CAR T cells and T cells phenotype and function are impacted by glucocorticoid exposure with different amplitude.

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Thomas Poiret, Sara Vikberg, Esther Schoutrop, Jonas Mattsson, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3366592/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 12 Mar, 2024 Read the published version in Journal of Translational Medicine → Version 1 posted 4 You are reading this latest preprint version Abstract Background. Chimeric antigen receptor (CAR) T cell therapy is associated with high risk of adverse events. Glucocorticoids (GCs) are cornerstone in the management of high-grade cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS). Given the potentially deleterious effects of GCs on CAR T cells anti-tumor activity, increasing our understanding of GCs impact on CAR T cells is crucial. Methods. Using several CAR T cells i.e., CD19, mesothelin (MSLN)-CD28 and MSLN-41BB CAR T cells (M28z and MBBz), we compared phenotypical, functional, changes and anti-tumor activity between i) transduced CD19 CAR T cells with untransduced T cells, ii) M28z with MBBz CAR T cells induced by Dexamethasone (Dx) or Methylprednisolone (MP) exposures. Results. Higher levels of GC receptor were found in less differentiated CAR T cells. Overall, Dx and MP showed a similar impact on CAR T cells. GC exposures induced more exhausted (LAG3+PD1+TIM3+) and dysfunctional (CD107a-INFγ-TNF-IL2-) untransduced T cells in comparison to CD19 CAR T cells. GC exposure impaired more CD4+ than CD8+ CD19 CAR T cells. GC exposures increased more PD-1 expression associated with reduced proliferative capacity and function of M28z as compared to MBBz CAR T cells. CAR T cells anti-tumor activity was greatly affected by repeated GC exposure but partly recovered within 48h after GCs withdrawal. Conclusions. In summary, GCs impacted phenotype and function of untransduced and CAR T cell with different amplitude. The nature of the CAR costimulatory domain influenced the amplitude of CAR T cell response to GCs. glucocorticoid CAR chimeric antigen receptor CD19 mesothelin 4-1-BB CD28 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Full Text Supplementary Files TPoiretsuppl.pdf TPoiretFiguressuppl.pdf Supplementary Figure Legends. Supplementary Figure 1. UT T cells phenotype. A. Representative plot of the differentiation subsets defined by CCR7 and CD45RA markers. B. Memory phenotype of CAR+ and untransduced (UT) fractions of CD3+ T cells in CD19, M28z and MBBz CAR T cell products.GR expression (MFI) in the naïve and TCM memory subsets of (C) UT (from CD19 CAR cell product) CD4+ and CD8+ T cells; (D) of the UT (from M28z and MBBz CAR cell products) CD4+ (left) and CD8+ (right) T cells. n=5 for CD19 CAR T cells and n=4 for M28z and MBBz CAR T cells. Student t test was used to compare GR in different subsets. Medians are represented. *p<0.05, **p<0.01. Supplementary Figure 2. Impact of GC exposure on UT T cells phenotype. Frequency of CD19 CAR T cells (A) and CD4/CD8 ratio in CD19 CAR T cells (B) overtime after Dx (green) or MP (blue) exposure. C. Frequency of LAG-3, PD-1, and TIM-3 in CD19 CAR+ T cells without or after 1 and 3 exposures with Dx or MP. Friedman test with Dunn’s correction was used to compare marker’s expression in No GC vs. Dx vs. MP conditions. Relative surface expression of LAG-3, PD-1, and TIM-3 in CAR+ and UT CD4+ (D) and CD8+ (E) T cells after 3 exposures with Dx (left) or MP (right). F. Relative expression of LAG-3, PD-1, and TIM-3 in CD4+ and CD8+ UT T cells after 3 exposures of Dx (left) or MP (right).n=6. Wilcoxon