SARS-CoV-2 mRNA vaccines sensitize tumours to immune checkpoint blockade

Abstract

Immune checkpoint inhibitors (ICIs) extend survival in many patients with cancer but are ineffective in patients without pre-existing immunity^1-9. Although personalized mRNA cancer vaccines sensitize tumours to ICIs by directing immune attacks against preselected antigens, personalized vaccines are limited by complex and time-intensive manufacturing processes^10-14. Here we show that mRNA vaccines targeting SARS-CoV-2 also sensitize tumours to ICIs. In preclinical models, SARS-CoV-2 mRNA vaccines led to a substantial increase in type I interferon, enabling innate immune cells to prime CD8⁺ T cells that target tumour-associated antigens. Concomitant ICI treatment is required for maximal efficacy in immunologically cold tumours, which respond by increasing PD-L1 expression. Similar correlates of vaccination response are found in humans, including increases in type I interferon, myeloid-lymphoid activation in healthy volunteers and PD-L1 expression on tumours. Moreover, receipt of SARS-CoV-2 mRNA vaccines within 100 days of initiating ICI is associated with significantly improved median and three-year overall survival in multiple large retrospective cohorts. This benefit is similar among patients with immunologically cold tumours. Together, these results demonstrate that clinically available mRNA vaccines targeting non-tumour-related antigens are potent immune modulators capable of sensitizing tumours to ICIs.

Fig. 1. COVID-19 mRNA vaccines are associated with improved survival in patients with NSCLC or metastatic melanoma receiving immunotherapy.

a–e, Survival for patients with NSCLC (a–c) or metastatic melanoma (d,e) treated with ICI who received a COVID-19 mRNA vaccine within 100 days of initiating ICI or did not receive a COVID-19 mRNA vaccine. Survival is shown for all patients with NSCLC (a), patients with unresectable stage III NSCLC (b), patients with stage IV NSCLC (c) and patients with metastatic melanoma (d and e). P values and HR_adj were calculated using two-sided Cox proportional hazards regression (Supplementary Tables 3, 5, 7, 9 and 11), including all variables that were significantly associated with survival on univariable analysis (Supplementary Tables 2, 4, 6, 8 and 10). The number of patients at risk at each timepoint is indicated below each graph. Source Data

Fig. 2. Spike RNA-LNPs prime anti-cancer immunity in an IFN-I dependent manner.

a, The experimental design and tumour volume of mice inoculated with B16F0 cells. Groups included untreated (UT; n = 7), anti-PD-1 (n = 8), RNA-LNPs (n = 8) and RNA-LNPs + anti-PD-1 (n = 8). mAb, monoclonal antibody. b, The experimental design and tumour volume for mice inoculated with LLC cells. Groups included untreated (n = 8), anti-PD-1 (n = 9), RNA-LNP (n = 9) and RNA-LNPs + anti-PD-1 (n = 9). c, The experimental design and tumour volume measurements for mice inoculated with LLC cells. Groups included untreated (n = 9), anti-PD-1 (n = 10), RNA-LNP (n = 7) and RNA-LNPs + PD-1 (n = 8). d, The experimental design and tumour volume for mice inoculated with B16F0 cells. n = 12 per group. e, IFNα plasma enzyme linked immunosorbent assay (ELISA) from B16F0-tumour-bearing mice (n = 8 per group) 24 h after one RNA-LNP vaccine (day 3). f–i, Cellular phenotyping within 24 h of vaccine 3 (days 3, 6 and 20) of cells from spleens of mice bearing B16F0 tumours (n = 5 per group), including the percentage of activated (CD80⁺CD86⁺) DCs (f) and macrophages (g). h,i, PD-L1 median fluorescence intensity (MFI) on activated mature DCs (h) and macrophages (i). j–o, Characterization of antigen presentation among myeloid cells in tumour draining lymph nodes (tdLNs) and spleens of mice bearing B16F10-ova tumours 24 h after vaccine 2 (days 10 and 13). The overall percentage of CD45⁺ cells that express MHC-II⁺ (j), the percentage of MHC-II⁺ cells presenting SIINFEKL (k), the percentage of SIINFEKL-presenting MHCII⁺ cells that express the activation marker CD86 (l), the percentage of CD45⁺ cells that are MHC-II⁺Ly6C⁺ (m), SIINFEKL⁺MHC-II⁺Ly6C⁺ cells as a percentage of all CD45⁺ cells (n) and Ly6C⁺MHCII⁺ cells as a percentage of total SIINFEKL-presenting cells (o) are shown. n = 4 biologically independent mice per group. Significance was determined using two-way analysis of variance (ANOVA)/mixed-effect analysis with Geisser–Greenhouse correction (a–d) and two-tailed unpaired t-tests (e–o). n indicates the number of biologically independent samples. For the box plots, the whiskers extend to the highest and lowest values, the box limits show the first and third quartiles and the centre line shows the median value. For a–d, data are mean ± s.e.m. Source Data

