Cross-competition shapes CD8+ T cell hierarchies and fate after cancer neoantigen RNA vaccination

Cross-competition shapes CD8+ T cell hierarchies and fate after cancer neoantigen RNA vaccination | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Cross-competition shapes CD8+ T cell hierarchies and fate after cancer neoantigen RNA vaccination Lélia Delamarre, Mark McCarron, Milena Hornburg, Thomas Wu, Ann-Jay Tong, and 24 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8304459/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Cancer patients typically mount T cell responses to only a small fraction of the neoantigens encoded in their individualized vaccine. Because each patient’s neoantigen set is unique, clinical studies cannot determine whether this restricted breadth reflects limitations of neoantigen prediction algorithms or intrinsic immunological constraints. Here, using a clinically relevant multi-neoantigen RNA–lipoplex vaccine in a preclinical model, we mechanistically dissected CD8+ T-cell breadth. We found that concurrently primed neoantigen-specific CD8+ T cells competed through a peptide-MHC-I-dependent cross-competition, with dominant responses suppressing both the magnitude and differentiation of subdominant responses. Dominant CD8+ T cell responses preferentially acquired a terminally differentiated effector phenotype, while sub-dominant responses adopted memory-precursor and stem-like features. Removing dominant responses enabled sub-dominant responses to expand and adopt terminally differentiated effector phenotypes. These findings provide the first mechanistic explanation for restricted CD8+ T cell breadth in multi-neoantigen vaccination and provide a framework for further optimizing cancer vaccine design. Biological sciences/Immunology/Adaptive immunity/Cellular immunity/Lymphocyte activation Biological sciences/Cancer/Tumour immunology Biological sciences/Immunology/Antigen processing and presentation/Cellular immunity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Full Text Additional Declarations Yes there is potential Competing Interest. All the authors were employees of Genentech at the time of the study, but DE, MV, LK and US who were employees of BioNTech Supplementary Files ExtendedDataFigure1.pdf Extended Data Fig. 1. Distinct hierarchy of CD8+ T cell responses following multi-neoantigen RNA-LPX vaccination a, C57BL/6 mice were immunized three times on days 0, 7 and 14 with heptatope RNA-LPX vaccine encoding all 7 neoantigens. Splenic T cells were harvested 5 days after the last immunization and restimulated with 27 mer peptides containing the neoantigen or non-peptide DMSO solvent controls. The magnitude of cytokine and CD107a responses were measured by flow cytometry and are shown as a percentage of total CD8+ T cells. Neoantigen specificities are indicated on the x-axis. n=3-5. b, As depicted, three different heptatope constructs were generated encoding 7 neoantigens in which the neoantigen position was varied across constructs. C57BL/6 mice were immunized three times on days 0, 7 and 14 and splenic T cells were harvested 5 days after the last immunization. Flow cytometric quantification of CD8+ T cells stained with pMHCI tetramers (specificities indicated on x-axis) was performed. n=5. c, Mice were immunized with monotope RNA-LPX vaccines encoding M16 or M10 alone or both monotype RNA-LPX vaccines were combined. Flow cytometric quantification of CD8+ T cells stained with pMHCI tetramers was performed in the blood 5 days after the third immunization. Clonal expansion was analyzed. n=5. d, Table depicts neoantigen and non-neoantigen wild type sequences of Nsun2 and Arvcf peptides identified in B16F10 tumors. e, Mice were immunized with monotope RNA-LPX vaccines encoding Nsun2, Arvcf or SIINFEKL in various combinations as depicted. Following three immunizations, splenocytes were assessed by IFNg ELISpot assay following peptide restimulation. One-way anova or unpaired t-test. ns not significant, *p<0.5, **p<0.01, ***p<0.001.<0.01, ***p<0.001. ExtendedDataFigure2.pdf Extended Data Fig. 2. T cell competition is not mediated by the limited bioavailability of IL-2 or limitations in CD4+ T cell help a, C57BL/6 mice were immunized with heptatope RNA-LPX in the presence and absence of exogenous IL-2 (15,000 units I.P daily). Following three immunizations, spleens were removed and neoantigen-specific T cell responses were evaluated by pMHC-I tetramer staining and IFN-gamma ELISpot. Data are shown as pMHCI tetramer+ cells as a percentage of total CD8+ T cells, total number of neoantigen-specific CD8+ T cells or IFNɣ spot count per 105 splenocytes. n=10.b Mice were immunized with monotype vaccines encoding M16 and SIINFEKL, either alone or mixed. Mice immunized with the M16 monotype or a mixture of M16 + SIINFEKL received half-life extended human IL-2 delivered in LNP (3ug) intravenously. M16 or SIINFEKL neoantigen-specific T cell responses were evaluated in the spleen 7 days after the last immunization using pMHCI tetramer+ staining and are shown as a percentage of total CD8+ T cells. c-e, the