Journal: bioRxiv

Article Title: Adjuvant conditioning shapes the adaptive immune response and promotes trained immunotolerance via NLRP3/IL-1

doi: 10.1101/2024.09.06.611736

Figure Lengend Snippet: Adjuvant conditioning (AC) shapes the adaptive immune response in vivo . A: Experimental design. 8-10-week-old male or female C57BL/6 mice were injected intraperitoneally (i.p.) with either Alum Imject™ (8mg in 200µL) or saline three times, every other day. 24 hours after the last injection, mice were adoptively transferred with 5×10 6 OT-II splenocytes intravenously, and 24 hours after the transfer, mice were immunized subcutaneously with 10 μg OVA adsorbed in 1.5mg of Alum Imject™, in a homologous prime (day 1) and boost (day 8) protocol. Seven days after the boost (day 15), mice were euthanized, and spleens were analyzed for OVA-specific Th1, Th2, Th17, and Treg total numbers. B: Flow cytometry plots and total number of OVA-specific T cells, evidenced by the expression of CD4 + Vα + in splenocytes from mice treated with either saline or alum before the immunization. Flow cytometry plots and total number of OVA-specific Th1 (C) , Th2 (D) , Th17 (E) , and Tregs (F) , evidenced by the expression of CD4 + Vα + Tbet + , CD4 + Vα + GATA3 + , CD4 + Vα + RORγT + , and CD4 + Vα + Foxp3 + in splenocytes from mice treated with either saline or alum before the immunization. G: Flow cytometry plots and total number (H) of OVA-specific Th17 and Tregs, evidenced by the expression of CD4 + Vα + IL-17A and CD4 + Vα + LAP + in splenocytes treated with either saline or alum before the immunization and further cultured with OVAp (2μg/mL) in vitro for 48h in the presence of brefeldin-A. I: OVA-specific IgG titer in serum from mice treated with either saline or alum before the immunization. Data shown represent three or more experiments and are expressed as mean±SEM; Student t-test was used for analysis; P values are indicated in each graph. All statistical analyses were performed using GraphPad Prism™.

Article Snippet: Single-cell suspensions were prepared from mouse splenocytes after RBC lysis using ACK lysis buffer (ThermoFisher Scientific).

Techniques: Adjuvant, In Vivo, Injection, Saline, Flow Cytometry, Expressing, Cell Culture, In Vitro

Journal: bioRxiv

Article Title: Adjuvant conditioning shapes the adaptive immune response and promotes trained immunotolerance via NLRP3/IL-1

doi: 10.1101/2024.09.06.611736

Figure Lengend Snippet: NLRP3 activation required for the effects of adjuvant conditioning in the adaptive immune response. A . Experimental design. 8-10-week-old male or female C57BL/6 or NLRP3 -/- mice were injected intraperitoneally (i.p.) with either Alum Imject™ (8mg in 200µL) or saline three times, every other day. 24 hours after the last injection, mice were adoptively transferred with 5×10 6 OT-II splenocytes intravenously, and 24 hours after the transfer, mice were immunized with 10 μg OVA adsorbed in Resiquimod (R848) (OVA-RSQ) (50µg), in a homologous prime (day 1) and boost (day 8) protocol. Seven days after the boost (day 15), mice were euthanized, and spleens were analyzed for OVA-specific Th1, Th2, Th17, and Treg total numbers. B: Total number of OVA-specific T cells, evidenced by the expression of CD4 + Vα + in splenocytes from mice treated with either saline or alum before the immunization. C: OVA-specific IgG titer in serum from mice treated with either saline or alum before the immunization. D: Flow cytometry plots of OVA-specific T cells, evidenced by the expression of CD4 + Vα + in splenocytes from mice treated with either saline or alum before the immunization. E: Total number of OVA-specific Th1, Th2, Th17 and Tregs, evidenced by the expression of CD4 + Vα + IFN-γ + , CD4 + Vα + IL-4 + , CD4 + Vα + IL-17A + and CD4 + Vα + LAP + respectively in splenocytes treated with either saline or alum before the immunization and further cultured with OVAp (2μg/mL) in vitro for 48h in the presence of brefeldin-A. Data shown represent three or more experiments, and are expressed as mean±SEM; Student t-test was used for analysis; P values are indicated in the graphs. All statistical analyses were performed using GraphPad Prism™.