matched-pairs signed rank test was used to compare marker’s expression in CAR+ vs. UT or CD4+ vs. CD8+ T cells. Medians are represented. * p<0.05, ** p<0.01, *** p<0.001. Supplementary Figure 3. Impact of GCs exposure on CD19 CAR+ T cells effector functions. A. Relative frequency of IFNγ+, TNF+ or IL2+ CD4+ or CD8+ CD19 CAR and UT after a single exposure with Dx (top) or MP (bottom) after PMA/ionomycin stimulation. Frequency of CD107a+, IFNγ+, TNF and IL-2+ CAR CD4+ and CD8+ T cells without GC exposure 1 exposure with low or high doses of GC (0.1 µg/ml and 10 µg/ml Dx and 0.5µg/ml, 50 µg/ml, and 100 µg/ml MP) following a single (B) or 3 (C) stimulations with CD19+ K562 tumor cells. D. Pie chart showing the number of functions of CD8+ CAR T cells without GC exposure compared to Dx or MP exposure following a single or 3 stimulations with CD19+ K562 tumor cells stimulation. n=8. Two-way ANOVA with Sidak’s correction was used to compare CD107a+, IFNγ+, TNF and IL-2+ cells frequency at different doses of Dx and MP. Friedman test with Dunn’s correction was used to compare multifunction in No GC vs. Dx vs. MP conditions. Medians are represented. * p<0.05, ** p<0.01, *** p<0.001. Supplementary Figure 4. Impact of GCs exposure on M28z and MBBz CAR+ T cells phenotype and functions. Frequency of M28z and MBBz CAR T cells (A) and CD4/CD8 ratio in CAR T cells (B) without GC exposure or after 3 exposures with low and high concentration of Dx or MP. Frequency of LAG-3 (C), PD-1 (D) and TIM-3 (E) in M28z and MBBz CAR T cells without GC exposure or after 3 exposures with low and high concentration of Dx or MP. Relative surface expression of LAG-3, PD-1 and TIM-3 in CAR+ and UT fractions of CD8+ M28z and MBBz T cells following 3 exposures with Dx (F) or MP (G). Frequency of CD107a+, IFNγ+, TNF and IL-2+ CD4+ (H) and CD8+ (I) M28z and MBBz CAR T cells without GC exposure compared to Dx or MP exposure following 3 stimulations with MSLN+ K562 tumor cells. A-E n=6, F&G n=11 and H&I n=5. Two-way ANNOVA with Sidak’s correction was used to compare CD107a+, IFNγ+, TNF and IL-2+ cells frequency at different doses of Dx and MP. Mann-Whitney test was used to the surface expressions and functions in M28z vs. MBBz. Medians are represented. * p<0.05, ** p<0.01, *** p<0.001. Supplementary Figure 5. M28z and MBBz CD4+ and CD8+ CAR T cells phenotype after GC removal. Relative frequency of LAG-3, PD-1 and TIM-3 in M28z and MBBz CD4+ (A&C) and CD8+ (B&D) CAR T cells after 3 exposures with Dx (A-B) or MP (C-D) and 48h rest in a GC-free medium. n=5. Student t test was used to compare surface marker expression in GC-exposed vs. GC-rested conditions. * p<0.05, ** p<0.01, *** p<0.001. Cite Share Download PDF Status: Published Journal Publication published 12 Mar, 2024 Read the published version in Journal of Translational Medicine → Version 1 posted Reviewers agreed at journal 04 Oct, 2023 Reviewers invited by journal 04 Oct, 2023 Editor assigned by journal 21 Sep, 2023 First submitted to journal 19 Sep, 2023 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-3366592","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":237868843,"identity":"58723f87-f7cf-45fd-a995-e6d8bbb2ca2b","order_by":0,"name":"Thomas Poiret","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2UlEQVRIiWNgGAWjYDACCRiDGURUJPAQ1MGDouXAGZK0gMDBtgTC7rKXbj664ccfhjx+dt6Hjz/OS5Nh4D98AL8tMsfSbva2MRRLNrMbGxzclsPDIJGG3yoeiRyzG7wNDIkbDrOxSRzcVgHUwmNAUMvNP3/AWth/HJwD1MJ//gNBLbd52CC2MBxsADqMIQevDgaeG2lpt2XbJIB+YWOWOHMsjYdNIg2/w9hnJB+7+eaPTR4//zHGDxU1yfb8/Icf4LcGAiQS4Ew2YtSDQAJBFaNgFIyCUTByAQCoPz9iN21mwAAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0003-1965-3522","institution":"Karolinska