heptatope RNA-LPX vaccine was modified to include 2W1S (“octatope”), or a non-immunogenic control antigen. Mice were immunized three times and neoantigen-specific T cell responses were evaluated in the spleen 5 days after the last immunization. c, the 2W1S CD4+ T cell response was confirmed by pMHC-II tetramer staining and production of IFNg after ex vivo restimulation of spleen cells by 2W1S peptide n=10. d, Neoantigen-specific CD8+ T cell responses were quantified in the spleen by pMHC tetramer+ staining in the presence and absence of the 2W1S CD4+ T cell responses. Data are shown as pMHCI tetramer+ cells as a percentage of total CD8+ T cells and total number of neoantigen-specific CD8+ T cells n=4-7. e, We generated a novel heptatope consisting of 6 neoantigens from the MC38 model that generate CD4+ T cell responses and a seventh CD4+ T cell antigen, 2W1S. Mice were immunized three times and neoantigen-specific CD8+ T cell responses were evaluated in the spleen 5 days later. Neoantigen-specific CD8+ T cell responses were quantified by IFNɣ ELISpot and are shown as IFNg spots per 105 splenocytes n=7. f, RNA-LPX monotype vaccines encoding either SIINFEKL or the M30 neoantigen, or both monotope vaccines combined were used to immunize mice 3 times. Neoantigen-specific T cell responses were quantified in the blood 5 days after the third immunization. n=6. Unpaired, two-tailed t-test. ns; not significant *p<0.5. ExtendedDataFigure3.pdf Extended Data Fig. 3. Neoantigen-MHC class I stability partially determines immunodominance a, pMHC affinity for each neoantigen was assessed by flow cytometric quantification of H2-Db or H2-Kb stabilization for both wild type (WT) or neoantigen mutant (MUT) peptides on TAP-deficient EL4 cells. Graph shows percent of maximum response. b, pMHC affinity for each neoantigen was assessed by flow cytometric quantification of H2-Kb stabilization of TAP-deficient EL4 cells. Percent of maximum response over a range of neoantigen concentrations is shown and the graph represents the calculated 1/EC50 for each specificity. A representative graph of three independent experiments is shown. c, pMHC complex stability (Koff) for each H2-Kb neoantigen was measured by differential scanning fluorimetry following thermal denaturation of soluble pMHC-I complexes. Melting curves were normalized to the minimum and maximum fluorescence values and Tm was calculated using the negative first derivative of RFU values over temperature. n=3.d, Differential scanning fluorimetry analysis of H-2Db/M16 (red) and H2-Db/M16-9L (blue) and buffer control (grey). Curves show the derivative of fluorescence over temperature, with dotted lines marking the melting temperature (Tm) on the x-axis. Each trace represents triplicate measurements. e, C57BL/6 mice were immunized four times on days 0, 7, 14 and 21 with either RNA-LPX monotope vaccines encoding a single neoantigen, decatope RNA-LPX vaccine encoding 10 neoantigens or RNA-LPX encoding F-luciferase was used as a control. Experimental design and flow cytometric quantification of CD8+ T cells with pMHCI-tetramer staining was performed in the blood 5 days after each immunization. Neoantigen specific CD8+ T cell responses as a percentage of total CD8+ T cells n=5. f, Neoantigen specific CD8+ T cell responses as a percentage of total CD8+ T cells is shown. Graph shows PD1 (gMFI) expression on neoantigen-specific CD8+ T cells=5. g, Naïve precursor CD8+ T cells (pCTL) were enriched from non-immunized mice and quantified. Graph shows the total number of pCTL for each specificity. Each dot represents an individual mouse n=5. h, pMHC complex stability (Koff) for each neoantigen was measured by differential scanning fluorimetry following thermal denaturation of soluble pMHC-I complexes. Melting curves were normalized to the minimum and maximum fluorescence values and Tm was calculated using the negative first derivative of RFU values over temperature. n=4 independent studies. i,j pMHC affinity for each neoantigen was assessed by flow cytometric quantification of H2-Db stabilization of TAP-deficient EL4 cells. Graph shows percent of maximum response and graph shows 1/EC50. ns not significant, *p<0.5, **p<0.01, ***p<0.001.<0.01, ***p<0.001. ExtendedDataFigure4.pdf Extended Data Fig. 4. a, C57BL/6 mice were immunized with RNA-LPX heptatope vaccine at four different concentrations, 0.4, 2, 10 or 50μg per mouse. In a second cohort, mice were immunized three times with the same four doses of heptatope RNA-LPX mixed with uncapped RNA to make a total RNA concentration of 50μg per mouse. Neoantigen-specific CD8+ T cell responses were quantified by flow cytometry in the spleen 5 days after the third immunization using pMHC-I tetramers. pMHC tetramer+ CD8+ T cells are shown as a percentage of total CD8+ T cells and the total number of neoantigen specific T cells (x106). n=5 mice.b, C57BL/6 mice were immunized once with either 30μg or 100μg of mScarlet RNA-LPX or N1psU-modified mScarlet RNA-LPX. CD11c+ DCs or a mixed population of monocytes and macrophages were examined in the spleen. Graphs show gMFI for activation