Deformability of t cells as a mechanical biomarker of immunosenescence and development of techniques for the clinical application

Resumen

The specific ways in which T cells self-regulate their migratory behaviors during various physiological processes, including immune activation, interstitial migration or immune synapses, greatly depend on cell mechanical properties and the mechanical constraints of the extracellular environment. During aging, changes across immune T cells overall contribute to a decrease in immunological competence, a progression broadly referred to as immunosenescence. The aging-dependent decline of T-cell competence has been mostly studied in terms of differentiation, proliferation, receptor expression, genetic and epigenetic alterations or metabolism. Yet, the question of how aging can affect biophysical markers of T cells potentially involved in cell migration remains an open question with partial answers. Characterizing the mechanical phenotype of primary T cells during aging of individuals has been at the center of this thesis project. To tackle such a large question, a first step was to investigate the links between T-cell mechanical properties and morphological characteristics of intracellular components, by performing multiple measurements on the same cells. Based on the results of this first study, showing that the relative nuclear size can account for variations of T-cell stiffness, a second step was to analyze whether aging prompted biophysical and biomolecular changes in T-cell phenotypes. This thorough analysis represents, to our knowledge, a first longitudinal study of biophysical, morphological, phenotypical and functional analysis (spontaneous migratory behavior) of the kind in primary mouse T cells (CD4+ and CD8+ T cells, in both naive and memory state). Several biophysical parameters that correlated with an age-related decline of T-cell migration were identified, including cell stiffness, DNA-methylation, Lamin B1 thickness and relative nuclear size. Based on these biophysical and biomolecular parameters, an estimation of chronological age was also developed, which represents another innovative aspect of this work. Finally, a biophysical model was proposed to explain the age-related changes of T cells. Several methodological and experimental enhancements were developed in the thesis to address current limitations of the methods and models based on micropipette aspiration (MA) for the analysis of cellular mechanics. In order to quantify mechanical properties at the cellular scale, MA has traditionally been considered a standard for the quantification of suspended cells. However, there are some drawbacks in the MA approach that require to be addressed in order to improve its efficiency and applicability, including the absence of an fully automated analysis, together with the inexistent use of the technique to investigate cells with rigid walls, such as infectious fungal cells, due to the lack of experimental and computational techniques appropriate for this sizable challenge. This dissertation addresses some of these limitations by developing a user-friendly ‘all-in-one’ routine for the image processing of MA microscopy data, a novel approach to quantitatively study multiple parameters on the same cells, and a novel methodology to probe cell mechanics for quantification of the effect of external pressure on the growth of fungal cell. Overall, these advances contribute to the acquisition of mechanophenotypic data which previously could not be obtained by applying the MA technique.