Institutet","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Thomas","middleName":"","lastName":"Poiret","suffix":""},{"id":237868844,"identity":"ffa90049-056e-40e6-929a-d790f6b6a294","order_by":1,"name":"Sara Vikberg","email":"","orcid":"","institution":"Karolinska Institutet","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Sara","middleName":"","lastName":"Vikberg","suffix":""},{"id":237868845,"identity":"11cba115-57a2-4a23-acff-da766e248f74","order_by":2,"name":"Esther Schoutrop","email":"","orcid":"","institution":"Karolinska Institutet","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Esther","middleName":"","lastName":"Schoutrop","suffix":""},{"id":237868846,"identity":"75fed901-5ffd-4b3e-8689-8a3d0f7679ad","order_by":3,"name":"Jonas Mattsson","email":"","orcid":"","institution":"Karolinska Institutet","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jonas","middleName":"","lastName":"Mattsson","suffix":""},{"id":237868847,"identity":"766ca3f6-bb90-4135-8439-a6fbe306bb9b","order_by":4,"name":"Isabelle Magalhaes","email":"","orcid":"","institution":"Karolinska Institutet","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Isabelle","middleName":"","lastName":"Magalhaes","suffix":""}],"badges":[],"createdAt":"2023-09-18 15:14:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3366592/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3366592/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12967-024-05063-4","type":"published","date":"2024-03-12T15:01:15+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":49546605,"identity":"e7d4aa3c-2a9f-4bfe-9324-1b729811fa77","added_by":"auto","created_at":"2024-01-12 19:03:40","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":67159,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhenotype of CD3+ T cells in CD19, M28z and MBBz CAR T cell products. A.\u003c/strong\u003e Differentiation subsets as defined by CD45RA and CCR7 expression \u003cstrong\u003eB.\u003c/strong\u003e Representative histogram of glucocorticoid receptor (GR) staining in CD19, M28z and MBBz CAR T cell product in comparison with isotype control staining. \u003cstrong\u003eC.\u003c/strong\u003e GR expression in CD3+ T cell subsets of CD19, M28z and MBBz CAR products. \u003cstrong\u003eD.\u003c/strong\u003e GR expression in the naïve and central memory (TCM) subsets of CD19 CAR+ and UT of CD4+ (left) and CD8+ (right) T cells. \u003cstrong\u003eE.\u003c/strong\u003e Comparison of GR expression between CD4+ and CD8+ CD19 CAR T cells in the naïve and TCM memory subsets. \u003cstrong\u003eF.\u003c/strong\u003e Comparison of GR expression between M28z and MBBz CAR T cells in the CD4+ (left) and CD8+ (right) naïve and TCM memory subsets. MFI: median fluorescence intensity. n=5 for CD19 CAR T cells and n=4 for M28z and MBBz CAR T cells. Friedman test was used to compare subsets within paired samples, Student t test was used to compare GR expression in different subsets. Medians are represented. *p\u0026lt;0.05, **p\u0026lt;0.01.\u0026nbsp;\u003c/p\u003e","description":"","filename":"fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-3366592/v1/18e8b6581a05e4f14a9dc0a1.png"},{"id":49546607,"identity":"57eeefab-f9bc-40ce-9b09-812dd2cea5e5","added_by":"auto","created_at":"2024-01-12 19:03:50","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":65089,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImpact of GCs exposure on untransduced (UT) and CD19 CAR+ T cells phenotype. A.