markers.n=3-5. c, Mice were immunized once with either 30μg or 100μg of heptatope RNA-LPX or N1psU-modified hepatope RNA-LPX. Six hours later, mice were bled and serum cytokines and chemokines were measured by luminex. Heatmap is based on the median expression of n=10 individual mice. Welch’s anova. *p<0.5, **p<0.01, ***p<0.001, ****p<0.0001 ExtendedDataFigure5.pdf Extended Data Fig. 5. Clonal expansion tracks with preferential differentiation of subdominant CD8+ T cells into a memory-precursor and progenitor-like phenotype a, Cell count fraction of neoantigen-specific tetramer-positive T cells after heptatope and heptatope3WT RNA-LPX immunization, as quantified by FACS and analyzed through single-cell RNA/ADT/TCR sequencing. b, Distribution of clonal expansion across T cell phenotypes, shown as the fraction of cells with an assigned clonotype expansion: single (≤ 1 cell with clonotype), expanded (≤ 500 cells with a clonotype), hyperexpanded (> 500 cells with a clonotype). c, Clonal expansion of cells with a specific T cell phenotype and neoantigen-specificity represented as the fraction of cells in each clonotype expansion group, T cell phenotype, and neoantigen-specificity relative to the total number of cells with a given neoantigen-specificity and T cell phenotype in a mouse. d, Distribution of neoantigen-specific clonotypes across T cell phenotypes as heatmap with columns representing each specific clonotype of a neoantigen-specificity and rows representing the T cell phenotypes. Colors indicate the fraction of cells of a specific clonotype in a T cell phenotype relative to all cells of that clonotype and neoantigen-specificity. Clonotypes are ordered by increasing clone size, with a scatterplot above the heatmap illustrating the log10 clone size of each clonotype. ExtendedDataTable1.xlsx Supplementary Table 1 ExtendedDataTable2.xlsx Supplementary Table 2 Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8304459","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":569689796,"identity":"0fb887b5-c042-43f0-9be5-2e62b60744f7","order_by":0,"name":"Lélia 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Schartner","email":"","orcid":"","institution":"Genentech","correspondingAuthor":false,"prefix":"","firstName":"Jill","middleName":"","lastName":"Schartner","suffix":""}],"badges":[],"createdAt":"2025-12-08 06:56:55","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8304459/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8304459/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":105310752,"identity":"235baad8-66e0-490e-b4f2-ecf6d5fbe094","added_by":"auto","created_at":"2026-03-24 15:12:18","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":159014,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDistinct hierarchy of CD8+ T cell responses following multi-neoantigen RNA-LPX vaccination\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ea,b C57BL/6 mice were immunized three times on days 0, 7 and 14 with either 50μg of RNA-LPX monotope encoding a single neoantigen or 50μg heptatope RNA-LPX vaccine encoding all 7 neoantigens. On day 19, splenic lymphocytes were quantified by flow cytometry with pMHCI tetramers (specificities indicated on x-axis). a, Neoantigen-specific CD8+ T cell responses as a percentage of total CD8+ T cells and the total number of neoantigen-specific CD8+ T cells (x106) are shown (n=5 individual mice). b, Fold change in the percentage and total number of neoantigen-specific CD8+ T cells following single neoantigen or multi-neoantigen RNA-LPX immunization (monotype/heptatope) is also demonstrated. c, Neoantigen specific CD8+ T cell responses were longitudinally tracked in the peripheral blood following heptatope RNA-LPX immunization. pMHCI tetramer staining during the primary and memory recall responses is shown. Dotted lines indicate the immunization schedule. pMHCI tetramer-stained CD8+ T cells were quantified by flow cytometry and are shown as a percentage of total CD8+ T cells and total number of neoantigen specific T cells. Median of n=10 for primary response, and n=5 for memory response. d, pMHCI tetramer staining was used to quantify neoantigen-specific populations in the blood 5 days after one or two heptatope RNA-LPX immunizations (n=5). e, Naïve precursor CD8+ T cells (pCTL) were enriched from non-immunized mice and quantified. Graph shows the total number of pCTL for each specificity. Each dot represents an individual mouse (n=3). f, C57BL/6 mice were immunized on days 0, 7 and 14 with RNA-LPX monotype vaccines that each encoded a single neoantigen. Functional avidity was assessed by IFNg Elispot assay using titrated peptides. The neoantigen peptide concentration (EC50) at which T cells showed 50% of maximal activity was calculated (n=5). Each dot represents an individual mouse n=5. One-way anova. *p\u0026lt;0.5, **p\u0026lt;0.01, ***p\u0026lt;0.001, ****p\u0026lt;0.0001\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8304459/v1/4fd1aea80e2a17917dd7e84f.png"},{"id":105310740,"identity":"f6e2dde4-fc91-40ec-9baf-15938d005371","added_by":"auto","created_at":"2026-03-24 