cytometry after 6 h of stimulation with 1 μg/mL S1 peptide pool. Representative FACS plots from two independent experiments are shown for CD4+ (left) and CD8+ T (right) cells, gated on live CD45+, CD3+ cells (S1E Fig, gating strategy). The graphic data of the frequencies of IFN-γ positive cells are representative from two independent experiments (n = 3/group). The mean ± SEM is shown. Data were analyzed using two-way ANOVA with Tukey’s multiple comparisons test.
Fig 6.
Transient Treg-cell depletion induces humoral and cellular immune responses to SARS-CoV-2 similar to poly IC, and the combination of both can enhance the response.
(A) A schematic diagram showing the experimental workflow. As in Fig 1A, but WT and Foxp3DTR mice were injected with DT i.p. and S1 s.c. with or without poly IC (50 μg) s.c. into four footpads on day 0. PBMCs were analyzed for Treg-cell depletion on day 2. On day 10, sera and draining lymph nodes (axillary and popliteal) were analyzed for antibodies and IFN-γ-secreting cells, respectively. (B) PBMCs were analyzed for Foxp3 on day 2. Representative FACS plots from a single independent experiment, gated on CD45+ CD4+ cells, are shown (S1A Fig for gating strategy). Data are presented as mean ± SEM (n = 3 /group). Data were analyzed using two-way ANOVA followed by Tukey’s multiple comparisons test. (C) Levels of whole S1 or RBD-specific IgM and IgG in sera on day 10 post-injection were determined by ELISA. Viral antibody endpoint titers against S1 and RBD were expressed as the reciprocal of the highest dilution with an optical density at 490 nm (OD490) cutoff value > 0.1. Sera obtained from a single experiment were analyzed. Data represents the mean ± SEM from a single experiment (n = 3 /group). Data were analyzed using two-way ANOVA followed by Tukey’s multiple comparisons test. The horizontal broken line indicates the detection limit. (D) The levels of neutralizing antibodies to authentic SARS-CoV-2 in sera on day 10 post-injection were analyzed by PRNT. The highest dilution reducing plaque numbers by 50% (PRNT50) titers were determined. Data represent the mean ± SEM from a single experiment (n = 3 /group). Data presented as mean ± SEM. Data were analyzed by two-way ANOVA followed by Tukey’s multiple comparisons test. The horizontal broken line indicates the detection limit. (E) Representative pictures of IFN-γ ELISpot assays from draining lymph nodes and spleens on day 10 following S1 and DT injection with or without poly IC are shown (left). The plates of the ELISpot Kit are from Abcam Inc.. IFN-γ-secreting spots were quantified after 36 h of stimulation with or without 1 μg/mL S1 peptide pool. The right graphics represent mean ±SEM from a single experiment (n = 3 /group). Data were analyzed using two-way ANOVA with Tukey’s multiple comparison.