Intracellular protein phosphorylation is a critical step in cellular activation stimulated by the binding of various ligands to cell surface receptors. This process is initiated by activation of specific protein-tyrosine kinases associated with intracellular domains of the respective ligand receptor. JAK-STATs pathway is one of the main pathways in the cell activation process and given their important role in various PIDs, STATs proteins have been extensively studied in immune function in health and disease.
Therefore, our work has focused on investigating and evaluating STATs activation and establishing flow cytometric methods to assess their phosphorylation to be a surrogate marker as a fast and sensitive diagnostic tool to current methods such as WB.
At the first, we studied STAT1 and STAT3 activation and established a flow cytometric procedure to analyze variations of INF-α- and IL-6-induced STAT1 and STAT3 phosphorylation in T cells from whole blood, respectively (publication ΙΙ). To examine whether our results were specific, the samples were also analyzed by WB in parallel. After that, we validated the normal values of pSTAT1 and pSTAT3 based on 21 healthy adult controls according to an appropriate validation process.
We showed that, in contrast to the conventional methods like WB, our assay offers a diagnostic benefit by avoiding labor and time consumption, with the advantage of achieving an earlier diagnosis, which potentially leads to improve treatment decisions; hence, patient’s outcome (publication ΙΙ).
Furthermore, we verified FCM-based pSTAT1 and pSTAT3 profiling established here in patients group suffering from CMC and HIES. Our results demonstrated that pSTAT1 and pSTAT3 assay is an effective tool to identify and characterize well-known PIDs such as CMC and HIES, respectively (publication ΙΙ).
Next, we introduced a fast and straightforward flow cytometric assay for the assessment of T cell proliferation, based on the staining of phosphorylated STAT5A (publication ΙΙΙ). We showed that pSTAT5A represents an appropriate approach to predict the behavior of T cells upon activation by CD3/CD28 and PHA. FCM-based pSTAT5A profiling is an intracellular flow cytometric method, enabling the early and reliable detection of T cell proliferation without long time incubation (within 24 h instead to 5 days). Importantly, measurement of pSTAT5A represents a new principle to assess T cell proliferation. It reveals important information on T cell biology by using series of kinetics and different kinds of T cell stimulation. For instance: [1] after stimulation via CD3/CD28 and negative pSTAT5A and T cell proliferation, the immune defect could be occurred in the whole signaling cascade (TCR-IL-2 transcription-JAK3-STAT5), [2] After stimulation via external IL-2 and negative pSTAT5A, the immune defect could be localized in the signaling cascade (IL-2R – JAK3 – STAT5), [3] After stimulation via external IL-2 and positive pSTAT5A, the immune defect could be localized in the signaling cascade (TCR - IL-2 transcription) (publication ΙΙΙ).
We showed a strong correlation between the STAT5A phosphorylation and the percentage of dividing cells (publication ΙΙΙ). Later on, we used the measurement established here to investigate whether the phosphorylation of STAT5A is an appropriate candidate for predicting CMV specific T cell proliferation.
It is well-known that CMV specific T cells expand with CMV reactivation and are probably prerequisite for control and protection. We demonstrated that CMV specific pSTAT5A detection represents a fast and straightforward diagnostic tool to assess CMV specific T cell proliferation without requisite several days’ culture (publication ΙV).
Furthermore, we showed a positive correlation between the percentage of pSTAT5A+ T cells vs. (1) CMV-IgG concentrations vs. (2) the percentage of expanded T cells and vs. (3) the percentage of initial CMV specific T cells (publication ΙV).
Finally, we evaluated the diagnostic value of pSTAT5 assay and determined the percentage of pSTAT5A+ T cells cut-off value at which pSTAT5 assay has the greatest diagnostic potency. Our data showed that a cut-off value of 9.1 % could be used to assess CMV specific T cell proliferation with a specificity and sensitivity of 100% and 73%, respectively.
We verified measurement established here by CMV specific T cells stimulation in three selected patients diagnosed with CARMIL2-mutation and suffering from chronic CMV
infection. Our results showed that the complete and the partial deficiency of CMV and CD3/CD28 stimulated pSTAT5A correlated with the complete and the partial deficiency of CMV and CD3/CD28 stimulated T cell proliferation, respectively (publication ΙV).
In conclusion, disorders in JAKs-STATs signal pathways in T cells may result in insufficient response to stimulants. Therefore, FCM-based pSTATs profiling is an effective tool for clinical laboratory diagnostics [1] to understand the susceptibility to recurrent opportunistic infections [2] to rapidly identify T cell proliferation [3] to investigate tumor-specific responses of CD8 T effector and memory cells (56) and finally [4] to identify and distinguish well-known PIDs like CARMIL-2 mutations, CMC, AD-HIES or AR-HIES.
| Identifer | oai:union.ndltd.org:DRESDEN/oai:qucosa:de:qucosa:72691 |
| Date | 04 November 2020 |
| Creators | Bitar, Michael |
| Contributors | Sack, Ulrich, Jassoy, Christian, Rodloff, Arne C, Universität Leipzig |
| Source Sets | Hochschulschriftenserver (HSSS) der SLUB Dresden |
| Language | German |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, doc-type:doctoralThesis, info:eu-repo/semantics/doctoralThesis, doc-type:Text |
| Rights | info:eu-repo/semantics/openAccess |
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