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Cited by 79Immunotherapy and Immune ResponsesCancer Immunotherapy and BiomarkersRNA Interference and Gene Delivery

mRNA vaccine with unmodified uridine induces robust type I interferon-dependent anti-tumor immunity in a melanoma model

Open access
Sittplangkoon, Chutamath, Alameh, Mohamad-Gabriel, Weissman, Drew, Lin, Paulo J. C., Tam, Ying K., Prompetchara, Eakachai, Palaga, Tanapat·2022·Frontiers in Immunology
DOI 10.3389/fimmu.2022.983000PMC PMC9614103
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Abstract

An mRNA with unmodified nucleosides induces type I interferons (IFN-I) through the stimulation of innate immune sensors. Whether IFN-I induced by mRNA vaccine is crucial for anti-tumor immune response remains to be elucidated. In this study, we investigated the immunogenicity and anti-tumor responses of mRNA encoding tumor antigens with different degrees of N1-methylpseudouridine (m1Ψ) modification in B16 melanoma model. Our results demonstrated that ovalbumin (OVA) encoding mRNA formulated in a lipid nanoparticle (OVA-LNP) induced substantial IFN-I production and the maturation of dendritic cells (DCs) with negative correlation with increasing percentages of m1Ψ modification. In B16-OVA murine melanoma model, unmodified OVA-LNP significantly reduced tumor growth and prolonged survival, compared to OVA-LNP with m1Ψ modification. This robust anti-tumor effect correlated with the increase in intratumoral CD40+ DCs and the frequency of granzyme B+/IFN-γ+/TNF-α+ polyfunctional OVA peptide-specific CD8+ T cells. Blocking type I IFN receptor completely reversed the anti-tumor immunity of unmodified mRNA-OVA reflected in a significant decrease in OVA-specific IFN-γ secreting T cells and enrichment of PD-1+ tumor-infiltrating T cells. The robust anti-tumor effect of unmodified OVA-LNP was also observed in the lung metastatic tumor model. Finally, this mRNA vaccine was tested using B16 melanoma neoantigens (Pbk-Actn4) which resulted in delayed tumor growth. Taken together, our findings demonstrated that an unmodified mRNA vaccine induces IFN-I production or the downstream signaling cascades which plays a crucial role in inducing robust anti-tumor T cell response for controlling tumor growth and metastasis.

AI snapshot

Written from the paper’s full text.

TL;DR

Unmodified (0% m1Ψ) mRNA-LNP encoding OVA induced strong type I IFN, dendritic cell maturation, and robust anti-tumor immunity in B16 melanoma models, whereas increasing m1Ψ modification improved translation but reduced immunogenicity. Blocking IFN-I signaling reversed the anti-tumor effect, and unmodified neoantigen mRNA also delayed B16F10 tumor growth.

Key findings

  • In vitro, 70–100% m1Ψ-modified mRNA gave significantly higher mCherry+ cells in BMDCs and BMDMs, but only 0% m1Ψ strongly induced IFN-I; unmodified mRNA significantly upregulated CD40 and CD86 on BMDCs.
  • In B16F0-OVA melanoma, unmodified OVA-LNP reduced tumor growth and prolonged survival: all unmodified OVA-LNP mice survived to day 31, all PBS or Luc-LNP controls died, and half of the 100% m1Ψ OVA-LNP mice survived.
  • Anti-IFNAR1 antibody treatment completely reversed tumor control by unmodified OVA-LNP, reduced OVA-specific IFN-γ-producing T cells, and increased PD-1+ tumor-infiltrating CD4+ and CD8+ T cells and CD206+ M2-like macrophages.
  • Unmodified OVA-LNP suppressed lung metastatic nodules, while 100% m1Ψ OVA-LNP did not; unmodified Pbk-Actn4 neoantigen mRNA delayed B16F10 tumor growth, with one third surviving to day 35 versus all control mice dead by day 29.

Methods

  • Female C57BL/6 mice (6–12 weeks) were used in B16F0-OVA and B16F10-Luc2 melanoma models; mice received intramuscular 10 µg mRNA-LNP doses on day 0 and 4 for immunogenicity or day 4 and 8 after tumor inoculation, with anti-IFNAR1 mAb at 400 µg/dose in blocking experiments.
  • mRNA was produced with varying m1Ψ percentages (0, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%); 0, 40, 70, and 100% were selected for LNP encapsulation. LNPs were ~80 nm with polydispersity index 0.02–0.06 and ~95% encapsulation efficiency.
  • Readouts included IFN-α/β ELISA, flow cytometry, intracellular cytokine staining, ELISpot, tumor volume, survival, and lung nodule counts; statistics used unpaired two-tailed Student’s t test, one-way or two-way ANOVA with Bonferroni correction, and log-rank Mantel-Cox tests.