\u003c/strong\u003e Viability of K562 tumor cells (n=3) and T cells (n=6) after 72h of exposure with different concentration of Dexamethasone (Dx, green) or Methylprednisolone (MP, blue). 2-way ANOVA with Sidak’s correction was used to do multiple comparisons between viability of cells exposed to different Dx and MP concentration. \u003cstrong\u003eB.\u003c/strong\u003e Experimental design. Relative surface expression of LAG-3, PD-1 and TIM-3 in CAR+ and UT T cells after one (1x, \u003cstrong\u003eC\u003c/strong\u003e) or 3 (3x, \u003cstrong\u003eD\u003c/strong\u003e) exposures with Dx (left) or MP (right). \u003cstrong\u003eE.\u003c/strong\u003e Relative expression of LAG-3, PD-1 and TIM-3 in CD4+ and CD8+ CAR+ T cells after 3 exposures of Dx (left) or MP (right). \u003cstrong\u003eF.\u003c/strong\u003e Comparison of LAG3+PD1+TIM3+ frequency between CD4+ and CD8+ CAR+ T cells without GC or after 3 exposures with Dx or MP. \u003cstrong\u003eC-F\u003c/strong\u003e, n=6. Wilcoxon matched-pairs signed rank test was used to compare expressions in CAR+ vs. UT or CD4+ vs. CD8+ T cells. Medians are represented. * p\u0026lt;0.05, ***p\u0026lt;0.001, ###p\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-3366592/v1/807a4e7533e494a651ddd992.png"},{"id":49546610,"identity":"0c2626b0-acf4-44dc-8d51-d7ea3d4db8e4","added_by":"auto","created_at":"2024-01-12 19:03:56","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":86621,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImpact of GCs exposure on CD19 CAR+ T cells effector functions. A. \u003c/strong\u003eRelative frequency of CD107a+ CD4+ (top) or CD8+ (bottom) CD19 CAR and UT T cells after one (1x) or 3 (3x) exposures with Dx or MP after PMA/ionomycin stimulation. \u003cstrong\u003eB.\u003c/strong\u003e Radar plot of relative frequency of IFNγ+, TNF+ or IL2+ CD4+ or CD8+ CD19 CAR and UT T cells after 3 exposures with Dx (top) or MP (bottom) after PMA/ionomycin stimulation. \u003cstrong\u003eC.\u003c/strong\u003e Relative frequency of CD107a- IFNγ- TNF- IL2- CD4+ and CD8+ CAR and UT T cells after 3 exposures with Dx or MP after PMA/ionomycin stimulation. \u003cstrong\u003eD.\u003c/strong\u003e Pie chart showing the number of functions of CD4+ CAR T stimulated 2 times with MSLN+ K562 tumor cells and then exposed or not to Dx or MP before the last (3rd) stimulation (stim) with CD19+ K562 tumor cells. \u003cstrong\u003eE.\u003c/strong\u003e Comparison of the relative frequency of CD107a- IFNγ- TNF- IL2- CD4 and CD8 CD19 CAR T cells exposed to Dx or MP following a single and 3 stimulations with CD19+ K562 tumor cells. \u003cstrong\u003eA-C\u003c/strong\u003e n=6 and \u003cstrong\u003eD-E\u003c/strong\u003e n=5. 2-way ANOVA with Sidak’s correction was used to compare CD107a+ cells frequency at different doses of Dx and MP. Mann-Whitney test was used to compare the functions in CAR vs. UT or 1x vs 3x. Friedman test with Dunn’s correction was used to compare multifunction in No GC vs. Dx vs. MP conditions. Student t test was used to compare CD107a expression between 1x vs. 3x (#) and CD107a- IFNγ- TNF- IL2- CD4 and CD8 relative frequency between CD4+ and CD8+ T cells. Medians are represented. * p\u0026lt;0.05, ** p\u0026lt;0.01, *** p\u0026lt;0.001. # p\u0026lt;0.05, ## p\u0026lt;0.01, ### p\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-3366592/v1/71a81886faf25bf942faf88c.png"},{"id":49546615,"identity":"2a627e1b-7d55-4b21-a28c-0e06cce6e34b","added_by":"auto","created_at":"2024-01-12 19:04:05","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":133220,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImpact of GCs exposure on M28z and MBBz CAR+ T cells phenotype and functions.