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- HLA‐A23/HLA‐A24 serotypes and dementia interaction in the elderly: Association with increased soluble HLA class I molecules in plasmaPublication . Cardoso, Elsa M.; Gomes, Vânia Lourenço; Esgalhado, André J.; Ferreira, Débora Reste; Oliveira, Nádia; Amaral, Ana Saraiva; Martinho, António; Gama, Jorge; Verde, Ignacio; Lourenço, Olga; Fonseca, Ana C.; Buchli, Rico; Arosa, Fernando A.MHC class I molecules regulate brain development and plasticity in mice and HLA class I molecules are associated with brain disorders in humans. We investigated the relationship between plasma-derived soluble human HLA class I molecules (sHLA class I), HLA class I serotypes and dementia. A cohort of HLA class I serotyped elderly subjects with no dementia/predementia (NpD, n = 28), or with dementia (D, n = 28) was studied. Multivariate analysis was used to examine the influence of dementia and HLA class I serotype on sHLA class I levels, and to compare sHLA class I within four groups according to the presence or absence of HLA-A23/A24 and dementia. HLA-A23/A24 and dementia, but not age, significantly influenced the level of sHLA class I. Importantly, the concurrent presence of HLA-A23/A24 and dementia was associated with higher levels of sHLA class I (p < 0.001). This study has shown that the simultaneous presence of HLA-A23/HLA-A24 and dementia is associated with high levels of serum sHLA class I molecules. Thus, sHLA class I could be considered a biomarker of neurodegeneration in certain HLA class I carriers.
- CD45RA, CD8β, and IFNγ Are Potential Immune Biomarkers of Human Cognitive FunctionPublication . Esgalhado, AJ; Reste-Ferreira, Débora; Albino, Stephanie; Sousa, Adriana; Amaral, Ana Paula; Martinho, António; Oliveira, Isabel Tomás; Verde, Ignacio; Lourenço, Olga; Fonseca, Ana M; Cardoso, Elsa M.; Arosa, FAThere is increasing evidence that in humans the adaptive immunological system can influence cognitive functions of the brain. We have undertaken a comprehensive immunological analysis of lymphocyte and monocyte populations as well as of HLA molecules expression in a cohort of elderly volunteers (age range, 64-101) differing in their cognitive status. Hereby, we report on the identification of a novel signature in cognitively impaired elderly characterized by: (1) elevated percentages of CD8+ T effector-memory cells expressing high levels of the CD45RA phosphate receptor (Temra hi); (2) high percentages of CD8+ T cells expressing high levels of the CD8β chain (CD8βhi); (3) augmented production of IFNγ by in vitro activated CD4+ T cells. Noteworthy, CD3+CD8+ Temra hi and CD3+CD8βhi cells were associated with impaired cognition. Cytomegalovirus seroprevalence showed that all volunteers studied but one were CMV positive. Finally, we show that some of these phenotypic and functional features are associated with an increased frequency of the HLA-B8 serotype, which belongs to the ancestral haplotype HLA-A1, Cw7, B8, DR3, DQ2, among cognitively impaired volunteers. To our knowledge, this is the first proof in humans linking the amount of cell surface CD45RA and CD8β chain expressed by CD8+ Temra cells, and the amount of IFNγ produced by in vitro activated CD4+ T cells, with impaired cognitive function in the elderly.