Limitations

  • Stated: the study did not distinguish adjuvant activity of mRNA from that of the LNP; whether unmodified mRNA-LNP shows enhanced stability upon lyophilization is unknown; neoantigen vaccine effects were less robust than OVA; the role of tumor-infiltrating CD4−CD8− double-negative T cells remains controversial and was not characterized.
  • Evident: all data are from mouse melanoma models, with no human data; several experiments used small group sizes (e.g., n = 4–10); the LNP composition is proprietary, which may limit direct generalizability.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.3389/fimmu.2022.983000

Cites

56 of 56 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

31 of 43 cited sources checked · 19 supported · 1 flagged · accuracy not yet measured

  • Premisecitation

    Unmodified mRNA induces type I interferons through stimulation of innate immune sensors.

    “An mRNA with unmodified nucleosides induces type I interferons (IFN-I) through the stimulation of innate immune sensors.”
    • Also stated in: § Introduction · plain
      “Purified unmodified IVT mRNA induces high level of type I interferon (IFN-I) through activation of toll-like receptors (TLRs).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [18] Vlatkovic, 2021, Non-immunotherapy application of LNP-mRNA: Maximizing efficacy and safety, Biomedicines source
  • Premisecitation

    M1 macrophages secrete CXCL9, CXCL10 and CXCL15 chemokines upon STAT1 signaling, which recruit cytotoxic T lymphocytes (CTLs) to the tumor.

    “In addition, M1 macrophages secrete CXCL9, CXCL10 and CXCL15 chemokines upon STAT1 signaling, which recruit cytotoxic T lymphocytes (CTLs) to the tumor (43).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [43] Liu, 2021, New insights into M1/M2 macrophages: key modulators in cancer progression, Cancer Cell Int source

      The Introduction’s “Major functional properties of M1 phenotype” section says M1 macrophages can strengthen CD8+ T-cell cytotoxic functions through IL-6, IL-12 and TNF. It does not say they secrete CXCL9, CXCL10 or CXCL15 upon STAT1 signaling, or that those chemokines recruit CTLs to tumors; searches for the chemokines and STAT1 found no supporting passage. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
  • Premisecitation

    IFN-I can act through DCs to promote T cell immunity.

    “This evidence strongly shows that IFN-I can act through DCs to promote T cell immunity (52).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [52] Fuertes, 2011, Host type I IFN signals are required for antitumor CD8+ T cell responses through CD8{alpha}+ dendritic cells, J Exp Med source
  • Premisecitation

    Unmodified mRNA itself provides adjuvant activity through binding and activation of the innate immune sensors, mainly TLRs 3, 7, and 8.

    “Unmodified mRNA itself provides adjuvant activity through binding and activation of the innate immune sensors, mainly TLRs 3, 7, and 8 (39).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [39] Dalpke, 2012, RNA Mediated toll-like receptor stimulation in health and disease, RNA Biol source

      The section on posttranscriptional modifications reports that RNA activates TLR3, TLR7 and TLR8, and that certain modifications suppress this activation. The section on synthetic RNA identifies potential vaccine-adjuvant activity, but it discusses RNA oligonucleotides rather than establishing that unmodified mRNA itself provides adjuvant activity mainly through those three receptors. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
  • Premisecitation

    Previous study showed that DC-specific Ifnar -/- mice were unable to reject highly immunogenic tumor cells due to the defects in antigen cross-presentation to CD8+ T cells.

    “Previous study showed that DC-specific Ifnar -/- mice were unble to reject highly immunogenic tumor cells due to the defects in antigen cross-presentation to CD8+ T cells.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [52] Fuertes, 2011, Host type I IFN signals are required for antitumor CD8+ T cell responses through CD8{alpha}+ dendritic cells, J Exp Med source

      The Results section on hematopoietic IFN signaling (page unavailable) reports that mice reconstituted with Stat1-deficient bone marrow failed to reject an immunogenic tumor. The paper also links type I IFN signaling and CD8α+ dendritic cells to antitumor CD8+ T-cell priming. It does not report tumor rejection in DC-specific Ifnar−/− mice, and its DC analysis found that APCs at the tumor site could process and present tumor antigen despite deficient host signaling; the claimed cross-presentation defect is therefore not established as the cause. (LLM, conf=0.89)

      Experimental — accuracy not yet measured
  • Classified as probably not a substantial claim. 25
  • May combine two claims. 33

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    cDC1 are critical for antigen cross-presentation required to prime CD8+ T cells for optimal anti-tumor immunity and priming of CD4+ T cells at early stages, partly because cDC1 provides antigen transportation to lymph nodes for processing by cDC2.