Fig. 3. Spike RNA-LNPs generate tumour-reactive T cells and increase PD-L1 expression on tumour cells.

a,b, The percentage of activated effector (a) and effector memory (b) T cells in the spleens of tumour-bearing mice on day 21 (vaccine days 3, 6, 17) (n = 5 per group). c, The percentage of tetramer⁺ cells of splenic CD8⁺ T cells collected from mice bearing B16F0 tumours on day 21 (vaccination days 14 and 17). Groups include untreated (n = 6), anti-PD-1 (n = 6), RNA-LNPs (n = 5) and RNA-LNPs + anti-PD-1 (n = 7). d, The normalized percentage of AIM⁺ T cells (n = 5 mice per peptide; exceptions are shown in the Supplementary Information) after splenocyte co-culture with overlapping peptide pools. e,f, Representative images (e) and blinded manual counting (n = 4 tumours per group with 4 counts per tumour) (f) of PD-1⁺CD3⁺ cells by immunofluorescence 24 h after vaccine 3 (days 3, 6 and 20) from s.c. tumors of B16F0-tumour-bearing mice treated with or without anti-PD-L1. For e, scale bars, 100 μm. AF647, Alexa Fluor 647. g, The percentage PD-1⁺CD8⁺ cells of CD3⁺ T cells in tumours of B16F0-bearing mice vaccinated with RNA-LNPs (days 14 and 17). Groups included untreated (n = 7), anti-PD-L1 (n = 8), RNA-LNPs (n = 9) and RNA-LNPs + anti-PD-L1 (n = 7). h, Pooled tetramer positivity (%) among CD8⁺ T cells in B16F0 tumours. Groups included untreated (n = 6), anti-PD-1 (n = 6), RNA-LNPs (n = 4) and RNA-LNPs + anti-PD-1 (n = 7) (RNA-LNPs days 14 and 17). i, PD-L1 expression on B16F0 tumour cells (CD45⁻FSC-A^high) isolated from mice 24 h after vaccine 3 (days 3, 6 and 17) as determined using flow cytometry. Groups included untreated (n = 4) and RNA-LNPs (n = 5). j,k, Blinded manual counting (n = 6 tumours per group with 4 counts per tumour) (j) and representative images (k) of PD-L1⁺ tumour cells (SOX10) by immunofluorescence 24 h after vaccine 3 (days 3, 6, 21) from B16F0-tumour-bearing mice. For k, scale bars, 50 μm. For j, the circle symbols indicate PBS treatment and the square symbols represent anti-PD-L1 treatment. For f and j, the colours represent individual tumours. Significance was determined using two-tailed unpaired t-tests (a–c and g–j), two-tailed Welch’s t-test (f), and two-tailed Brown–Forsythe and Welch ANOVA, followed by Dunnett’s T3 multiple-comparison test (d). For the box plots, the whiskers extend to the highest and lowest values, the box limits show the first and third quartiles and the centre line shows the median value. n values indicate biologically independent samples unless indicated otherwise. Source Data

Fig. 4. COVID-19 mRNA vaccines generate a surge in IFNα, innate immune activation and adaptive immunity in humans.