15:12:13","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":118593,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImmunodominance is mediated by T cell cross-competition\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ea-b, C57BL/6 mice were immunized three times on days 0, 7 and 14 with either RNA-LPX heptatope vaccine encoding encoding all 7 neoantigens or vaccine constructs in which a single neoantigen (Heptatope1WT) or three neoantigens (Heptatope3WT) were replaced by their non-immunogenic, non-neoantigen wild-type counterparts. Flow cytometric quantification of splenic CD8+ T cells was performed with pMHC tetramers (specificities indicated on x-axis) on day 19 (n=10-15). Fold change and statistical significance is denoted below the neoantigen name. Unpaired, two-tailed t-test. ns; not significant (P\u0026gt;0.5), *p\u0026lt;0.5, **p\u0026lt;0.01, ***p\u0026lt;0.001, ****p\u0026lt;0.0001. na; not applicable. c, Experimental design. d, C57BL/6 mice were immunized three times on days 0, 7 and 14 with heptatope3WT RNA-LPX. After a rest period of 40 days, mice were boost immunized with the RNA-LPX heptatope weekly over four weeks. pMHC-I tetramers were used to quantify neoantigen-specific populations in the blood 5 days after each immunization.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8304459/v1/ab7b9c8f2fe15c017db4b37c.png"},{"id":105310728,"identity":"ce6df84a-cbf7-448a-abb6-6ee7737f0ee8","added_by":"auto","created_at":"2026-03-24 15:12:11","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":118782,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNeoantigen-MHC class I stability partially determines immunodominance\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ea, C57BL/6 mice were immunized three times on days 0, 7 and 14. To assess competition for H2-Db loading, mice were immunized with either a non-competitive heptatope RNA-LPX vaccine construct encoding M10 and 6 copies of M152WT or a competitive construct encoding M10 and 6 copies of M16WT. To assess competition for H2-Kb loading, mice were immunized with either a non-competitive heptatope RNA-LPX vaccine construct encoding M172 with 6 copies of M16WT or a competitive construct encoding M172 with 6 copies of M152WT. Neoantigen-specific CD8+ T cell responses were measured using two independent assays, pMHCI tetramer+ cells as a percentage of total CD8+ T cells or IFNg spot count per 105 splenocytes. Immunization with RNA-LPX encoding F-luciferase was used as a control. n=5. b, pMHC affinity for each neoantigen was assessed by flow cytometric quantification of H2-Db stabilization on TAP-deficient EL4 cells. Percent of maximum response over a range of neoantigen concentrations is shown and the graph represents the calculated 1/EC50 for each specificity. Data is representative of three individual experiments. c, pMHC complex stability (Koff) for each H2-Db neoantigen was measured by differential scanning fluorimetry following thermal denaturation of soluble pMHC-I complexes. Melting curves were normalized to the minimum and maximum fluorescence values and Tm was calculated using the negative first derivative of RFU values over temperature. n=3 independent studies. d-e, C57BL/6 mice were immunized three times on days 0, 7 and 14 with either 50μg of RNA-LPX monotope encoding a single neoantigen (M16 or M16-9L) or 50μg heptatope RNA-LPX vaccine encoding either the M16 neoantigen (heptatope (M16) or a mutated M16 variant (Heptatope (M16-9L) with the other 6 neoantigens.. d, On day 19, splenic lymphocytes were quantified by flow cytometry with pMHCI tetramers. Neoantigen-specific CD8+ T cell responses as a percentage of total CD8+ T cells and the total number of neoantigen-specific CD8+ T cells are shown (n=5-10 individual mice). e, Neoantigen-specific CD8+ T cell responses following were measured using IFNg spot count per 105 splenocytes. The dotted line indicates the maximum number of spots that can be counted. Immunization with RNA-LPX encoding F-luciferase was used as a control. Unpaired, two-tailed t-test or two-way anova. ns; not significant (P\u0026gt;0.5), **p\u0026lt;0.01, ***p\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8304459/v1/95a077bb539131c81f2599b1.png"},{"id":105310743,"identity":"cfc8c4ff-2fea-42ea-a694-c52de353a7aa","added_by":"auto","created_at":"2026-03-24 15:12:14","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":182164,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIncreasing antigen expression can mitigate immunodominance hierarchies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ea, C57BL/6 mice were immunized with 2, 10 or 50μg of RNA-LPX vaccine encoding GFP and DCs were evaluated in the spleen 18 h after immunization. DCs that were positive for GFP, indicating they had taken up the mRNA LPX complex were quantified by flow cytometry. The percent and MFI of the activation marker CD80 on CD11c+MHC-II+ DCs is also shown. n=5 per group. b,c C57BL/6 mice were immunized with RNA-LPX heptatope vaccine at four different concentrations, 0.4, 2, 10 or 50μg per mouse. In a second cohort, mice were immunized