    “cDC1 are critical for antigen cross-presentation required to prime CD8+ T cells for optimal anti-tumor immunity and priming of CD4+ T cells at early stages, partly because cDC1 provides antigen transportation to lymph nodes for processing by cDC2 (36).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [36] Ferris, 2020, cDC1 prime and are licensed by CD4(+) T cells to induce anti-tumour immunity, Nature source

      Split into 2 atoms — 0 confirmed, 1 partially supported, 1 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

      Checked by a reasoning model — may be wrong.

      Experimental — accuracy not yet measured

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    Incorporation of naturally occurring nucleosides and stringent purification to remove double-stranded RNA contaminants can circumvent the high innate immunogenicity of purified unmodified IVT mRNA.

    “This can be circumvented by the incorporation of naturally occurring nucleosides and by applying stringent purification to remove double-stranded RNA contaminants (19–21).”
    Quote in the paperSubstantial claim2 of 3 sources checked
    • cites [19] Andries, 2015, N(1)-methylpseudouridine-incorporated mRNA outperforms pseudouridine-incorporated mRNA by providing enhanced protein expression and reduced immunogenicity in mammalian cell lines and mice, J Control Release source
    • cites [20] Kariko, 2008, Incorporation of pseudouridine into mRNA yields superior nonimmunogenic vector with increased translational capacity and biological stability, Mol Ther source

      The Introduction and Results report that incorporating pseudouridine, a naturally occurring modified nucleoside, suppresses immune activation compared with unmodified mRNA. The Methods describe transcript purification by DNase digestion, LiCl precipitation, and an ethanol wash, but do not say that this removed double-stranded RNA contaminants or reduced immunogenicity. Page numbers are not available in our copy. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites [21] Anderson, 2010, Incorporation of pseudouridine into mRNA enhances translation by diminishing PKR activation, Nucleic Acids Res source

      The Discussion (page not provided) says that naturally occurring nucleoside modifications reduce PKR activation and that modified mRNA can have low immunogenicity. The mRNA synthesis methods describe precipitation and washing, but neither they nor the retrieved passages say that stringent purification removes double-stranded RNA contaminants or that such removal circumvents immunogenicity. (LLM, conf=0.87)

      Experimental — accuracy not yet measured

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    mRNA with nucleoside modifications is insensitive to RNase L degradation, does not activate TLRs and protein kinase R, and has improved translational efficiency and stability compared to unmodified mRNA.

    “Karikó et al. reported that mRNA with nucleoside modifications were insensitive to ribonuclease L (RNase L) degradation, did not activate TLRs and protein kinase R, which subsequently resulted in improved translational efficiency and stability, compared to unmodified mRNA (20).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [20] Kariko, 2008, Incorporation of pseudouridine into mRNA yields superior nonimmunogenic vector with increased translational capacity and biological stability, Mol Ther source

      The Results report greater translation and persistence of pseudouridine-modified mRNA than unmodified mRNA in mice, and the Discussion reports reduced TLR stimulation. But the paper does not establish resistance to RNase L degradation; it offers reduced protein kinase R activation only as a possible explanation for improved translation, not a demonstrated cause. It also shows that improved translation does not apply to every nucleoside modification tested. (LLM, conf=0.90)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    CD40 signaling in cDC1 is required for tumor rejection by playing a key role in augmenting the proliferation of antigen-specific CD8+ T cells.

    “CD40 signaling in cDC1 is required for tumor rejection by playing a key role in augmenting the proliferation of antigen-specific CD8+ T cells (36).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [36] Ferris, 2020, cDC1 prime and are licensed by CD4(+) T cells to induce anti-tumour immunity, Nature source

      In the section “CD40 signaling in cDC1 is required for optimal activation of both CD8 and CD4 T cells” (page not provided), mice lacking CD40 on cDC1 failed to reject tumours and showed reduced expansion of antigen-specific CD8 T cells. The paper reports reduced CD8 T-cell proliferation after immunization with cell-associated antigen, but not in tumour-bearing mice; it therefore describes the tumour effect as enhanced expansion and/or persistence, rather than specifically enhanced proliferation. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    Antitumor innate immune signals, particularly type I IFNs, which are the main cytokines secreted from DCs upon mRNA transfection, play an important role in antigen presentation and T cell differentiation into cytolytic effector cells.