a, Schematic of the experimental design in which blood was drawn from five healthy individuals at baseline and 6 h, 24 h, 7 days and 14 days after Spikevax (mRNA-1273) COVID-19 mRNA immunization. b,c, Individual datapoints highlighting changes in expression of IFNα from baseline to 24 h for each of five healthy volunteers. Data are expressed as the fold change measured using the NULISAseq Inflammation Panel (b). The concentration was also measured separately with NULISAseq absolute quantification (AQ) (c). d, Dynamic expression of the cytokines that are significantly elevated at 24 h at 6 h, 24 h, 7 days and 14 days after COVID-19 mRNA vaccination. Significant variables were defined as those with P < 0.05 and a log₂-transformed fold change with an absolute value of greater than 0.5 after linear modelling with fixed effects. Adjusted P values were calculated using moderated two-tailed t-tests with false-discovery rate (FDR) correction for multiple testing. e,f, PD-L1 expression on circulating myeloid cells (CD3⁻CD19⁻CD56⁻CD11b⁺) (n = 5) (e) and DCs (CD3⁻CD19⁻CD56⁻CD11c⁺MHC-II⁺) (n = 5) (f) at 6 h, 24 h and 7 days after immunization. g,h, Activation of natural killer cells (CD56⁺; n = 5) (g), and T cells expressed as numbers of CD69⁺ cells of CD8⁺CD3⁺ cells (n = 5) (h) at 6 h, 24 h, 7 days and 14 days after immunization. Data are mean ± s.e.m. P values were calculated using two-tailed paired t-tests. Source Data

Fig. 5. COVID-19 mRNA vaccines are associated with increased PD-L1 expression on tumours and improved clinical outcomes across a broad set of tumour histologies.

a, Schematic of patients with NSCLC biopsies documenting PD-L1 TPS. b, TPS stratified by COVID-19 mRNA vaccination timing. c, The distribution of samples with TPS ≥ 50%. d, TPS stratified by influenza (left) or pneumonia (right) vaccination timing. e, Schematic of biopsies documenting TPS or combined positive score (CPS) of PD-L1 at our institution (January 2020 to October 2023). f, Primary tumour locations from this diverse cohort. g, TPS in the tissue-agnostic cohort stratified by COVID-19 mRNA immunization timing. h, TPS stratified by timing of influenza vaccination. P values were calculated using two-tailed unpaired t-tests (b, d and h), two-tailed unpaired t-tests with Welch’s correction for unequal variance (g) and two-sided Fisher’s exact test evaluating the likelihood of TPS greater than 50% (c). The violin plots show the distribution of data with individual datapoints included. i, Survival of patients in the tissue-agnostic cohort treated with ICI who received any COVID-19 vaccine within 100 days of initiating ICI or did not receive any COVID-19 vaccine. j, Survival of patients in i stratified by receipt of COVID-19 vaccine before ICI. k, Survival for patients in i who started ICI in the pandemic era (since 2 September 2020, 100 days before mRNA vaccine approval). Survival analyses in the tissue-agnostic cohort were not limited to only those patients with a clear TPS value. l–o, The OS for patients with metastatic stage IV NSCLC treated with ICI who received a COVID-19 mRNA vaccine within 100 days of initiating ICI or did not receive a COVID-19 vaccine who had baseline PD-L1 expression at baseline biopsy TPS < 1% (l), 1–49.9% (m) or ≥50% (n). To evaluate the impact of vaccination in each clinical setting, patients were excluded if they received a COVID-19 mRNA vaccine before their biopsy. o, OS of unvaccinated patients with stage IV NSCLC stratified by era of ICI start who had baseline TPS < 1% at biopsy. P values and HRs were calculated using log-rank (Mantel–Cox, two-sided) tests (i–o). Source Data

Extended Data Fig. 1. COVID mRNA Vaccines are associated with improved survival in NSCLC patients who receive ICI.