\u003c/strong\u003e Relative surface expression of LAG-3, PD-1 and TIM-3 in CAR+ and UT fractions of CD4+ M28z and MBBz T cells following 3 exposures with Dx (\u003cstrong\u003eA\u003c/strong\u003e) or MP (\u003cstrong\u003eB\u003c/strong\u003e). \u003cstrong\u003eC.\u003c/strong\u003e Representative histogram of M28z and MBBz CAR T cells proliferation after 3 exposures with or without Dx over 6 days. \u003cstrong\u003eD.\u003c/strong\u003e Relative frequency of low proliferative cells in the CAR+ and UT fractions of M28z and MBBz T cells. \u003cstrong\u003eE.\u003c/strong\u003e Pie chart showing the number of functions of CD4+ and CD8+ M28z and MBBz CAR T cells stimulated 2 times with MSLN+ K562 tumor cells and then exposed or not to Dx or MP before the last (3rd) stimulation with MSLN+ K562 tumor cells. Relative frequency of CD107a+ (\u003cstrong\u003eF\u003c/strong\u003e), TNF (\u003cstrong\u003eG\u003c/strong\u003e), IFNγ+ (\u003cstrong\u003eH\u003c/strong\u003e) and IL-2+ (\u003cstrong\u003eI\u003c/strong\u003e) CD4+ and CD8+ M28z and MBBz CAR T cells without GC exposure compared to Dx or MP exposure after MSLN+ K562 tumor cells stimulation \u003cstrong\u003eA\u0026amp;B\u003c/strong\u003e n=11, \u003cstrong\u003eD\u003c/strong\u003e n=3, \u003cstrong\u003eF\u003c/strong\u003e n=5 and \u003cstrong\u003eE\u003c/strong\u003e n=5. Mann-Whitney test and Student t test were used to compare the functions in M28z vs. MBBz. Friedman test with Dunn’s correction was used to compare multifunction in No GC vs. Dx vs. MP conditions. Medians are represented. * p\u0026lt;0.05, ** p\u0026lt;0.01.\u0026nbsp;\u003c/p\u003e","description":"","filename":"fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-3366592/v1/acff2c3f1a8e2c348ac3b6de.png"},{"id":49546620,"identity":"35cf02dc-c25b-4231-b116-ac196a6f5a7c","added_by":"auto","created_at":"2024-01-12 19:04:12","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":126701,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRecovery of M28z and MBBz CAR T cells functions after GC exposure. A.\u003c/strong\u003e Representative flow cytometry analysis used for FACS-based killing assay. \u003cstrong\u003eB.\u003c/strong\u003e Frequency of viable GFP+ MSLN+ K562 target cells after 24h incubation with GC-exposed M28z or MBBz CAR T cells. \u003cstrong\u003eC.\u003c/strong\u003e Frequency of killing reduction of M28z and MBBz induced by 3 exposures with Dx or MP. Relative frequency of LAG-3, PD-1 and TIM-3 in M28z and MBBz CAR T cells after 3 exposures with Dx (\u003cstrong\u003eD\u003c/strong\u003e) or MP (\u003cstrong\u003eE\u003c/strong\u003e) and 48h rest in a GC-free medium. \u003cstrong\u003eF.\u003c/strong\u003e Frequency of killing reduction of M28z and MBBz CAR T cells induced by 3 exposures with Dx or MP and 48h rest in a GC-free medium. n=5. Friedman test with Dunn’s correction was used to compare K562 viability in No GC vs. Dx vs. MP conditions. Student t test was used to compare surface marker expression in GC-exposed vs. GC-rested conditions. Medians are represented. * p\u0026lt;0.05, **p\u0026lt;0.01, *** p\u0026lt;0.001.\u0026nbsp;\u003c/p\u003e","description":"","filename":"fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-3366592/v1/d7b9021ee277820fb5f55c6a.png"},{"id":52907310,"identity":"ef146d3b-4daf-4e93-a986-ef869f41ca51","added_by":"auto","created_at":"2024-03-18 15:11:51","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":755887,"visible":true,"origin":"","legend":"","description":"","filename":"TPoiretmain.