    “Antitumor innate immune signals, particularly type I IFNs, which are the main cytokines secreted from DCs upon mRNA transfection (35) play an important role in antigen presentation and T cell differentiation into cytolytic effector cells.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [35] Pollard, 2013, Type I IFN counteracts the induction of antigen-specific immune responses by lipid-based delivery of mRNA vaccines, Mol Ther source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Adjuvant activity of the LNP critically relies on IL-6 and its constituent ionizable lipid.

    “Adjuvant activity of the LNP critically relies on IL-6 and its constituent ionizable lipid.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [38] Alameh, 2021, Lipid nanoparticles enhance the efficacy of mRNA and protein subunit vaccines by inducing robust T follicular helper cell and humoral responses, Immunity source

  • May combine two claims.

    Premisecitation

    Substitution of uridine with m1Ψ in mRNA significantly improves translation efficiency and decreases innate immunogenicity.

    “Substitution of uridine with m1Ψ in mRNA significantly improves the translation efficiency and decreases innate immunogenicity (20).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [20] Kariko, 2008, Incorporation of pseudouridine into mRNA yields superior nonimmunogenic vector with increased translational capacity and biological stability, Mol Ther source

      The Introduction reports that pseudouridine (Ψ) in mRNA suppresses immune activation and enhances translation; the Results also report greater translation of Ψ-containing mRNA in cultured cells. The claim specifies N1-methylpseudouridine (m1Ψ), a different modification that the paper does not test, so its findings do not establish the claimed effects for m1Ψ. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    LNP exerts potent stimulatory activity against T follicular helper cells and immune induction superior to AddaVax formulated vaccine.

    “When used in mRNA and protein subunit vaccines, LNP exerts potent stimulatory activity against T follicular helper cell and the immune induction was superior to what induced by AddaVax formulated vaccine.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [38] Alameh, 2021, Lipid nanoparticles enhance the efficacy of mRNA and protein subunit vaccines by inducing robust T follicular helper cell and humoral responses, Immunity source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Engagement of CD40 with its ligand induces recruitment of TRAFs and initiates signaling cascades that activate genes involved in cytokine production, as well as upregulation of co-stimulatory molecules such as CD80 and CD86.

    “Engagement of CD40 with its ligand induces recruitment of TNF receptor-associated factor family of proteins (TRAFs) and initiates signaling cascades that activate genes involved in cytokine production, as well as upregulation of co-stimulatory molecules such as CD80 and CD86 (37).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [37] Ma, 2009, The role of CD40 and CD154/CD40L in dendritic cells, Semin Immunol source

  • May combine two claims.

    Premisecitation

    The mutated epitopes of Pbk and Actn4 were recognized and reacted to by CD8+ or CD4+ T cells, respectively, upon RNA monotope vaccinations and showed good MHC class I binding scores (low score 0.1 and 0.2, respectively).

    “These mutated epitopes of Pbk and Actn4 were recognized and reacted to by CD8+ or CD4+ T cells, respectively, upon RNA monotope vaccinations and showed good MHC class I binding scores (‘low score’ 0.1 and 0.2, respectively) (25).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [25] Kreiter, 2015, Mutant MHC class II epitopes drive therapeutic immune responses to cancer, Nature source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Ligation of PD-1 with its ligands PD-L1 or PD-L2 induces tyrosine phosphorylation of the PD-1 cytoplasmic domain by phosphorylating kinase Lck and subsequent recruitment of cytosolic tyrosine phosphatase SHP-2 and PD-1-associated SHP-2 preferentially dephosphorylates CD28 and suppresses CD28 costimulatory signaling leading to restrained effector T cell function.

    “Ligation of PD-1 with its ligands programmed death-ligand 1 or 2 (PD-L1 or PD-L2) induces tyrosine phosphorylation of the PD-1 cytoplasmic domain by phosphorylating kinase Lck and subsequent recruitment of cytosolic tyrosine phosphatase SHP-2 and PD-1-associated SHP-2 preferentially dephosphorylates CD28 and suppresses CD28 costimulatory signaling leading to restrained effector T cell function (49”
    Quote in the paperNot a substantial claim2 of 3 sources checked
    • cites [49] Hui, 2017, T Cell costimulatory receptor CD28 is a primary target for PD-1-mediated inhibition, Science source

      Split into 2 atoms — 1 confirmed, 1 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

      Experimental — accuracy not yet measured
    • cites [50] Yokosuka, 2012, Programmed cell death 1 forms negative costimulatory microclusters that directly inhibit T cell receptor signaling by recruiting phosphatase SHP2, J Exp Med source

      Split into 2 atoms — 0 confirmed, 2 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

      Experimental — accuracy not yet measured
    • cites [51] Sheppard, 2004, PD-1 inhibits T-cell receptor induced phosphorylation of the ZAP70/CD3zeta signalosome and downstream signaling to PKCtheta, FEBS Lett source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Replacing of uridine by pseudouridine into mRNA gives superior nonimmunogenic mRNA with increased translational capacity and biological stability.