a-c, Overall survival for NSCLC patients who received immune therapy and obtained a COVID mRNA vaccine differentiated by vaccine manufacturer (a), whether the patient received their first vaccine during this period (“Prime only”), a booster (“Boost only”), or both a priming vaccine and a booster vaccine within the 100-day period (“Prime and Boost”) (b), and number of vaccines received within 100 days of ICI initiation (c). One patient who received 3 vaccines within 100 days is not represented. d, Overall survival among patients with NSCLC receiving their first round of ICI, stratified by receipt of COVID mRNA vaccine in the 100 days prior to ICI initiation. e, Overall survival for NSCLC patients who received immune therapy and obtained a COVID mRNA vaccine within 50 days of initiating immunotherapy. f, Overall survival for NSCLC patients receiving ICI starting on or after 9/2/2020, stratified by receipt of COVID mRNA vaccination within 100 days surrounding ICI initiation. g, Overall survival for NSCLC patients stratified by receipt of COVID mRNA vaccines with all events occurring in the first 100 days after initiating ICI removed to correct for immortal time bias. h-i, Propensity score matching for overall survival in patients with Stage III Unresectable NSCLC (h) and metastatic NSCLC (i) treated with ICI who received a COVID mRNA vaccine within 100 days of initiating ICI or did not receive a COVID mRNA vaccine. Hazard ratios and p values were calculated by log-rank (Mantel-Cox, two-sided) tests. Source Data

Extended Data Fig. 2. COVID mRNA vaccines are uniquely associated with improved survival in patients with NSCLC treated with ICI.

a, Overall survival for NSCLC patients who did not receive immune checkpoint inhibition and received a COVID vaccine within 100 days of initiating chemotherapy or did not receive a COVID vaccine. b-e, Overall survival for NSCLC patients stratified by receipt of influenza vaccines (b-c), or pneumonia vaccines (d-e) with all events included (b, d) or, to correct for immortal time bias, including only events that occured greater than 100 days after initiating ICI (c, e). Patients who also received COVID vaccination were excluded from the influenza and pneumonia vaccine analyses. f-g, Overall survival for (f) all patients with Stage III NSCLC and (g) patients with Resectable Stage III NSCLC treated with ICI who received a COVID mRNA vaccine within 100 days of initiating ICI or did not receive a COVID mRNA vaccine. Hazard ratios and p values were calculated by log-rank (Mantel-Cox, two-sided) tests. Source Data

Extended Data Fig. 3. COVID mRNA Vaccines are uniquely associated with improved survival in melanoma patients who are receiving their first round of ICI.

a-c, Overall survival for Stage IV melanoma patients who received their first round of immune therapy and obtained a COVID mRNA vaccine differentiated by vaccine manufacturer (a), whether the patient received their first vaccine during this period (“Prime only”), a booster (“Boost only”), or both a priming vaccine and a booster vaccine within the 100-day period (“Prime and Boost”) (b), and the number of vaccines received within 100 days of ICI initiation (c). d, Overall survival among patients with Stage IV melanoma who are receiving their first round of ICI stratified by receipt of COVID mRNA vaccine in the 100 days prior to ICI initiation. Hazard ratios are reported using log-rank tests. e-f, Overall survival for Stage IV Melanoma receiving ICI starting on or after 9/2/2020, stratified by receipt of COVID mRNA vaccination within 100 days surrounding ICI initiation. For crossing survival curves as in e, RMST was calculated rather than logrank (Mantel-Cox) testing (see Methods). f, Restricted Mean Survival Time (RMST) at 12 and 24 months. Absolute differences between arms are compared with a two-tailed non-parametric area under the curve (AUC) analysis. g-i, Overall survival for patients in the Melanoma dataset treated with ICI. g, Survival for patients in the Melanoma cohort treated with ICI who received a COVID mRNA vaccine within 100 days of initiating any line of ICI or did not receive a COVID mRNA vaccine. h, Survival for Stage III patients in the Melanoma cohort treated with ICI who received a COVID mRNA vaccine within 100 days of initiating any line of ICI or did not receive a COVID mRNA vaccine. i, Survival for all Stage IV patients in the Melanoma cohort treated with ICI who received a COVID mRNA vaccine within 100 days of initiating any line of ICI or did not receive a COVID mRNA vaccine. j-k, Propensity score matching for overall survival (j) and progression-free survival (k) in patients with metastatic melanoma treated with ICI who received a COVID mRNA vaccine within 100 days of initiating ICI or did not receive a COVID mRNA vaccine. Matching was performed using all variables significantly associated with survival on multivariable analysis. Hazard ratios and p values were calculated by log-rank (Mantel-Cox, two-sided) tests unless otherwise specified. Numbers underneath the graph indicate the number of patients at each timepoint. Source Data

