responder t cell population  (Jackson Laboratory)

Journal: Nano letters

Article Title: Personalized Tumor RNA Loaded Lipid-Nanoparticles Prime the Systemic and Intratumoral Milieu for Response to Cancer Immunotherapy

doi: 10.1021/acs.nanolett.8b02179

Figure Lengend Snippet: PD-L1+ APCs do not suppress, but mediate enhancement of antigen-specific T cell immunity. (A, B) OVA RNA-NPs were injected iv into C57Bl/6 mice, and spleens were harvested the next day; spleens were FACS sorted for CD11c+MHCII+CD86+PD-L1+ cells (PD-L1+ APCs) (40 000 cells) which were added to a standard coculture assay (3 replicates/group) of OVA-mRNA electroporated primary murine bone marrow derived DCs (40 000 cells) and/or OTI derived splenocytes (400 000 cells) followed by serum assessment of IFN-γ by ELISA (**p < 0.01, unpaired t test). (C) OVA RNA-NPs were injected iv into C57Bl/6 mice, and spleens were harvested the next day; spleens were FACS sorted for CD11c+MHCII+CD86+PD-L1+ (PD-L1+ APCs) cells which were adoptively transferred to naïve C57Bl/6 mice (n = 5/group) “spiked” with a responder T cell population from OTI transgenic mice (1 × 107 OTI splenocytes per mouse) (*p < 0.05, Mann–Whitney test). (D, E) OVA RNA-NPs were injected iv into tumor-bearing C57Bl/6 mice. Tumors were harvested within 3 weeks, FACS sorted for CD11c+MHCII+CD86+PD-L1+ cells (PD-L1+ intratumoral APCs) or CD 11 c+MHCII+CD86+PDL1- cells (PD-L1- intratumoral APCs) (40 000 cells) which were added to a standard coculture assay (n = 3 replicates/group) of OVA-mRNA electroporated primary murine bone marrow derived DCs (40 000 cells) and OVA mRNA activated T cells (400 000 cells). T cell activation was assessed by ELISA for IFN-γ production after 48 h (*p < 0.05, **p <0.01, unpaired t test). (F, G) PD-L1 expression by flow cytometry from splenocytes of C57Bl/6 mice (n = 3–5/group) vaccinated with iv GFP RNA-NPs, CFA-OVA peptide (administered intradermal), NPs alone, or iv GFP RNA-NPs with anti-PD-L1 mAbs or isotype mAbs (administered intraperitoneal (ip) 24 h prior to RNA-NPs) (*p < 0.05, **p < 0.01, Mann–Whitney test). (H) C57Bl/6 mice (n = 4–5/group) were vaccinated with pp65 RNA-NPs iv once weekly (×3) before spleens were harvested a week later for analysis of IFN-γ by ELISA (from soups of restimulated splenocytes with overlapping pp65 peptide pool) and by flow cytometric analysis ofpercent PD-1+ CD8+cells (**p < 0.01, Mann–Whitney test). (I) B16F10-OVA(1 000 000 cells) was implanted subcutaneously in the flanks of C57Bl/6 mice (n = 5–9/ group), and PD-1 mAbs, PD-L1 mAbs, OVA RNA-NPs, or OVA RNA-NP+ PD-L1 mAbs were administered the following day. OVA RNA-NPs were injected iv once weekly (×3), and PD-1/PD-L1 mAbs were injected ip twice weekly (×3 weeks). Survival is plotted on a Kaplan–Meier curve (*p < 0.05, **p < 0.01, Gehan–Breslow–Wilcoxon test).

Article Snippet: In some experiments, mice receiving OVA RNA-NPs were “spiked” with a responder T cell population from OTI mice (C57Bl/6-Tg(TcraTcrb)l100Mjb/J, Jackson Laboratory) containing transgenic T cell receptors recognizing OVA peptide epitopes 257–264.

Techniques: Injection, Co-culture Assay, Derivative Assay, Enzyme-linked Immunosorbent Assay, Transgenic Assay, MANN-WHITNEY, Activation Assay, Expressing, Flow Cytometry

Journal: Nano letters

Article Title: Personalized Tumor RNA Loaded Lipid-Nanoparticles Prime the Systemic and Intratumoral Milieu for Response to Cancer Immunotherapy

doi: 10.1021/acs.nanolett.8b02179

Figure Lengend Snippet: Type I IFN drives synergistic activity from RNA-NP and ICIs. (A, B) NPs alone, GFP RNA-NPs, or GFP RNA-NPs with IFNAR1 blocking antibodies (500 μg administered ip) were administered systemically into naïve C57Bl/6 mice (n = 4–5/group), and spleens were harvested the next day for flow cytometric analysis of CD86, PD-L1, and CD11c expression (**p < 0.01, Mann–Whitney test). (C) RNA-NPs were administered iv to naïve C57Bl/6 mice (n = 4–5/group), and spleens were harvested the next day for assessment of F4/80 expression. (D, E) NPs alone, luciferase RNA-NPs, or luciferase RNA-NPs with PDCA-1 blocking antibodies were administered systemically into naïve C57Bl/6 mice (n = 4/group). Within 24 h, serum was collected for IFN-α detection by ELISA (overflow values assigned concentration of 2,000 pg/mL), and spleens were harvested for flow cytometric analysis of PDCA-1, MHCII, CD86, and PD-L1 expression (*p < 0.05, Mann–Whitney test). (F) B16F10-OVA melanomas (1 000 000 cells) were implanted in the flanks of C57Bl/6 mice (n = 7–8), and the following day, all mice received 1 × 107 splenocytes iv obtained from OTI mice. Animals were left unvaccinated or received treatment with PD-L1 mAbs, OVA RNA-NPs, or OVA RNA-NP+PD-L1 mAbs with or without concomitant IFNAR1 mAbs. PD-L1 and IFNAR1 mAbs were administered ip twice weekly, and RNA-NPs were administered iv on day 1. Early time-point measurements of tumor volumes were plotted (day 22 tumor volumes: RNA-NP+PD-L1 mAb versus RNA-NP+ PD-L1 mAb + IFNAR1 mAb, **p< 0.01, Mann–Whitney test). (G) Spleens were harvested from all groups on day 25 for assessment of PD-1 expression on CD8+ splenocytes (**p < 0.01, Mann–Whitney test).

Article Snippet: In some experiments, mice receiving OVA RNA-NPs were “spiked” with a responder T cell population from OTI mice (C57Bl/6-Tg(TcraTcrb)l100Mjb/J, Jackson Laboratory) containing transgenic T cell receptors recognizing OVA peptide epitopes 257–264.

Techniques: Activity Assay, Blocking Assay, Expressing, MANN-WHITNEY, Luciferase, Enzyme-linked Immunosorbent Assay, Concentration Assay