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3366592/v1_covered_4727176b-a88c-4c32-81c6-ae566519f0dc.pdf"},{"id":44335317,"identity":"c00e5352-9b0f-42ec-a64c-11919225bd31","added_by":"auto","created_at":"2023-10-10 04:08:37","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":186138,"visible":true,"origin":"","legend":"","description":"","filename":"TPoiretsuppl.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3366592/v1/de0ea13feaaf76d76b1ede46.pdf"},{"id":44335318,"identity":"9f57a11b-79ee-4db5-a33e-a2e56e9b3b30","added_by":"auto","created_at":"2023-10-10 04:08:37","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":2963998,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure Legends.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Figure 1.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eUT T cells phenotype. A. \u003c/strong\u003eRepresentative plot of the differentiation subsets defined by CCR7 and CD45RA markers. \u003cstrong\u003eB.\u003c/strong\u003e Memory phenotype of CAR+ and untransduced (UT) fractions of CD3+ T cells in CD19, M28z and MBBz CAR T cell products.GR expression (MFI) in the naïve and TCM memory subsets of (\u003cstrong\u003eC\u003c/strong\u003e) UT (from CD19 CAR cell product) CD4+ and CD8+ T cells; (\u003cstrong\u003eD\u003c/strong\u003e) of the UT (from M28z and MBBz CAR cell products) CD4+ (left) and CD8+ (right) T cells. n=5 for CD19 CAR T cells and n=4 for M28z and MBBz CAR T cells. Student t test was used to compare GR in different subsets. Medians are represented. *p\u0026lt;0.05, **p\u0026lt;0.01.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Figure 2.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImpact of GC exposure on UT T cells phenotype.\u003c/strong\u003e Frequency of CD19 CAR T cells (\u003cstrong\u003eA\u003c/strong\u003e) and CD4/CD8 ratio in CD19 CAR T cells (\u003cstrong\u003eB\u003c/strong\u003e) overtime after Dx (green) or MP (blue) exposure. \u003cstrong\u003eC.\u003c/strong\u003e Frequency of LAG-3, PD-1, and TIM-3 in CD19 CAR+ T cells without or after 1 and 3 exposures with Dx or MP. Friedman test with Dunn’s correction was used to compare marker’s expression in No GC vs. Dx vs. MP conditions. Relative surface expression of LAG-3, PD-1, and TIM-3 in CAR+ and UT CD4+ (\u003cstrong\u003eD\u003c/strong\u003e) and CD8+ (\u003cstrong\u003eE\u003c/strong\u003e) T cells after 3 exposures with Dx (left) or MP (right). \u003cstrong\u003eF.\u003c/strong\u003e Relative expression of LAG-3, PD-1, and TIM-3 in CD4+ and CD8+ UT T cells after 3 exposures of Dx (left) or MP (right).n=6. Wilcoxon matched-pairs signed rank test was used to compare marker’s expression in CAR+ vs. UT or CD4+ vs. CD8+ T cells. Medians are represented. * p\u0026lt;0.05, ** p\u0026lt;0.01, *** p\u0026lt;0.001.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Figure 3.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImpact of GCs exposure on CD19 CAR+ T cells effector functions. A.\u003c/strong\u003e Relative frequency of IFNγ+, TNF+ or IL2+ CD4+ or CD8+ CD19 CAR and UT after a single exposure with Dx (top) or MP (bottom) after PMA/ionomycin stimulation. Frequency of CD107a+, IFNγ+, TNF and IL-2+ CAR CD4+ and CD8+ T cells without GC exposure 1 exposure with low or high doses of GC (0.1 µg/ml and 10 µg/ml Dx and 0.5µg/ml, 50 µg/ml, and 100 µg/ml MP) following a single (\u003cstrong\u003eB\u003c/strong\u003e) or 3 (\u003cstrong\u003eC\u003c/strong\u003e) stimulations with CD19+ K562 tumor cells. \u003cstrong\u003eD.