    “Replacing of uridine by pseudouridine into mRNA gives superior nonimmunogenic mRNA with increased translational capacity and biological stability (20).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [20] Kariko, 2008, Incorporation of pseudouridine into mRNA yields superior nonimmunogenic vector with increased translational capacity and biological stability, Mol Ther source

  • Classified as probably not a substantial claim.

    Premisecitation

    In the tumor microenvironment, immunosuppression and tumor evasion strategies cause an inability of the immune cells to detect and eliminate with subsequent exhaustion.

    “In the tumor microenvironment, immunosuppression and tumor evasion strategies cause an inability of the immune cells to detect and eliminate with subsequent exhaustion (47).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [47] Yang, 2019, Tumor-intrinsic signaling pathways: key roles in the regulation of the immunosuppressive tumor microenvironment, J Hematol Oncol source

      The Background and ‘Effective immunocyte exclusion and dysfunction’ sections describe tumor immune escape through reduced immune-cell infiltration, impaired function, and immunosuppression. The ‘KRAS signaling’ section reports increased T-cell exhaustion markers in a specific mouse lung-cancer model, but the review does not establish subsequent exhaustion as a general consequence for immune cells in the tumor microenvironment. Page numbers are unavailable in our copy. (LLM, conf=0.82)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    CD4 CTL recognizes target cells via peptide-MHC II complex on APCs.

    “CD4 CTL recognizes target cells via peptide-MHC II complex on APCs (53).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [53] Takeuchi, 2017, CD4 CTL, a cytotoxic subset of CD4(+) T cells, their differentiation and function, Front Immunol source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationcitation

    Additional neoantigens formulated in the mRNA vaccine may help improve the anti-tumor response of mRNA vaccines using neoantigens.

    “Additional neoatigens formulated in the mRNA vaccine may help improve the anti-tumor response of mRNA vaccines using neoantigens, such as demonstrated by Kreiter et al. (25).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [25] Kreiter, 2015, Mutant MHC class II epitopes drive therapeutic immune responses to cancer, Nature source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    CD4+ CTLs are characterized by their cytotoxic functions to secrete granzyme B and perforin, two major tools to directly kill the target cells.

    “CD4+ CTLs are characterized by their cytotoxic functions to secrete granzyme B and perforin, two major tools to directly kill the target cells.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [53] Takeuchi, 2017, CD4 CTL, a cytotoxic subset of CD4(+) T cells, their differentiation and function, Front Immunol source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Tumor regression was dependent on class II-restricted recognition of tumors by tumor-reactive CD4+ CTLs which developed cytotoxic activity and kill tumor.

    “Tumor regression was dependent on class II-restricted recognition of tumors by tumor-reactive CD4+ CTLs which developed cytotoxic activity and kill tumor (54).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [54] Quezada, 2010, Tumor-reactive CD4(+) T cells develop cytotoxic activity and eradicate large established melanoma after transfer into lymphopenic hosts, J Exp Med source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Potent immune responses from a single immunization of LNP loaded non-inflammatory nucleoside-modified mRNA was related to LNP adjuvanticity.

    “Remarkably, potent immune responses from a single immunization of LNP loaded non-inflammatory nucleoside-modified mRNA was related to LNP adjuvanticity (38).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [38] Alameh, 2021, Lipid nanoparticles enhance the efficacy of mRNA and protein subunit vaccines by inducing robust T follicular helper cell and humoral responses, Immunity source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Lyophilization of modified mRNA-LNP provides long-term stability at room temperature.

    “Furthermore, lyophilization of modified mRNA-LNP provides long-term stability at room temperature (56).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [56] Muramatsu, 2022, Lyophilization provides long-term stability for a lipid nanoparticle-formulated, nucleoside-modified mRNA vaccine, Mol Ther source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The use of DN T cells for cancer immunotherapy against blood and solid tumor were reported.

    “Althought the roles of these cells in tumors are still controversial, the use of DN T cells for cancer immunotherpy against blood and solid tumor were reported (46).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [46] Wu, 2022, CD3(+)CD4(-)CD8(-) (Double-negative) T cells in inflammation, immune disorders and cancer, Front Immunol source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Modifying the structural elements of mRNA including the 5′ cap, 5′-and 3′-untranslated regions, the coding region, and polyadenylation tail help improved the intracellular stability of mRNA.

