Figure S5.
IFNAR inhibition alongside mRNA-LNP vaccination promotes exclusive generation of TSCMcells, driving increased proliferation of specific T cells upon viral rechallenge. Related to Fig. 9. (A–D) Draining lymph node P14 cells from mice d8 following GP33-encoding mRNA-LNP vaccination in combination with IFNAR and/or IFNγ blockade at d0–1. Data are representative of two independent experiments with four to five mice per group in each experiment. Each dot in C represents a single mouse. Data are the mean ± SEM. Statistical differences were analyzed using one-way ANOVA tests. (A) Experimental scheme. Naïve P14 cells were adoptively transferred into wild-type host mice 24 h prior to mRNA-LNP vaccines, which encode the GP33 epitope. Cohorts did not receive further treatment or received treatments of either IFNAR blocking, IFNγ blocking, or a combination at d0–1 following vaccination. (B) Representative flow cytometry plots showing TSCM (TCF-1+SLAMF6+) cell populations of P14 cells within each condition. (C) Graph summarizing frequencies in B. Each dot represents a single mouse sample. (D) Representative histograms of TEFF (KLRG1+SLAMF6−), TSCM (KLRG1−SLAMF6+), and TEX (KLRG1−SLAMF6−) P14 cell populations from control, IFNAR-blocked, IFNγ-blocked, or combined IFNAR- and IFNγ-blocked mice for expression of CD127, CD62L, PD-1, and TIM-3. Average gMFI ± SEM for each graph are indicated. (E and F) Graphs summarizing (E) CD61+CD55− and (F) CD61−CD55+ P14 cell frequencies. Analysis of P14 cells from days 4 to 12 following mRNA-LNP vaccination. Data are the mean ± SEM. Each dot represents a single mouse. 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. (G) Graphs summarizing the total P14 cell and TSCM (TCF-1+SLAMF6+) cell numbers within control and d0–1 IFNAR-blocked mice 28 days following mRNA-LNP vaccination. (H–O) Comparison of P14 cell 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 as described in Fig. 9 I. (H) Representative plots of P14 cells showing TEFF (KLRG1+SLAMF6−) and TSCM (KLRG1−SLAMF6+) cell populations. (I) Graphs summarizing population frequencies in H. (J) Cell counts of TEFF (KLRG1+SLAMF6−) and TSCM (KLRG1−SLAMF6+) cell populations in each experimental condition. (K) Representative flow cytometry plots of TSCM (TCF-1+SLAMF6+) P14 cell populations. (L) Graph summarizing frequencies in K. (M) TSCM (TCF-1+SLAMF6+) cell counts. (N) Representative flow cytometry plots of TIM-3 expression on P14 cells within each condition. (O) TIM-3+ P14 cell counts. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

IFNAR inhibition alongside mRNA-LNP vaccination promotes exclusive generation of T SCM cells, driving increased proliferation of specific T cells upon viral rechallenge. Related to Fig. 9. (A–D) Draining lymph node P14 cells from mice d8 following GP33-encoding mRNA-LNP vaccination in combination with IFNAR and/or IFNγ blockade at d0–1. Data are representative of two independent experiments with four to five mice per group in each experiment. Each dot in C represents a single mouse. Data are the mean ± SEM. Statistical differences were analyzed using one-way ANOVA tests. (A) Experimental scheme. Naïve P14 cells were adoptively transferred into wild-type host mice 24 h prior to mRNA-LNP vaccines, which encode the GP33 epitope. Cohorts did not receive further treatment or received treatments of either IFNAR blocking, IFNγ blocking, or a combination at d0–1 following vaccination. (B) Representative flow cytometry plots showing TSCM (TCF-1+SLAMF6+) cell populations of P14 cells within each condition. (C) Graph summarizing frequencies in B. Each dot represents a single mouse sample. (D) Representative histograms of TEFF (KLRG1+SLAMF6−), TSCM (KLRG1−SLAMF6+), and TEX (KLRG1−SLAMF6−) P14 cell populations from control, IFNAR-blocked, IFNγ-blocked, or combined IFNAR- and IFNγ-blocked mice for expression of CD127, CD62L, PD-1, and TIM-3. Average gMFI ± SEM for each graph are indicated. (E and F) Graphs summarizing (E) CD61+CD55− and (F) CD61−CD55+ P14 cell frequencies. Analysis of P14 cells from days 4 to 12 following mRNA-LNP vaccination. Data are the mean ± SEM. Each dot represents a single mouse. 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. (G) Graphs summarizing the total P14 cell and TSCM (TCF-1+SLAMF6+) cell numbers within control and d0–1 IFNAR-blocked mice 28 days following mRNA-LNP vaccination. (H–O) Comparison of P14 cell 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 as described in Fig. 9 I. (H) Representative plots of P14 cells showing TEFF (KLRG1+SLAMF6−) and TSCM (KLRG1−SLAMF6+) cell populations. (I) Graphs summarizing population frequencies in H. (J) Cell counts of TEFF (KLRG1+SLAMF6−) and TSCM (KLRG1−SLAMF6+) cell populations in each experimental condition. (K) Representative flow cytometry plots of TSCM (TCF-1+SLAMF6+) P14 cell populations. (L) Graph summarizing frequencies in K. (M) TSCM (TCF-1+SLAMF6+) cell counts. (N) Representative flow cytometry plots of TIM-3 expression on P14 cells within each condition. (O) TIM-3+ P14 cell counts. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

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