Article Snippet: Single-cell suspensions were prepared from mouse splenocytes after RBC lysis using ACK lysis buffer (ThermoFisher Scientific).

Techniques: Activation Assay, Adjuvant, Injection, Saline, Expressing, Flow Cytometry, Cell Culture, In Vitro

Journal: bioRxiv

Article Title: Adjuvant conditioning shapes the adaptive immune response and promotes trained immunotolerance via NLRP3/IL-1

doi: 10.1101/2024.09.06.611736

Figure Lengend Snippet: NLRP3-dependent IL-1 signaling is involved in the effects of adjuvant conditioning in the adaptive immune response. A: Flow cytometry plots and total number (B) of OVA-specific T cells, evidenced by the expression of CD4 + Vα + in splenocytes from mice treated with either saline, alum or alum + Anakinra (30mg/kg) (AC + AK) before the immunization. C: OVA-specific IgG titer in serum from mice treated with either saline, alum or alum + Anakinra (30mg/kg) (AC+AK) before the immunization. D: Total number of OVA-specific Th1, Th2, Th17 and Tregs, evidenced by the expression of CD4 + Vα + IFN -γ + , CD4 + Vα + IL-4 + , CD4 + Vα + IL-17A + and CD4 + Vα + LAP + in splenocytes treated with either saline, alum or alum + Anakinra (30mg/kg) (AC+AK) before the immunization and further cultured with OVAp (2μg/mL) in vitro for 48h in the presence of brefeldin-A. Data shown represent three or more experiments, and are expressed as mean±SEM; Student t-test was used for analysis; P values are indicated in the graphs. All statistical analyses were performed using GraphPad Prism™.

Article Snippet: Single-cell suspensions were prepared from mouse splenocytes after RBC lysis using ACK lysis buffer (ThermoFisher Scientific).

Techniques: Adjuvant, Flow Cytometry, Expressing, Saline, Cell Culture, In Vitro

Journal: bioRxiv

Article Title: Adjuvant conditioning shapes the adaptive immune response and promotes trained immunotolerance via NLRP3/IL-1

doi: 10.1101/2024.09.06.611736

Figure Lengend Snippet: Adjuvant conditioning induces the reprogramming of myeloid cells to a trained immunosuppression phenotype. A: Flow cytometry plots of CD11b + Ly6C + and CD11b + Ly6G + cells in the spleen 24h after saline or alum injections. B: TNF-α, IL-6 (C) , IL-10 (D) , and nitric oxide (E) production from CD11b + GR1 + cells isolated from saline or AC-treated mice cultured with either medium or LPS (200ng/mL) for 24h. F: Histograms of CD4 + Vα + cells in a suppression assay using isolated CD11b + GR1 + cells. Isolated MDSCs from spleens from AC-treated mice were cultured with OVAp (1µg/mL), C57BL/6 splenocytes (1×10 5 ) and isolated OT-II naïve CD4 T cells (2×10 5 ) in the ratios (MDSC : T cell) indicated in the graph. G: Percentage of CD4 + Vα + CTV low cells in a suppression assay using isolated CD11b + GR1 + cells and OT-II CD4 T cells. H: Percentage of CD4 + Vα + FVD + cells in a suppression assay using isolated CD11b + GR1 + cells and OT-II CD4 T cells. I: Flow cytometry plots of CD11b + Ly6C + and CD11b + Ly6G + cells in the spleen 1 day, 7 days, and 14 days after saline or alum injections. J: Total count of CD11b + Ly6C + and CD11b + Ly6G + cells in the spleen 1 day, 7 days, and 14 days after saline or alum injections. K: TNF-α, and nitric oxide (L) production from CD11b + GR1 + cells isolated 1 day, 7 days, and 14 days after saline or alum injections cultured with either medium or LPS (200ng/mL) for 24h. Data shown represent three or more experiments and are expressed as mean±SEM; Student t-test was used for analysis; P values are indicated in each graph. All statistical analyses were performed using GraphPad Prism™.

Article Snippet: Single-cell suspensions were prepared from mouse splenocytes after RBC lysis using ACK lysis buffer (ThermoFisher Scientific).