\u003c/strong\u003e Pie chart showing the number of functions of CD8+ CAR T cells without GC exposure compared to Dx or MP exposure following a single or 3 stimulations with CD19+ K562 tumor cells stimulation. n=8. Two-way ANOVA with Sidak’s correction was used to compare CD107a+, IFNγ+, TNF and IL-2+ cells frequency at different doses of Dx and MP. Friedman test with Dunn’s correction was used to compare multifunction in No GC vs. Dx vs. MP conditions. Medians are represented. * p\u0026lt;0.05, ** p\u0026lt;0.01, *** p\u0026lt;0.001.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Figure 4.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImpact of GCs exposure on M28z and MBBz CAR+ T cells phenotype and functions.\u003c/strong\u003e Frequency of M28z and MBBz CAR T cells (\u003cstrong\u003eA\u003c/strong\u003e) and CD4/CD8 ratio in CAR T cells (\u003cstrong\u003eB\u003c/strong\u003e) without GC exposure or after 3 exposures with low and high concentration of Dx or MP. Frequency of LAG-3 (\u003cstrong\u003eC\u003c/strong\u003e), PD-1 (\u003cstrong\u003eD\u003c/strong\u003e) and TIM-3 (\u003cstrong\u003eE\u003c/strong\u003e) in M28z and MBBz CAR T cells without GC exposure or after 3 exposures with low and high concentration of Dx or MP. Relative surface expression of LAG-3, PD-1 and TIM-3 in CAR+ and UT fractions of CD8+ M28z and MBBz T cells following 3 exposures with Dx (\u003cstrong\u003eF\u003c/strong\u003e) or MP (\u003cstrong\u003eG\u003c/strong\u003e). Frequency of CD107a+, IFNγ+, TNF and IL-2+ CD4+ (\u003cstrong\u003eH\u003c/strong\u003e) and CD8+ (\u003cstrong\u003eI\u003c/strong\u003e) M28z and MBBz CAR T cells without GC exposure compared to Dx or MP exposure following 3 stimulations with MSLN+ K562 tumor cells. \u003cstrong\u003eA-E\u003c/strong\u003e n=6, \u003cstrong\u003eF\u0026amp;G\u003c/strong\u003e n=11 and \u003cstrong\u003eH\u0026amp;I\u003c/strong\u003e n=5. Two-way ANNOVA with Sidak’s correction was used to compare CD107a+, IFNγ+, TNF and IL-2+ cells frequency at different doses of Dx and MP. Mann-Whitney test was used to the surface expressions and functions in M28z vs. MBBz. Medians are represented. * p\u0026lt;0.05, ** p\u0026lt;0.01, *** p\u0026lt;0.001.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Figure 5.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eM28z and MBBz CD4+ and CD8+ CAR T cells phenotype after GC removal. \u003c/strong\u003eRelative frequency of LAG-3, PD-1 and TIM-3 in M28z and MBBz CD4+ (\u003cstrong\u003eA\u0026amp;C\u003c/strong\u003e) and CD8+ (\u003cstrong\u003eB\u0026amp;D\u003c/strong\u003e) CAR T cells after 3 exposures with Dx (\u003cstrong\u003eA-B\u003c/strong\u003e) or MP (\u003cstrong\u003eC-D\u003c/strong\u003e) and 48h rest in a GC-free medium. n=5. Student t test was used to compare surface marker expression in GC-exposed vs. GC-rested conditions. * p\u0026lt;0.05, ** p\u0026lt;0.01, *** p\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"TPoiretFiguressuppl.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3366592/v1/920420690a181aa6f31da35a.pdf"}],"financialInterests":"","formattedTitle":"CAR T cells and T cells phenotype and function are impacted by glucocorticoid exposure with different amplitude.","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-translational-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jtrm","sideBox":"Learn more about [Journal of Translational Medicine](http://translational-medicine.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/jtrm/default.aspx","title":"Journal of Translational Medicine","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"glucocorticoid, CAR, chimeric antigen receptor, CD19, mesothelin, 4-1-BB, CD28","lastPublishedDoi":"10.21203/rs.3.rs-3366592/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3366592/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground.