Priming of antigen-specific CD8+ T cells by vaccination is key for subsequent viral clearance. However, to date, CD8+ T-cell characterization is performed under the assumption that cells of a particular type are identical, and not acknowledging their heterogeneous nature. It has recently been proven, in many different studies, that cells do differ quite significantly and can drive different responses. In order to characterize the heterogeneity of vaccine-induced CD8+ T cells, we performed gene expression profiling and protein characterization at the single-cell level, after delivery of influenza RNA-based vaccine (SAM (H1) (A/California/07/2009 (H1/N1)) or MF59TM -adjuvanted Monovalent Influenza Vaccine (aMIV (A/California/07/2009 (H1N1)). CD8+ T cells were isolated from mouse splenocytes, stained with MHC class-I HA533-554 pentamer and either single-cell sorted, lysed, and processed for reverse transcription- quantitative real-time polymerase chain reaction (RT-qPCR) analysis of 96 individual genes, or characterized by mass cytometry for the expression of 32 biomarkers. In parallel, the functionality of the CD8+ T cells was assessed in an in vivo cytotoxic assay. CD8+ T cells induced by the SAM(H1) vaccine showed increased antigen-specific lysis activity in vivo, compared to cells induced by aMIV. Ex vivo, higher frequencies of HA533-541-pentamer+ CD8+ T cells were induced by SAM(H1) compared to aMIV at all time points tested, and pentamer+ cells were single-cell sorted for gene expression analysis. Refined analysis of TEFF (il-7rα -- cd62l- ), TEM (il-7rα +- cd62l- ) and TCM (il-7rα +- cd62l+ )) highlighted significant differences in gene expression profiles between and within the vaccines. Overall, SAM(H1) induced mostly TEM cells, while aMIV induced equal number of TCM and TEM cells. Both vaccines induced few TEFF cells, and only CD8+ T cells from SAM(H1) showed TEFF terminally differentiated short-lived effector cells (SLEC) (klrg1+ il-7rα - cd62lcxcr3- tbet+ blimp-1 + ). Ten days after the second immunization, TEM cells, unlike TCM cells, showed a transcriptional difference between the vaccines. SAM(H1) and aMIV, with up regulation of members of the cytolytic/Fas Ligans (FasL) and tumor necrosis factor (TNF) Superfamily pathways, respectively. A specific molecular signature of 12 differentially expressed genes (DEG) was identified and further evaluated at the protein expression level using mass cytometry. Most of the proteins corresponding to the 12 DEG list were also expressed. The aim of this study was to apply state-of-the-art technology platforms to the study of gene and protein expression by immune cells in response to vaccination. With this approach, we identified distinct HA533-541- pentamer+ CD8+ T-cell subsets elicited by RNA- and adjuvanted protein-based vaccines, which revealed a particular biomarker signature associated to each vaccine. In conclusion, our results showed that these novel technology platforms might be extremely useful for the in-depth characterization of immune responses to vaccination and can be extended to other medical applications.
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|Titolo:||New technology platforms applied to the profiling of gene and protein expression at the single T-cell level|
|Appare nelle tipologie:||8.1 Tesi Dottorato|
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