Extended Data Fig. 4. Synthesis and characterization of RNA-LNPs approximating BNT162b2.

a, Sequence map of mRNA. b, Quality of mRNA assessed on a BioAnalyzer. c, Total anti-Spike IgG generated by C57Bl/6 mice after 3 doses of vaccine (n = 3 biological replicates). Data are displayed as mean with SEM. d, Visualization of mRNA loading in LNPs via gel electrophoresis. e, Size distribution of LNPs assessed by DLS with 3 technical replicates. Data are displayed as mean with SD. f, Size distribution determined via nanoparticle tracking analysis. g, Table of LNP properties. h, pH and zeta potential at biologically relevant levels of sodium bicarbonate. Source Data

Extended Data Fig. 5. Spike RNA-LNPs prime anti-cancer immunity in preclinical models.

a, Graphical experimental design and individual tumour growth curves of C57Bl6 mice inoculated with B16F0 (50,000 cells) and vaccinated with RNA-LNP i.m. (Day 14,17) with and without anti-PD1. In this experiment, groups included untreated (n = 7), PD1 mAb alone (n = 8), RNA-LNP alone (n = 8), and RNA-LNP and anti-PD1 mAb (n = 8). b, Graphical experimental design and individual tumour growth curves for C57Bl/6 mice inoculated with LLC (200,000 cells) and vaccinated with spike RNA-LNP (Day 3,6) with and without anti-PD1 mAbs. In this experiment, groups included untreated (n = 8), anti-PD1 mAb alone (n = 9), RNA-LNP alone (n = 9), and RNA-LNP and anti-PD1 mAb (n = 9). c, Graphical experimental design of individual tumour growth curves (c), boxplots of tumours weights (d) and counts of metastatic tumours in lungs on Day 17 (e) in C57Bl/6 mice inoculated with LLC (200,000 cells) and vaccinated with spike RNA-LNP i.m. (Day 9, 12) with and without anti-PD1. In this experiment, groups included untreated (n = 9), anti-PD1 mAb alone (n = 10), RNA-LNP alone (n = 7), and RNA-LNP and anti-PD1 mAb (n = 7). f, Tumour growth for C57Bl6 mice inoculated with B16F0 (50,000 cells) and vaccinated with RNA-LNP i.m. (Day 3, 6) with and without anti-PD1 (Day 6, 10, 13, 17) (n = 9). g, Lung weight in orthotopic LLC tumours (100,000 cells) treated with RNA-LNPs (Days 3 and 6) with or without anti-PD1. In this experiment, groups included untreated (n = 9), anti-PD1 mAb alone (n = 10), RNA-LNP alone (n = 9), and RNA-LNP and anti-PD1 mAb (n = 10). Significance was determined by two-tailed Mann-Whitney U test (d, e, g) and two-way ANOVA/mixed-effect analysis with Geisser-greenhouse correction (f). All p values are two-tailed. For d-e and g, whiskers extend to highest and lowest values from a box drawn between 1st and 3rd quartiles with a line centred at the median. f, data are represented as mean +/- SEM. Source Data

Extended Data Fig. 6. Antitumor effects of RNA-LNPs are mediated by Type I IFN.