    “For mRNA vaccine, modifying the structural elements of mRNA including the 5′ cap, 5′-and 3′-untranslated regions, the coding region, and polyadenylation tail help improved the intracellular stability of mRNA (55).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [55] Kim, 2022, Modifications of mRNA vaccine structural elements for improving mRNA stability and translation efficiency, Mol Cell Toxicol source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    CD4+ T cells also contribute to direct tumor killing besides their supporting role as cytokine producers.

    “Although CD8+ T cells are known to play a pivotal role in antitumor immunity, CD4+ T cells also contribute to direct tumor killing besides their supporting role as cytokine producers.”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites [53] Takeuchi, 2017, CD4 CTL, a cytotoxic subset of CD4(+) T cells, their differentiation and function, Front Immunol source
    • cites [54] Quezada, 2010, Tumor-reactive CD4(+) T cells develop cytotoxic activity and eradicate large established melanoma after transfer into lymphopenic hosts, J Exp Med source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Upon transferring of naive tumor reactive CD4+ T cells into lymphopenic recipients, substantial T cell expansion and differentiation were observed.

    “Upon transferring of naive tumor reactive CD4+ T cells into lymphopenic recipients, substantial T cell expansion and differentiation were observed.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [54] Quezada, 2010, Tumor-reactive CD4(+) T cells develop cytotoxic activity and eradicate large established melanoma after transfer into lymphopenic hosts, J Exp Med source

  • May combine two claims.

    Findingown data

    CD40 upregulation in LN cDC1 was equally observed in all types of mRNA, regardless of m1ψ modification, and was higher than untreated control.

    “CD40 upregulation in LN cDC1 was equally observed in all types of mRNA, regardless of m1ψ modification that is higher than the untreated control ( Figure 2C ; Figure S1C ).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Cells transfected with unmodified mRNA significantly upregulated CD40 and CD86 expression, suggesting DC maturation.

    “In contrast, cells transfected with unmodified mRNA significantly upregulated CD40 and CD86 expression, suggesting DC maturation ( Figure 1F ; Figure S1A ).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    After the second dose, the level of IFN-α induced by unmodified mRNA was much lower than after the first dose but remained detectable.

    “After the second dose, the level of IFN-α induced by unmodified mRNA was much lower compared to that from the first dose but remained at detectable level.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Modified mRNA with 100% m1ψ substitution did not significantly induce maturation of BMDCs despite efficient protein translation.

    “Although modified mRNA with 100% of m1ψ substitution showed efficient protein translation, this treatment did not significantly induce maturation of BMDCs.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    LNP-formulated mRNA with 100% m1ψ substitution resulted in significantly higher percentages and MFI of mCherry+ cells than other conditions in both BMDCs and BMDMs, with 77% and 39% mCherry+ cells respectively.

    “Similar to the results obtained by TransIT reagent, LNP formulated modified mRNA with 100% of m1ψ substitution resulted in a significantly higher percentages and median fluorescence intensity (MFI) of mCherry+ cells than other conditions in both BMDCs and BMDMs with 77% and 39% of mCherry+ cells, respectively ( Figure 1E ; Figures S1A, B ).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Intramuscular administration of 10 μg mRNA-LNP with 100% m1ψ substitution was efficiently taken up and translated into proteins by cDC1, cDC2 and macrophages in both LNs and spleens, with highest percentages of mCherry+ cells at 48 hr.

    “Intramuscular administration (i.m.) of 10 µg mRNA-LNP with 100% of m1ψ substitution was efficiently taken up and translated into proteins by cDC1, cDC2 and macrophages in both LNs and spleens with the highest percentages of mCherry+ cells observed after 48 hr ( Figures 2A, B ; Figure S1C ).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Mice immunized with unmodified OVA-LNP survived until the end of the 31-day experimental period, while all mice in PBS or Luc-LNP control groups were dead.

    “Mice immunized with unmodified OVA-LNP survived until the end of the experimental period of 31 days while all mice in the PBS or Luc-LNP control group were dead ( Figure 4B ).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The majority of tumor-infiltrating CD3+ T cell population in the unmodified OVA-LNP group were CD4-CD8- T cells, while the group receiving OVA-LNP with m1Ψ modification of 100% had CD8+ T cells as the major population.

    “The majority of tumor-infiltrating CD3+ T cell population in the unmodified OVA-LNP group were CD4-CD8- T cells, while the group receiving OVA-LNP with m1Ψ modification of 100% had CD8+ T cells as the major population ( Figure 4D ; Figure S3 ).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    There was a significant increase in PD-1+ CD8+ T cells in the group with OVA-LNP with m1Ψ modification of 100%, whereas PD-1+ CD4+ T cells increased in the unmodified OVA-LNP group.