three times with the same four doses of heptatope RNA-LPX mixed with uncapped RNA to make a total RNA concentration of 50μg per mouse. Neoantigen-specific CD8+ T cell responses were quantified by flow cytometry in the spleen 5 days after the third immunization using pMHC-I tetramers. pMHC tetramer+ CD8+ T cells are shown as a percentage of total CD8+ T cells and the total number of neoantigen specific T cells (x106). n=5 mice. d,e C57BL/6 mice were immunized once with either 30μg or 100μg of mScarlet RNA-LPX or N1psU-modified mScarlet RNA-LPX. Mice were euthanized 17 or 40 hours after immunization and CD11c+ cDCs or a mixed population of monocytes and macrophages were examined in the spleen. n=3-5 d, Cells expressing mScarlet protein were quantified by flow cytometry. Both the percentage and total number of scarlet + cells in the spleen are shown. e, Graphs show gMFI for activation markers. f, C57BL/6 mice were immunized weekly for 4 weeks with 30μg or 100μg of either heptatope RNA-LPX or N1psU-modified hepatope RNA-LPX. Flow cytometric quantification of CD8+ T cells with pMHC tetramers was performed in the blood and spleen 4 days after the last immunization. Neoantigen-specific CD8+ T cell responses as a percentage of total CD8+ T cells and the total number of neoantigen-specific CD8+ T cells (x106) are shown (n=10 blood and n=5 spleen). Each dot represents an individual mouse. Welch’s anova. *p\u0026lt;0.5, **p\u0026lt;0.01, ***p\u0026lt;0.001, ****p\u0026lt;0.0001\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8304459/v1/3d6ff359c97c68cbfbd603fe.png"},{"id":105310781,"identity":"196466e3-1efe-4ebe-a064-a7afdee5e919","added_by":"auto","created_at":"2026-03-24 15:12:22","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":283291,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eT cell cross-competition results in sub-dominant CD8+ T cells adopting a memory-precursor and stem-like phenotype\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ea, Experimental design for single-cell RNA, ADT, and TCR sequencing of heptatope RNA-LPX and heptatope3WT RNA-LPX vaccinated mice. Mice received four weekly vaccinations, followed by isolation and pMHC tetramer labeling of splenic CD8+ T cells for single-cell library preparation. n-5 mice per group. b, Clonal expansion beeswarm plot of neoantigen-specific T cell clones post-vaccination with heptatope RNA-LPX and heptatope3WT RNA-LPX. Each dot represents a unique clonotype based on nucleotide sequence. Clonotypes with a single count have been excluded. c, UMAP visualization of splenic tetramer-positive T cells post-heptatope and heptatope3WT immunization, colored by T cell phenotype. Dotplot shows z-scored expression of selected T cell phenotype markers, dot size denotes percentage of cells with gene expression. d, Proportion of cells in each T cell phenotype relative to total cells per mouse. e, Clonal interactions among T cell phenotypes for dominant (M16, M2) and subdominant (M10, M152) neoantigen-specific T cells post-Heptatope immunization. Boxplot displays distribution of T cell phenotype fractions across mice, and link widths indicate shared clonotypes weighted by clone size. f, Differentially expressed genes between T_Gzma_Klrg1 M16, M2 cells and T_Gzma_Klf2 M10, M152 cells after heptatope immunization, identified by pseudobulk differential expression analysis. Colors represent scaled expression values, with hierarchical clustering defining row and column order within the comparison groups. g, C57BL/6 mice were immunized three times on days 0, 7 and 14 with heptatope RNA-LPX vaccine encoding all 7 neoantigens. Flow cytometric quantification of CD127 and KLRG1 expression on neoantigen-specific CD8+ T cells from each specificity (indicated on x-axis) in the spleen on day 19 is shown. The ratio of SLEC (CD127-KLRG1+) to MPEC (CD127+KLRG1-) is shown for each neoantigen specificity. n=5 mice per grooup. h-i, Proportion of cells in each T cell phenotype relative to total cells per mouse and clonal interactions among T cell phenotypes for M10, M152 neoantigen-specific T cells post-heptatope3WT immunization\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8304459/v1/cf17339326bb0ce35973c9e7.png"},{"id":105310928,"identity":"4d06fc54-ad71-4537-9120-1a4906fbc587","added_by":"auto","created_at":"2026-03-24 15:13:04","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1253830,"visible":true,"origin":"","legend":"Article File","description":"","filename":"McCarronManuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8304459/v1_covered_954fd935-c6f3-4225-a7ab-17826a522191.pdf"},{"id":105310749,"identity":"2e889928-a638-4905-ae3b-8b6a2f971ac5","added_by":"auto","created_at":"2026-03-24 15:12:15","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":707498,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExtended Data Fig. 1. Distinct hierarchy of CD8+ T cell responses following multi-neoantigen RNA-LPX vaccination\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ea, C57BL/6 mice were immunized three times on days 0, 7 and 14 with heptatope RNA-LPX vaccine encoding all 