\u0026nbsp;\u003c/strong\u003e Chimeric antigen receptor (CAR) T cell therapy is associated with high risk of adverse events. Glucocorticoids (GCs) are cornerstone in the management of high-grade\u0026nbsp;cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS). Given the potentially deleterious effects of GCs on CAR T cells anti-tumor activity, increasing our understanding of GCs impact on CAR T cells is crucial.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods.\u003c/strong\u003e Using several CAR T cells i.e., CD19, mesothelin (MSLN)-CD28 and MSLN-41BB CAR T cells (M28z and MBBz), we compared phenotypical, functional, changes and anti-tumor activity between i) transduced CD19 CAR T cells with untransduced T cells, ii) M28z with MBBz CAR T cells induced by Dexamethasone (Dx) or Methylprednisolone (MP) exposures.\u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults.\u003c/strong\u003e Higher levels of GC receptor were found in less differentiated CAR T cells. Overall, Dx and MP showed a similar impact on CAR T cells. GC exposures induced more exhausted (LAG3+PD1+TIM3+) and dysfunctional (CD107a-INFγ-TNF-IL2-) untransduced T cells in comparison to CD19 CAR T cells. GC exposure impaired more CD4+ than CD8+ CD19 CAR T cells. GC exposures increased more PD-1 expression associated with reduced proliferative capacity and function of M28z as compared to MBBz CAR T cells. CAR T cells anti-tumor activity was greatly affected by repeated GC exposure but partly recovered within 48h after GCs withdrawal.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions.\u003c/strong\u003e In summary, GCs impacted phenotype and function of untransduced and CAR T cell with different amplitude. The nature of the CAR costimulatory domain influenced the amplitude of CAR T cell response to GCs.\u003c/p\u003e","manuscriptTitle":"CAR T cells and T cells phenotype and function are impacted by glucocorticoid exposure with different amplitude.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-10-10 04:08:32","doi":"10.21203/rs.3.rs-3366592/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2023-10-04T15:42:10+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2023-10-04T14:18:35+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-09-21T10:26:24+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Translational Medicine","date":"2023-09-20T02:02:51+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-translational-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jtrm","sideBox":"Learn more about [Journal of Translational Medicine](http://translational-medicine.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/jtrm/default.aspx","title":"Journal of Translational Medicine","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"baa341da-5a33-42d9-8536-5d4c50362852","owner":[],"postedDate":"October 10th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-03-18T15:05:46+00:00","versionOfRecord":{"articleIdentity":"rs-3366592","link":"https://doi.org/10.1186/s12967-024-05063-4","journal":{"identity":"journal-of-translational-medicine","isVorOnly":false,"title":"Journal of Translational Medicine"},"publishedOn":"2024-03-12 15:01:15","publishedOnDateReadable":"March 12th, 2024"},"versionCreatedAt":"2023-10-10 04:08:32","video":"","vorDoi":"10.1186/s12967-024-05063-4","vorDoiUrl":"https://doi.org/10.1186/s12967-024-05063-4","workflowStages":[]},"version":"v1","identity":"rs-3366592","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3366592","identity":"rs-3366592","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}