    “There was a significant increase in PD-1+ CD8+ T cells in the group with OVA-LNP with m1Ψ modification of 100% whereas the PD-1+ CD4+ T cells increased in the unmodified OVA-LNP group ( Figure 5B ; Figure S4 ).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    One third of the Neo-LNP treated mice survived until day 35, while all mice in the control group died by day 29.

    “One third of the Neo-LNP treated mice survived until day 35, while all mice in the control group died by day 29 ( Figure 8F ).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Unmodified mRNA compromises translation efficiency but induces high levels of type I IFN and robust expression of costimulatory molecules in major APCs.

    “Although unmodified mRNA compromises the translation efficiency, it induces high levels of type I IFN and robust expression of costimulatory molecules in major APCs.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Modified mRNA with 100% m1ψ substitutions significantly improves translation efficiency and decreases innate immunogenicity.

    “Taken together, these results indicated that modified mRNA with 100% m1ψ substitutions significantly improves translation efficiency and decreases innate immunogenicity.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    The vaccination schedule used prevents the typical post-expansion T-cell retraction phase and maintains high frequencies of antigen-specific T cells.

    “The schedule was designed to prevent the typical post-expansion T-cell retraction phase and maintain high frequencies of antigen-specific T cells.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [34] Fotin-Mleczek, 2011, Messenger RNA-based vaccines with dual activity induce balanced TLR-7 dependent adaptive immune responses and provide antitumor activity, J Immunother source
    • cites [30] Kranz, 2016, Systemic RNA delivery to dendritic cells exploits antiviral defence for cancer immunotherapy, Nature source

  • May combine two claims.

    Interpretationown data

    Administration of OVA-LNP robustly activates OVA-specific CD8+ T cell responses, and unmodified mRNA effectively stimulates CTL responses.

    “Taken together, these results demonstrated that administration of OVA-LNP robustly activates OVA-specific CD8+ T cell responses, particularly unmodified mRNA effectively stimulates CTL responses.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Unmodified mRNA encoding tumor neoantigen formulated in LNP induced a strong anti-tumor immune response that retarded tumor growth and partially prolonged survival of tumor-bearing mice.

    “Taken together, we confirmed that unmodified mRNA encoding tumor neoantigen formulated in LNP induced a strong anti-tumor immune response that retarded tumor growth and partially prolonged survival of tumor-bearing mice.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Unmodified mRNA induces more efficient DC activation that may augment anti-tumor immunity and skews toward Th1 in the tumor microenvironment.

    “This result strongly supports that unmodified mRNA induces more efficient DC activation that may augment the anti-tumor immunity and skews toward Th1 in the tumor microenvironment.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    While unmodified mRNA/LNP compromises the translation efficiency of mRNA into protein antigen, its superior impact on DC maturation is beneficial for anti-tumor immunity.

    “While unmodified mRNA/LNP compromises the translation efficiency of mRNA into protein antigen, its superior impact on DC maturation is beneficial for anti-tumor immunity.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductnone given

    In our study, we did not distinguish adjuvant activity of mRNA from LNP and the impact on anti-tumor responses may derive from LNP and/or mRNA.

    “In our study, we did not distinguish adjuvant activity of mRNA from LNP and the impact on anti-tumor responses may derive from LNP and/or mRNA.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationreasoning

    Differences in the use of whole protein rather than peptide antigens, the LNP formulation and its size may result in the modest differences observed between the two reports.

    “Although unmodified mRNA was used in the current work and that by Kranz et al., differences in the use of whole protein rather than peptide antigens, the LNP formulation and its size may result in the modest differences observed between the two reports.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    PD-1 expression on cell surface of activated T cells is induced after T cell receptor (TCR) activation.

    “Generally, PD-1 expression on cell surface of activated T cells is induced after T cell receptor (TCR) activation (48).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [48] Meng, 2018, FBXO38 mediates PD-1 ubiquitination and regulates anti-tumour immunity of T cells, Nature source

  • May combine two claims.

    Premisecitation

    M1 macrophages possess phagocytic capacity and the ability to secrete reactive nitrogen and oxygen species and pro-inflammatory cytokines such as IL-6, IL-12, IL-23 and TNF-α, which in turn promote CD8+ T cell and NK cell cytotoxicity.

    “which possesses phagocytic capacity, and the ability to secrete reactive nitrogen and oxygen species and pro-inflammatory cytokines such as IL-6, IL-12, IL-23 and TNF-α, which in turn promote CD8+ T cell and NK cell cytotoxicity.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [42] Chistiakov, 2018, The impact of interferon-regulatory factors to macrophage differentiation and polarization into M1 and M2, Immunobiology source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Their OVA mRNA construct encoded for the H-2Kb-restricted immunodominant epitope OVA 257-264 and the lipid formulation contained DOTAP and DOPE with the mean diameter of mRNA-LNP was 200 nm.