7 neoantigens. Splenic T cells were harvested 5 days after the last immunization and restimulated with 27 mer peptides containing the neoantigen or non-peptide DMSO solvent controls. The magnitude of cytokine and CD107a responses were measured by flow cytometry and are shown as a percentage of total CD8+ T cells. Neoantigen specificities are indicated on the x-axis. n=3-5. b, As depicted, three different heptatope constructs were generated encoding 7 neoantigens in which the neoantigen position was varied across constructs. C57BL/6 mice were immunized three times on days 0, 7 and 14 and splenic T cells were harvested 5 days after the last immunization. Flow cytometric quantification of CD8+ T cells stained with pMHCI tetramers (specificities indicated on x-axis) was performed. n=5. c, Mice were immunized with monotope RNA-LPX vaccines encoding M16 or M10 alone or both monotype RNA-LPX vaccines were combined. Flow cytometric quantification of CD8+ T cells stained with pMHCI tetramers was performed in the blood 5 days after the third immunization. Clonal expansion was analyzed. n=5. d, Table depicts neoantigen and non-neoantigen wild type sequences of Nsun2 and Arvcf peptides identified in B16F10 tumors. e, Mice were immunized with monotope RNA-LPX vaccines encoding Nsun2, Arvcf or SIINFEKL in various combinations as depicted. Following three immunizations, splenocytes were assessed by IFNg ELISpot assay following peptide restimulation. One-way anova or unpaired t-test. ns not significant, *p\u0026lt;0.5, **p\u0026lt;0.01, ***p\u0026lt;0.001.\u0026lt;0.01, ***p\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"ExtendedDataFigure1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8304459/v1/603d160f8bdf8ce81b3aede7.pdf"},{"id":105310741,"identity":"6d7fa2c5-00a1-4c73-9b2e-d53be5f70047","added_by":"auto","created_at":"2026-03-24 15:12:13","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":674481,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExtended Data Fig. 2. T cell competition is not mediated by the limited bioavailability of IL-2 or limitations in CD4+ T cell help\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ea, C57BL/6 mice were immunized with heptatope RNA-LPX in the presence and absence of exogenous IL-2 (15,000 units I.P daily). Following three immunizations, spleens were removed and neoantigen-specific T cell responses were evaluated by pMHC-I tetramer staining and IFN-gamma ELISpot. Data are shown as pMHCI tetramer+ cells as a percentage of total CD8+ T cells, total number of neoantigen-specific CD8+ T cells or IFNɣ spot count per 105 splenocytes. n=10.b Mice were immunized with monotype vaccines encoding M16 and SIINFEKL, either alone or mixed. Mice immunized with the M16 monotype or a mixture of M16 + SIINFEKL received half-life extended human IL-2 delivered in LNP (3ug) intravenously. M16 or SIINFEKL neoantigen-specific T cell responses were evaluated in the spleen 7 days after the last immunization using pMHCI tetramer+ staining and are shown as a percentage of total CD8+ T cells. c-e, the heptatope RNA-LPX vaccine was modified to include 2W1S (“octatope”), or a non-immunogenic control antigen. Mice were immunized three times and neoantigen-specific T cell responses were evaluated in the spleen 5 days after the last immunization. c, the 2W1S CD4+ T cell response was confirmed by pMHC-II tetramer staining and production of IFNg after ex vivo restimulation of spleen cells by 2W1S peptide n=10. d, Neoantigen-specific CD8+ T cell responses were quantified in the spleen by pMHC tetramer+ staining in the presence and absence of the 2W1S CD4+ T cell responses. Data are shown as pMHCI tetramer+ cells as a percentage of total CD8+ T cells and total number of neoantigen-specific CD8+ T cells n=4-7. e, We generated a novel heptatope consisting of 6 neoantigens from the MC38 model that generate CD4+ T cell responses and a seventh CD4+ T cell antigen, 2W1S. Mice were immunized three times and neoantigen-specific CD8+ T cell responses were evaluated in the spleen 5 days later. Neoantigen-specific CD8+ T cell responses were quantified by IFNɣ ELISpot and are shown as IFNg spots per 105 splenocytes n=7. f, RNA-LPX monotype vaccines encoding either SIINFEKL or the M30 neoantigen, or both monotope vaccines combined were used to immunize mice 3 times. Neoantigen-specific T cell responses were quantified in the blood 5 days after the third immunization. n=6. Unpaired, two-tailed t-test. ns; not significant *p\u0026lt;0.5.