Figure 9.
IFNAR blocking in combination with mRNA-LNP vaccination promotes TSCMdifferentiation conferring enhanced protective capacity. (A and B) P14 cells from mice that received intramuscular vaccinations of GP33-encoding mRNA-LNP and d0–1 treatment of IFNAR and/or IFNγ blocking. Draining lymph node P14 cells were analyzed at d8 following vaccination. Data are representative of two independent experiments with four to five mice per group in each experiment. Each dot in B represents a single mouse. Data are the mean ± SEM. Statistical differences were analyzed using one-way ANOVA tests. (A) Representative plots of P14 cells showing TEFF (KLRG1+SLAMF6−), TSCM (KLRG1−SLAMF6+), and TEX (KLRG1−SLAMF6−TIM-3+) populations. (B) Graphs summarizing frequencies in A. (C) Graph summarizing the persistence of antigen (NLuc) within draining lymph nodes from NLuc-encoding mRNA-LNP–vaccinated mice with or without d0–1 IFNAR blocking treatments over time. Data are the mean ± SEM. The dashed line indicates NLuc LOD from blank tissue. (D–H) Analysis of antigen-specific P14 cells from days 4 to 12 following mRNA-LNP vaccination. Data are the mean ± SEM. Data are representative of two independent experiments with five to six mice per group. Statistical differences were analyzed using unpaired t tests at each time point after vaccination. (D and E) Graphs summarizing (D) CX3CR1+SLAMF6− (TEFF) cells and (E) CX3CR1−SLAMF6+ (TSCM) cells. (F) Representative flow cytometry plots of gating CD61+ TPEX cells and CD55+ TSCM cells. Cells are pre-gated on TCF-1+SLAMF6+CXCR6−CD62L+ cells as per Fig. S2 F. (G and H) Graphs summarizing (G) CD61+CD55− and (H) CD61−CD55+ cell frequencies. (I–M) Comparison of response to chronic LCMV infection rechallenge in mice that received d0–1 IFNAR blocking following mRNA-LNP vaccination with unvaccinated mice and mice that received only vaccination. Data are the mean ± SEM. Each dot in K–M represents a single mouse. Data are representative of three independent experiments with four to five mice per group. Statistical differences were analyzed using one-way ANOVA. (I) Schematic illustration of the experiment timeline. P14 cells were transferred into wild-type hosts prior to intramuscular vaccination of GP33-encoding mRNA-LNP. Half of the vaccinated mice immediately received IFNAR blocking followed by secondary treatment a day later. Mice were left for 30 days to establish memory. Another cohort of naïve mice received adoptive cell transfer of naïve P14 cells a day prior to all mice being infected with chronic LCMV. Mice were weighed daily following rechallenge, and lymph nodes were collected at d8. (J) Graph summarizing average proportion of weight change over the course of chronic LCMV infection within each group. (K) Graph of total P14 cell count in each indicated experimental group. (L) Expression of exhaustion marker TIM-3 on the surface of P14 cells. (M) Graph summarizing the PFU from viable virus in spleens of mice in each experimental group. The dashed line indicates viral plaque LOD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Fig. S5 show additional supporting data. LOD, limit of detection.

IFNAR blocking in combination with mRNA-LNP vaccination promotes T SCM differentiation conferring enhanced protective capacity. (A and B) P14 cells from mice that received intramuscular vaccinations of GP33-encoding mRNA-LNP and d0–1 treatment of IFNAR and/or IFNγ blocking. Draining lymph node P14 cells were analyzed at d8 following vaccination. Data are representative of two independent experiments with four to five mice per group in each experiment. Each dot in B represents a single mouse. Data are the mean ± SEM. Statistical differences were analyzed using one-way ANOVA tests. (A) Representative plots of P14 cells showing TEFF (KLRG1+SLAMF6−), TSCM (KLRG1−SLAMF6+), and TEX (KLRG1−SLAMF6−TIM-3+) populations. (B) Graphs summarizing frequencies in A. (C) Graph summarizing the persistence of antigen (NLuc) within draining lymph nodes from NLuc-encoding mRNA-LNP–vaccinated mice with or without d0–1 IFNAR blocking treatments over time. Data are the mean ± SEM. The dashed line indicates NLuc LOD from blank tissue. (D–H) Analysis of antigen-specific P14 cells from days 4 to 12 following mRNA-LNP vaccination. Data are the mean ± SEM. Data are representative of two independent experiments with five to six mice per group. Statistical differences were analyzed using unpaired t tests at each time point after vaccination. (D and E) Graphs summarizing (D) CX3CR1+SLAMF6− (TEFF) cells and (E) CX3CR1−SLAMF6+ (TSCM) cells. (F) Representative flow cytometry plots of gating CD61+ TPEX cells and CD55+ TSCM cells. Cells are pre-gated on TCF-1+SLAMF6+CXCR6−CD62L+ cells as per Fig. S2 F. (G and H) Graphs summarizing (G) CD61+CD55− and (H) CD61−CD55+ cell frequencies. (I–M) Comparison of response to chronic LCMV infection rechallenge in mice that received d0–1 IFNAR blocking following mRNA-LNP vaccination with unvaccinated mice and mice that received only vaccination. Data are the mean ± SEM. Each dot in K–M represents a single mouse. Data are representative of three independent experiments with four to five mice per group. Statistical differences were analyzed using one-way ANOVA. (I) Schematic illustration of the experiment timeline. P14 cells were transferred into wild-type hosts prior to intramuscular vaccination of GP33-encoding mRNA-LNP. Half of the vaccinated mice immediately received IFNAR blocking followed by secondary treatment a day later. Mice were left for 30 days to establish memory. Another cohort of naïve mice received adoptive cell transfer of naïve P14 cells a day prior to all mice being infected with chronic LCMV. Mice were weighed daily following rechallenge, and lymph nodes were collected at d8. (J) Graph summarizing average proportion of weight change over the course of chronic LCMV infection within each group. (K) Graph of total P14 cell count in each indicated experimental group. (L) Expression of exhaustion marker TIM-3 on the surface of P14 cells. (M) Graph summarizing the PFU from viable virus in spleens of mice in each experimental group. The dashed line indicates viral plaque LOD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Fig. S5 show additional supporting data. LOD, limit of detection.

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