a, b, Graphical experimental design, individual tumour growth curves (a) and tumour volume (b) for C57Bl6 mice inoculated with B16F0 (50,000 cells) and vaccinated with RNA-LNPs (Day 3, 6, 20) with and without anti-PD-L1 and anti-IFNAR1 or anti-IL-1R mAbs (n = 12/group). c, Tumour growth for C57Bl/6 mice with subcutaneous B16F0 tumours (50,000 cells) treated with anti-PD-L1 and either RNA-LNPs or exogenous IFN-α (Days 3, 6, and 20) (n = 8/group). Early differences in tumour growth volumes were lost by day 20 without continued treatment. d, C57Bl/6 mice with s.c. B16F0 tumours (50,000 cells) are treated with anti-PD1 starting on Day 14/17/20 with or without RNA-LNPs or Poly I:C (Days 14,17) (n = 8/group). e, Tumour growth for C57Bl/6 mice with B16F0 tumours (50,000 cells) treated with anti-PD-L1(Days 3/6/10/13/17/20) with or without RNA-LNPs (Days 3,6,20) containing mRNA coding for the Spike or the CMV antigen pp65 incorporating N1-methyl pseudouridine (“modified”) or wild-type uridine (“unmodified”) and (f) boxplots of day 17 and day 20 tumour volumes (n = 8/group). Tumour measurements from mice that met humane end points prior to each measurement day are excluded (Day 17: n = 1 (Untreated), Day 20: n = 2 (Untreated (n = 1) and Modified Spike RNA-LNP + PD-L1 (n = 1)) (see data file). Significance was determined by two-tailed Mann-Whitney U test (b,f) and two-way ANOVA/mixed-effect analysis with Geisser-Greenhouse correction (c,d,e). Data are displayed as means with standard error. Boxplots in b and f display whiskers extending to the highest and lowest values from a box drawn between the 1st and 3rd quartiles with a line centred at the median. For c-e, data are represented as mean +/- SEM. Source Data

Extended Data Fig. 7. ssRNA RNA extracted from RNA-LNPs contains high molecular weight secondary structures.

a, QC analysis of dsRNA contamination before and after purification (analysis performed by Genscript’s dsRNA residue assay). b, Tumour growth for mice with B16F0 tumours (50,000 cells) treated with anti-PDL1 mAbs with or without RNA-LNPs versus anionic (LPA) versus ssRNA (Days 3,6, 17) (n = 12). In this experiment, anionic LPA was synthesized by mixing DOTAP liposomes with mRNA at a 1:1 mass to mass ratio to formulate lipid particle aggregates (see methods). c, ELISA for IFN-α in serum collected from wildtype C57Bl/6 mice (n = 5) 24 h after treatment with PBS (WT PBS) or RNA-LNP (WT RNA-LNP), or RIG-I null mice (n = 3) treated with RNA-LNP (RIG-I -/- RNA-LNP). d, Non-complexed RNA (A1, B1) and LNP extracted RNA (A2,B2) analysed on a tape station with or without heating. Data are displayed as means with standard error. Significance was determined by two-way ANOVA/mixed-effect analysis with Geisser-greenhouse correction (b), and two-tailed unpaired t tests (c). For b, data are represented as mean +/- SEM. For c, the height of the bars represents mean and error bars represent +SEM. Source Data

Extended Data Fig. 8. RNA-LNPs elicit a dramatic shift in systemic cytokines/chemokines.

a-l, Cytokine/chemokine multiplex panel. Values are represented as pg/mL (a-j) for samples within the standard curve, and as fluorescent intensity values for cytokines above the standard curve (k-l). Plasma from subcutaneous B16F0 (50,000 cells) bearing C57Bl/6 animals (n = 5/group) 24 h after one RNA-LNP vaccine i.m. Whiskers extend to highest and lowest values, with a box shown between 1^st and 3^rd quartiles with a line centred at median. Significance was determined by one-way ANOVA analysis followed by two-tailed Šidák correction. Source Data

Extended Data Fig. 9. Spike RNA-LNP prime anti-cancer immunity in an IFN-I dependent manner.