\u003c/p\u003e","description":"","filename":"ExtendedDataFigure2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8304459/v1/c2c548e3e43a4c62c894710a.pdf"},{"id":105310745,"identity":"91f58a19-c963-46ce-92ca-cd27f09c39d0","added_by":"auto","created_at":"2026-03-24 15:12:14","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":817619,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExtended Data Fig. 3. Neoantigen-MHC class I stability partially determines immunodominance\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ea, pMHC affinity for each neoantigen was assessed by flow cytometric quantification of H2-Db or H2-Kb stabilization for both wild type (WT) or neoantigen mutant (MUT) peptides on TAP-deficient EL4 cells. Graph shows percent of maximum response. b, pMHC affinity for each neoantigen was assessed by flow cytometric quantification of H2-Kb stabilization of TAP-deficient EL4 cells. Percent of maximum response over a range of neoantigen concentrations is shown and the graph represents the calculated 1/EC50 for each specificity. A representative graph of three independent experiments is shown. c, pMHC complex stability (Koff) for each H2-Kb neoantigen was measured by differential scanning fluorimetry following thermal denaturation of soluble pMHC-I complexes. Melting curves were normalized to the minimum and maximum fluorescence values and Tm was calculated using the negative first derivative of RFU values over temperature. n=3.d, Differential scanning fluorimetry analysis of H-2Db/M16 (red) and H2-Db/M16-9L (blue) and buffer control (grey). Curves show the derivative of fluorescence over temperature, with dotted lines marking the melting temperature (Tm) on the x-axis. Each trace represents triplicate measurements. e, C57BL/6 mice were immunized four times on days 0, 7, 14 and 21 with either RNA-LPX monotope vaccines encoding a single neoantigen, decatope RNA-LPX vaccine encoding 10 neoantigens or RNA-LPX encoding F-luciferase was used as a control. Experimental design and flow cytometric quantification of CD8+ T cells with pMHCI-tetramer staining was performed in the blood 5 days after each immunization. Neoantigen specific CD8+ T cell responses as a percentage of total CD8+ T cells n=5. f, Neoantigen specific CD8+ T cell responses as a percentage of total CD8+ T cells is shown. Graph shows PD1 (gMFI) expression on neoantigen-specific CD8+ T cells=5. g, Naïve precursor CD8+ T cells (pCTL) were enriched from non-immunized mice and quantified. Graph shows the total number of pCTL for each specificity. Each dot represents an individual mouse n=5. h, pMHC complex stability (Koff) for each neoantigen was measured by differential scanning fluorimetry following thermal denaturation of soluble pMHC-I complexes. Melting curves were normalized to the minimum and maximum fluorescence values and Tm was calculated using the negative first derivative of RFU values over temperature. n=4 independent studies. i,j pMHC affinity for each neoantigen was assessed by flow cytometric quantification of H2-Db stabilization of TAP-deficient EL4 cells. Graph shows percent of maximum response and graph shows 1/EC50. ns not significant, *p\u0026lt;0.5, **p\u0026lt;0.01, ***p\u0026lt;0.001.\u0026lt;0.01, ***p\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"ExtendedDataFigure3.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8304459/v1/603ea45c9ef91a1418b09da9.pdf"},{"id":105310780,"identity":"26242a55-0b2b-4f10-8468-15aae7754b62","added_by":"auto","created_at":"2026-03-24 15:12:22","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":774204,"visible":true,"origin":"","legend":"\u003cp\u003eExtended Data Fig. 4.\u003c/p\u003e\n\u003cp\u003ea, C57BL/6 mice were immunized with RNA-LPX heptatope vaccine at four different concentrations, 0.4, 2, 10 or 50μg per mouse. In a second cohort, mice were immunized three times with the same four doses of heptatope RNA-LPX mixed with uncapped RNA to make a total RNA concentration of 50μg per mouse. Neoantigen-specific CD8+ T cell responses were quantified by flow cytometry in the spleen 5 days after the third immunization using pMHC-I tetramers. pMHC tetramer+ CD8+ T cells are shown as a percentage of total CD8+ T cells and the total number of neoantigen specific T cells (x106). n=5 mice.b, C57BL/6 mice were immunized once with either 30μg or 100μg of mScarlet RNA-LPX or N1psU-modified mScarlet RNA-LPX. CD11c+ DCs or a mixed population of monocytes and macrophages were examined in the spleen. Graphs show gMFI for activation markers.n=3-5. c, Mice were immunized once with either 30μg or 100μg of heptatope RNA-LPX or N1psU-modified hepatope RNA-LPX. Six hours later, mice were bled and serum cytokines and chemokines were measured by luminex. Heatmap is based on the median expression of n=10 individual mice. Welch’s anova. *p\u0026lt;0.5, **p\u0026lt;0.01, ***p\u0026lt;0.001, ****p\u0026lt;0.0001\u003c/p\u003e","description":"","filename":"ExtendedDataFigure4.