- Characterization of Effector-Memory CD8+ T cells and their Association with Human Cognitive FunctionPublication . Esgalhado, André João Gabriel ; Arosa, Fernando Aguilar; Uhrberg, MarkusHuman effector-memory CD8+ T cells consist of highly differentiated cells that differ in the expression of the tyrosine phosphatase isoform CD45RA, being designated as CD8+ TEM and CD8+ TEMRA cells. These highly heterogeneous and polyfunctional cells possess cytotoxic, regulatory, and suppressive features, and are capable of migrating to non-lymphoid tissues and organs, including the brain under certain conditions. Expansions of CD8+ TEM and CD8+ TEMRA cells have been described in chronic inflammatory diseases, tumors and viral infections, as well as in healthy elderly individuals, including centenarians. Over the past few decades, the role of CD8+ T cells, particularly CD8+ TEMRA cells, has been the subject of several studies in the context of aging, cognition, and neurodegeneration, where they have been generally regarded as detrimental to the central nervous system (CNS), though recent investigations have challenged this view. These highly differentiated CD8+ T cells are known to arise through TCR-dependent and TCR-independent mechanisms, such as cytokine-driven proliferation via interleukin (IL)-15. Intriguingly, chronic antigenic stimulation has been shown to drive the generation of CD8+ T cells expressing low levels of the CD8β chain, though antigen-independent mechanisms remain poorly understood. Herein, we have performed a comprehensive characterization of peripheral blood mononuclear cells (PBMC) as well as of human leukocyte antigen (HLA) molecules in a cohort of elderly volunteers differing in their cognitive status. A detailed analysis of the level of expression of CD45RA in the CD8+ TEMRA compartment revealed the presence of two distinct populations: CD8+ TEMRAlow and CD8+ TEMRAhigh cells. Notably, CD8+ TEMRAhigh cells formed a well-defined and sharply delineated population that was significantly expanded in cognitively impaired volunteers, whereas cognitively unimpaired volunteers were enriched in CD8+ TEMRAlow cells. Further analysis of CD8α and CD8β expression also identified the existence of two distinct CD8+ T cells subsets based on the expression of CD8β: CD8αβlow and CD8αβhigh T cells, with the former being more prevalent among cognitively unimpaired individuals. Moreover, stimulation with PMA and Ionomycin revealed significantly increased IFN-γ production by CD4+ T cells from cognitively impaired elderly. Noteworthy, all but one of the volunteers studied were cytomegalovirus (CMV) seropositive. Finally, a higher prevalence of the HLA-B8 serotype, which belongs to the ancestral haplotype HLA-A1, Cw7, B8, DR3, DQ2, was found among the cognitively impaired elderly. Additionally, we assessed the impact of IL-15 on the cell surface expression of CD8β using CFSE-labeled purified human naïve CD8+ T cells cultured for 12 days. IL-15 induced a robust proliferation and differentiation, resulting in a cell cycle-dependent down-modulation of CD8β from the cell surface, while CD8α expression remained stable or increased slightly. This led to the generation of CD8αβlow and CD8αβ– (i.e., CD8αα) T cells. In contrast, IL-2 and IL-7 alone were unable to replicate this effect. Determination of mRNA levels for CD8α and CD8β isoforms by qPCR revealed that IL-15 promoted a significant decrease in mRNA levels of the CD8β M-4 isoform, while increasing the levels of the M-1 and M-2 isoforms alongside with CD8α. Remarkably, analysis of the level of the tyrosine kinase Lck showed a significant increase in CD8+ T cell blasts after culture of CD8+ T cells with IL-15, when compared to CD8+ T cells at the beginning of the culture. Our findings show an association with certain CD8+ T cell subsets that is compatible with a protective role in cognition and neurodegenerative diseases by identifying novel markers that define discrete subsets of highly differentiated CD8+ T cells expanded in cognitively unimpaired elderly individuals and identify IL-15 as a factor involved in the generation of these subsets. In-depth phenotypic, functional, and transcriptomic characterization of ex vivo and in vitro obtained CD8+ T cell subset is warranted to further elucidate their unique functional properties.
- Regulação da Expressão de Moléculas MHC Classe I em Linfócitos T em DivisãoPublication . Esgalhado, André João Gabriel ; Arosa, Fernando AguilarMHC Class I molecules are transmembrane glycoproteins composed by a 44-49 kDa heavy chain, a 12 kDa light chain named ß2-microglobulin and a 9-11 amino acid peptide. These molecules present peptides originating manly from intracellular peptides to cytotoxic T cells and are mandatory for the induction and development of adaptive immune responses. MHC Class I molecules are expressed by all nucleated cells. However, these molecules can be expressed in a different conformation, where MHC class I heavy chains are not associated with peptide and/or ß2-microglobulin (called open conformers) that can associate with a variety of cells surface receptors, resulting in modulation of their activation. Previous studies showed that open conformers are expressed at the cell surface of proliferating lymphoid cells. In this work, we isolated peripheral blood mononuclear cells, from blood samples of healthy patients and iron overload and erythrocytosis patients, lysed red blood cells, labeled cells with CFSE, added different stimulus and cultured the cells for 6 days. After this period of time cells were labeled with different antibodies and analyzed by flow cytometry. Whole blood cells from Polycythemia Vera patients were isolated from 3 mL blood samples, red blood cells were lysed and cells were labelled with W6/32 and HC-10 antibodies. Our results clearly demonstrate that the stimulus applied to the cells resulted in different proliferation rates and that in dividing T cells there’s a tendency for the maintenance or a slightly decrease in the expression of closed MHC Class I molecules and an increase in the expression of open conformers, for both CD4+ and CD8+ T lymphocytes. Regarding, Polycythemia Vera patients, expression of closed MHC class I molecules is increased, while the levels of open conformers expression are increased in monocytes and granulocytes.