a-b, Box-Plots of cellular phenotyping within 24 h of 3^rd RNA-LNP vaccine i.m. (Days 3, 6, 20) from spleens of C57Bl/6 animals bearing subcutaneous B16F0 (50,000 cells, n = 5/group). a, Percentage of activated (MHCII + CD86 + ) Ly6C+ cells. b, MHCII and PD-L1 positive of Ly6C+ cells (%). c, Prevalence of CD86 + Ly6C+ cells of CD45+ cells in tumours. d-f, Box-plots of cellular phenotyping within 24 h of 3^rd RNA-LNP vaccine i.m. (Days 3, 6, 20) from spleens of subcutaneous B16F0 (50,000 cells) bearing C57Bl/6 animals (n = 5/group) for (d) percentage of CD44 + T cells in the CD8+ compartment, (e) percentage of CD44+PD1+ among CD3+ cells, and (f) median fluorescence intensity (MFI) of PD1 on effector CD8 T cells. g,h. Upregulation of PDL1 on tumour cells is dependent on Type I IFN. Wild type and IFN-gamma KO mice with s.c. B16F0 tumours (50,000 cells) were treated with three doses of mRNA vaccines (Days 3, 6, and 17) with or without twice weekly antibodies blocking the IFN-a receptor (IFNAR1) (n = 4 untreated, n = 5 for all other groups). PDL1 expression on tumour cells was evaluated on Day 18 with flow cytometry. Whiskers extend to highest and lowest values from a box drawn between 1^st and 3^rd quartiles with a line centred at median. Significance was determined using two-tailed unpaired t tests. Source Data

Extended Data Fig. 10. COVID mRNA vaccines generate a surge in IFN-α, innate immune activation and adaptive immunity in humans.

a, Schematic depicting the experimental design wherein blood was drawn from eleven healthy subjects at baseline and 6 h, 24 h, and 48 h after BNT162b COVID mRNA immunization. b, Heat map displaying dynamic expression of the cytokines that are significantly elevated at 24 h at the following time points: 6 h, 24 h, and 48 h after COVID mRNA vaccination. Significant variables were defined as those with p < .05 and log2-Fold-Change with absolute value greater than 0.5 following linear modelling with fixed effect. Adjusted p values were calculated using moderated two-tailed t-tests with FDR correction for multiple testing. c-d, Individual data points highlighting changes in expression of IFN-α from baseline to 24 h for healthy volunteers (n = 11) expressed as fold change from baseline (c) and concentration (d). e-h, PD-L1 expression on circulating myeloid cells (e) and dendritic cells (f), activation of NK cells (g), and activation of T cells expressed at percentage of CD69 + CD8+ cells (h) at baseline, 6 h (6 h), 24 h (24 h), and 48 h (48 h) after immunization (n = 7). Data are presented as means with standard error. p values in e-h are results of two-tailed paired t tests. i, Heatmap displaying differentially expressed cytokines for patients receiving Spikevax (2023-2024 formulation, 50 µg mRNA) relative to the Comirnaty COVID mRNA vaccine (2024-2025 formulation, 30 µg mRNA). Moderated t tests were performed on per-patient log₂ fold change differences between cytokines at baseline vs 6 h or 24 h, with direct comparison of fold change from baseline in volunteers treated with either Moderna or Pfizer at each timepoint. Relative fold change for Moderna compared to Pfizer was displayed for differences that were significant with |log₂FC | > 0.5 and p < 0.05 at either 6 h or 24 h after multiple comparisons testing. j,k. Cumulative moving average of PDL1 expression for patients in the NSCLC (j) and Tissue Agnostic (k) cohorts stratified by the time from each patient’s most recent COVID mRNA vaccine. Data indicates the average of all TPS measurements from patients who received biopsy within each period from COVID mRNA immunization. Blue lines indicate unvaccinated patient average TPS. Source Data

Extended Data Fig. 11. Schematic describing how mRNA vaccines sensitize immunologically “cold” tumours.

RNA-LNPs stimulate production of IFN-α, leading APCs to prime T cells in lymphoid organs. These primed, tumour-reactive T cells then infiltrate tumours and begin killing tumour cells. Tumour cells respond by expressing PD-L1. Combination therapy with RNA-LNPs and ICIs overcomes this resistance mechanism, leading to tumour rejection. Image created in BioRender (Grippin, A. (2025) https://BioRender.com/zcaaisj).