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8304459/v1/08923d5ed6aed21553bd7c95.pdf"},{"id":105310821,"identity":"06215d47-46ba-462f-8b29-4dd4b36cbc24","added_by":"auto","created_at":"2026-03-24 15:12:34","extension":"pdf","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":492285,"visible":true,"origin":"","legend":"\u003cp\u003eExtended Data Fig. 5. Clonal expansion tracks with preferential differentiation of subdominant CD8+ T cells into a memory-precursor and progenitor-like phenotype\u003c/p\u003e\n\u003cp\u003ea, Cell count fraction of neoantigen-specific tetramer-positive T cells after heptatope and heptatope3WT RNA-LPX immunization, as quantified by FACS and analyzed through single-cell RNA/ADT/TCR sequencing. b, Distribution of clonal expansion across T cell phenotypes, shown as the fraction of cells with an assigned clonotype expansion: single (≤ 1 cell with clonotype), expanded (≤ 500 cells with a clonotype), hyperexpanded (\u0026gt; 500 cells with a clonotype). c, Clonal expansion of cells with a specific T cell phenotype and neoantigen-specificity represented as the fraction of cells in each clonotype expansion group, T cell phenotype, and neoantigen-specificity relative to the total number of cells with a given neoantigen-specificity and T cell phenotype in a mouse. d, Distribution of neoantigen-specific clonotypes across T cell phenotypes as heatmap with columns representing each specific clonotype of a neoantigen-specificity and rows\u003c/p\u003e\n\u003cp\u003erepresenting the T cell phenotypes. Colors indicate the fraction of cells of a specific clonotype in a T cell phenotype relative to all cells of that clonotype and neoantigen-specificity. Clonotypes are ordered by increasing clone size, with a scatterplot above the heatmap illustrating the log10 clone size of each clonotype.\u003c/p\u003e","description":"","filename":"ExtendedDataFigure5.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8304459/v1/87c098f559de5dc8590964f3.pdf"},{"id":105310760,"identity":"a4b1e063-5df0-4571-832a-00b0ade287d3","added_by":"auto","created_at":"2026-03-24 15:12:18","extension":"xlsx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":1101164,"visible":true,"origin":"","legend":"Supplementary Table 1","description":"","filename":"ExtendedDataTable1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8304459/v1/f1c235dd601dd66659176f32.xlsx"},{"id":105310733,"identity":"d079174a-d58f-48ad-a3aa-fc61255cd066","added_by":"auto","created_at":"2026-03-24 15:12:11","extension":"xlsx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":754373,"visible":true,"origin":"","legend":"Supplementary Table 2","description":"","filename":"ExtendedDataTable2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8304459/v1/10868105482392e04ef0326c.xlsx"}],"financialInterests":"\u003cb\u003eYes\u003c/b\u003e there is potential Competing Interest.\nAll the authors were employees of Genentech at the time of the study, but DE, MV, LK and US who were employees of BioNTech","formattedTitle":"Cross-competition shapes CD8+ T cell hierarchies and fate after cancer neoantigen RNA vaccination","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-8304459/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8304459/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Cancer patients typically mount T cell responses to only a small fraction of the neoantigens encoded in their individualized vaccine. Because each patient’s neoantigen set is unique, clinical studies cannot determine whether this restricted breadth reflects limitations of neoantigen prediction algorithms or intrinsic immunological constraints. Here, using a clinically relevant multi-neoantigen RNA–lipoplex vaccine in a preclinical model, we mechanistically dissected CD8+ T-cell breadth. We found that concurrently primed neoantigen-specific CD8+ T cells competed through a peptide-MHC-I-dependent cross-competition, with dominant responses suppressing both the magnitude and differentiation of subdominant responses. Dominant CD8+ T cell responses preferentially acquired a terminally differentiated effector phenotype, while sub-dominant responses adopted memory-precursor and stem-like features. Removing dominant responses enabled sub-dominant responses to expand and adopt terminally differentiated effector phenotypes. These findings provide the first mechanistic explanation for restricted CD8+ T cell breadth in multi-neoantigen vaccination and provide a framework for further optimizing cancer vaccine design.","manuscriptTitle":"Cross-competition shapes CD8+ T cell hierarchies and fate after cancer neoantigen RNA vaccination","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-24 15:09:19","doi":"10.21203/rs.3.rs-8304459/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-cancer","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"natcancer","sideBox":"Learn more about [Nature Cancer](http://www.nature.com/natcancer/)","snPcode":"","submissionUrl":"","title":"Nature Cancer","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Research","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"b2129b72-845d-4d1f-995f-3fcae6487dde","owner":[],"postedDate":"March 24th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":60616118,"name":"Biological sciences/Immunology/Adaptive immunity/Cellular immunity/Lymphocyte activation"},{"id":60616119,"name":"Biological sciences/Cancer/Tumour immunology"},{"id":60616120,"name":"Biological sciences/Immunology/Antigen processing and presentation/Cellular immunity"}],"tags":[],"updatedAt":"2026-03-24T15:09:22+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-24 15:09:19","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8304459","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8304459","identity":"rs-8304459","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}