Techniques: Adjuvant, Flow Cytometry, Saline, Isolation, Cell Culture, Suppression Assay

Journal: bioRxiv

Article Title: Adjuvant conditioning shapes the adaptive immune response and promotes trained immunotolerance via NLRP3/IL-1

doi: 10.1101/2024.09.06.611736

Figure Lengend Snippet: AC-induced MDSCs inhibition of the adaptive immune response in vivo requires NLRP3 activation and IL-1 signaling. A: Experimental design. Briefly, 2×10 6 CD11b + GR1 + cells isolated from either AC, alum + Anakinra (30mg/kg) (AC+AK) or saline-injected C57BL/6 or NLRP3 -/- mice were adoptively transferred to C57BL/6 mice before the immunization protocol. B: Flow cytometry plots and total number of OVA-specific T cells (C) , evidenced by the expression of CD4 + Vα + in splenocytes from mice adoptively transferred with CD11b + GR1 + cells isolated from either AC, or saline-injected C57BL/6 before the immunization protocol. D: OVA-specific IgG titer in serum from mice adoptively transferred with CD11b + GR1 + cells isolated from either AC, or saline-injected C57BL/6 before the immunization protocol. E: Flow cytometry plots and total number of OVA-specific T cells (F) , evidenced by the expression of CD4 + Vα + in splenocytes from mice adoptively transferred with CD11b + GR1 + cells isolated from either alum + Anakinra (30mg/kg) (AC+AK) or saline-injected C57BL/6 or NLRP3 -/- mice before the immunization protocol. G: OVA-specific IgG titer in serum from mice adoptively transferred with CD11b + GR1 + cells isolated from either alum + Anakinra (30mg/kg) (AC + AK) or saline-injected C57BL/6 or NLRP3 -/- mice before the immunization protocol. Data shown represent three or more experiments and are expressed as mean±SEM; Student t-test was used for analysis; P values are indicated in the graphs. All statistical analyses were performed using GraphPad Prism™.

Article Snippet: Single-cell suspensions were prepared from mouse splenocytes after RBC lysis using ACK lysis buffer (ThermoFisher Scientific).

Techniques: Inhibition, In Vivo, Activation Assay, Isolation, Saline, Injection, Flow Cytometry, Expressing

Journal: bioRxiv

Article Title: Adjuvant conditioning shapes the adaptive immune response and promotes trained immunotolerance via NLRP3/IL-1

doi: 10.1101/2024.09.06.611736

Figure Lengend Snippet: Adjuvant conditioning promotes allogeneic tolerance dependent on NLRP3. A: Allogeneic pancreatic islet transplantation experimental design. Briefly, alum or saline-treated C57BL/6 or NLRP3 -/- mice were treated with streptozotocin (STZ) 4 days before the transplantation with Balb/c isolated pancreatic islets. Survival of the graft was assessed daily by checking blood glucose levels. B: Survival curve of alum or saline-treated C57BL/6 or NLRP3 -/- mice that underwent allogeneic pancreatic islet transplantation. C57BL/6 saline (n=5), AC (n=6), NLRP3 -/- saline (n=5), AC (n=6). Graft survival significance was assessed by Kaplan-Meier/Mantel-Cox log-rank test. C: Cutaneous hypersensitivity experimental design. Briefly, alum or saline-treated C57BL/6 or NLRP3 -/- mice were subcutaneously immunized with 20× 10 6 Balb/c splenocytes on the neck. Seven days later, mice were challenged with 20×10 6 Balb/c splenocytes injected subcutaneously into the base of the ear, as described by . D: Cutaneous allogeneic response was assessed by measuring ear pinna swelling (mm) at 2-, 4- and 24- hours post-challenge. P-values show the comparison between ear swelling at 24-hours post-challenge. n=10 for each group. Data shown represent three or more experiments and are expressed as mean±SEM; Student t-test was used for analysis; P values are indicated in each graph. All statistical analyses were performed using GraphPad Prism™.

Article Snippet: Single-cell suspensions were prepared from mouse splenocytes after RBC lysis using ACK lysis buffer (ThermoFisher Scientific).

Techniques: Adjuvant, Transplantation Assay, Saline, Isolation, Injection, Comparison