    “Their OVA mRNA construct encoded for the H-2Kb-restricted immunodominant epitope OVA 257-264 and the lipid formulation contained DOTAP and DOPE with the mean diameter of mRNA-LNP was 200 nm (30).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [30] Kranz, 2016, Systemic RNA delivery to dendritic cells exploits antiviral defence for cancer immunotherapy, Nature source

  • May combine two claims.

    Interpretationcitation

    The prominent therapeutic efficacy of unmodified mRNA is possibly due to activation of endosomal TLR7/8 and subsequently causes pro-inflammatory cytokine secretion via MyD88-dependent IRF-5 phosphorylation.

    “The prominent therapeutic efficacy of unmodified mRNA is possibly due to activation of endosomal toll-like receptor 7/8 (TLR7/8) and subsequently causes pro-inflammatory cytokine secretion via MyD88-dependent IRF-5 phosphorylation (41).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [41] Schoenemeyer, 2005, The interferon regulatory factor, IRF5, is a central mediator of toll-like receptor 7 signaling, J Biol Chem source
46 of 46

  • The paper relies on this without support.

    The impact of different degrees of nucleoside modifications on mRNA-LNP cancer vaccine-induced anti-tumor immunity has not been investigated.

    “However, the impact of different degrees of nucleoside modifications on mRNA-LNP cancer vaccine-induced anti-tumor immunity has not been investigated.”
1 of 1

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • CD8+ T-cell responses induced by LNP-formulated mRNAs with unmodified nucleotides can exceed those induced by LNP-formulated mRNAs with modified nucleotides.

    “They primarily induce CD8+ T-cell responses [22], which can exceed those induced by LNP-formulated mRNAs with modified nucleotides [34, 35], as well as CD4+ T-cell responses.”

    From Preclinical development of an mRNA-based multiepitope immunotherapeutic for glioblastoma (2025) · cites this paper as [35]

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Figure 5 response is called both double- and triple-positive

    Minor

    The results describe IFN-γ/TNFα double-producing cells in Figure 5D, whereas the figure caption identifies the corresponding category as IFN-γ/TNF-α/GzmB triple-producing cells.

  • Translation-modification relationship is described in opposite directions

    Minor

    The section heading says translation efficiency is inversely related to the level of nucleoside modification, but the results say 100% modification gives higher translation than the other conditions.

2 of 2

Cites

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1 of the 57 references couldn’t be matched to a known paper.

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Related

Authors
Sittplangkoon, C., Alameh, M.-G., Weissman, D., Lin, P. J. C., Tam, Y. K., Prompetchara, E., & Palaga, T.
Affiliations
  • Chulalongkorn University (TH)
  • University of Pennsylvania (US)
  • Acuitas Therapeutics (Canada) (CA)
Corresponding author
Palaga, Tanapat
ORCID
Venue
Frontiers in Immunology
Publisher
Frontiers Media SA
Published
2022-10-14
Volume
13
Pages
983000-983000
ISSN
1664-3224 (ISSN-L)
DOI
10.3389/fimmu.2022.983000
PMC
PMC9614103
OpenAlex
W4306157635
Semantic Scholar
64a133c8835e71526c15993ed5e01970a8e06b0c
Cited by
79
References
57
Citations by year
2026: 13 · 2025: 28 · 2024: 28 · 2023: 9 · 2022: 1
FWCI
5.10
Type
journal article
Language
en
Open access
Licence
Locations
  • Frontiers in Immunology (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • PubMed Central (submittedVersion, cc-by)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
National Research Council of Thailand (811/2563)
Topics
  • Life Sciences › Immunology and Microbiology › Immunology › Immunotherapy and Immune Responses
  • Health Sciences › Medicine › Oncology › Cancer Immunotherapy and Biomarkers
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
Keywords
mRNA vaccines, type I interferon, antitumor immunity, melanoma, lipid nanoparticles, ovalbumin, dendritic cell maturation, CD8+ T cells, type I interferon receptor, PD-1, neoantigens, lung metastasis, innate immune recognition
MeSH terms
mRNA Vaccines, Animals, Antigens, Neoplasm, Interferon Type I, Liposomes, Melanoma, Experimental, Ovalbumin, RNA, Messenger, Uridine, CD8-Positive T-Lymphocytes, Mice, Nanoparticles

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-11.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-11.

Cited sources are checked by gpt-6-sol.

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