Journal: The Journal of Clinical Investigation

Article Title: Antigen-loaded monocyte administration induces potent therapeutic antitumor T cell responses

doi: 10.1172/JCI128267

Figure Lengend Snippet: (A) Frequency of OVA-specific (tetramer+) CD8+ T cells among total CD8+ T cells in the spleen on day 7 after IV injection of the indicated doses of OVA-monocytes. (B) Representative dot plots gated on live CD8+ T cells showing frequency of OVA-specific CD8+ T cells among total CD8+ T cells in the spleen on day 7 after SQ 200 μg OVA/CFA, IV OVA-monocytes (3 × 106; OVA-mono), or IV monocytes alone (Mono). (C) Frequency of OVA-specific CD8+ T cells derived from B. (D) Expression of cytotoxicity-related markers on splenic OVA-specific CD8+ T cells presented as MFI and percentages of marker-positive cells among OVA-specific CD8+ T cells on day 7 after SQ OVA/CFA, or IV OVA-mono. Eomes, eomesodermin; GZB, granzyme B. (E) In vivo CTL activity of splenocytes toward SIINFEKL-pulsed targeted cells (specific lysis) 7 days after SQ PBS/CFA, OVA/CFA and IV OVA-mono. (F) Representative dot plots showing frequency of TRP2-specific (tetramer+) CD8+ T cells among total blood CD8+ T cells 2 days after the last dose of 5 IV injections of TRP2180-188–monocytes (TRP2-mono) or unloaded monocytes (Mono) (106/injection, every other day). (G) Frequency of TRP2-specific CD8+ T cells derived from F. (H) Frequency of OVA-specific CD8+ T cells among total CD8+ T cells in the spleen on day 7 after the indicated OVA-loaded cellular vaccination. (I) Representative dot plots showing frequency of OVA-specific CD8+ T cells among total CD8+ T cells in the spleen and LN on day 7 after SQ or IV OVA-monocyte vaccination. (J) Frequency of OVA-specific CD8+ T cells derived from I. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. One-way ANOVA with Tukey’s test (A, C, E, H); 2-way ANOVA with Bonferroni’s test (D and J); and unpaired 2-tailed Student’s t test (G). Data represent mean ± SEM.

Article Snippet: For peptide loading, purified monocytes (10 6 /mL) were incubated with TRP2 180-188 , SIINFEKL (OVA 257-264 ) (both at 250 μg/mL; AnaSpec), or 5-carboxyfluorescein–labeled (FAM-labeled) SIINFEKL (10–500 μg/mL; GenScript) in cRPMI-20 medium for 2 hours with 5% CO 2 at 37°C.

Techniques: IV Injection, Derivative Assay, Expressing, Marker, In Vivo, Activity Assay, Lysis, Injection

Journal: The Journal of Clinical Investigation

Article Title: Antigen-loaded monocyte administration induces potent therapeutic antitumor T cell responses

doi: 10.1172/JCI128267

Figure Lengend Snippet: (A and B) Growth of SQ B16/F10-OVA melanoma tumors (2 × 105) in mice untreated (no treatment) or vaccine treated beginning 8 days after tumor inoculation. (A) Vaccines: 106 OVA-monocytes IV weekly × 3 (OVA-mono 3×) or 106 OVA RNA–DCs SQ weekly × 3 (OVA-DC 3×) with tetanus-diphtheria toxoid immunization (Td) and adoptive OT-I cell transfer (OT-I). (B) Vaccines: 3 × 106 OVA-monocytes IV × 1 (OVA-mono 1x), 106 OVA RNA-pulsed DCs SQ × 3 (OVA-DC 3×) + Td, or 106 OVA RNA-pulsed DCs SQ × 3 (OVA-DC 3×) + Td + OT-I. (C) Growth of SQ B16/F10 melanoma tumors (5 × 104) in mice untreated (no treatment) or vaccinated every other day beginning 2 days after tumor inoculation for 5 doses of SQ 106 TRP2180-188–loaded (250 μg/mL) DCs (TRP2-DC SQ), IV 106 TRP2180-188–loaded (250 μg/mL) DCs (TRP2-DC IV), IV 106 SIINFEKL-loaded monocytes (SIINFEKL-mono IV), or IV 106 TRP2180-188–loaded monocytes (TRP2-mono IV). Tumor size comparison: *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 (unpaired 2-tailed Student’s t test). (D) Frequency of TRP2-specific (TRP2180-188-H-2Kb tetramer+) CD8+ T cells among total blood CD8+ T cells on day 16 after tumor inoculation in the experiment of C. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 (1-way ANOVA with Tukey’s test). (E) Survival of the mice IC inoculated with TRP2-expressing CT-2A astrocytoma cells (5 × 104) either untreated (no treatment) or treated with 5 doses of TRP2180-188–loaded DCs (TRP2-DC SQ or IV) or monocyte (TRP2-mono IV) vaccination every other day beginning on day 2 after tumor inoculation. Median, median survival days. Survival curve comparison: *P < 0.05, and **P < 0.01 (log-rank test). For A–E, n = 8 per group. Data represent mean ± SEM.

Article Snippet: For peptide loading, purified monocytes (10 6 /mL) were incubated with TRP2 180-188 , SIINFEKL (OVA 257-264 ) (both at 250 μg/mL; AnaSpec), or 5-carboxyfluorescein–labeled (FAM-labeled) SIINFEKL (10–500 μg/mL; GenScript) in cRPMI-20 medium for 2 hours with 5% CO 2 at 37°C.

Techniques: Vaccines, Comparison, Expressing