Phenotypic and Functional Characterization of Memory CD4+ and CD8+ T Cells After Antigenic Stimulation.

The encounter of T cells with the antigen through the interaction of T cell receptors with peptides and major histocompatibility complex (MHC) molecules on the surface of antigen-presenting cells (APCs) can generate effector response and memory T cells. Memory T cells developed following infections or vaccination may persist, leading to the generation of a specific immune response upon reexposure to the same pathogen through rapid clonal proliferation and activation of effector functions. T cell memory subsets can be identified based on the expression of several membrane markers such as CCR7, CD27, and CD45RA. Using fluorescent antibodies against these markers and a flow cytometer, it is possible to perform immunophenotyping via the analysis of cell surface expression of proteins by different subpopulations such as the subsets of naïve, effector, and memory T cells as well as via the analysis of functional markers that further characterize each sample. Intracellular cytokine staining allows for the evaluation of intracellular proteins expressed in T cells in response to antigenic stimulation. This chapter presents the phenotypic and functional characterization of memory T cells after antigenic stimulation, detailing the procedures for identifying intracellular and surface protein markers. Herein, we review and present a reproducible standardized protocol using antibodies for specific markers and applying flow cytometry.

CYTOSPOT: Intracellular Staining and Detection of Cytokines by Flow Cytometer, an Alternative to LYMESPOT.

Intracellular cytokine staining is a versatile technique used to analyze cytokine production in individual cells by flow cytometry. This methodology has the specific advantage of enabling the simultaneous assessment of multiple phenotypic, differentiation, and functional parameters pertaining to responding T cells. This methodology applied after short-term culture of cells, followed by fixation and permeabilization make this technique ideal for the assessment of T-cell immune responses induced by different challenges. Here we describe an intracellular staining method followed by flow cytometry after cell stimulation with immune-relevant antigens for Lyme disease.

Regulatory/inflammatory cellular response discrimination in operational tolerance.

BACKGROUND: Antigen-specific cellular response is essential in immune tolerance. We tested whether antigen-specific cellular response is differentially modulated in operational tolerance (OT) in renal transplantation with respect to critical antigenic challenges in allotransplantation-donor antigens, pathogenic antigens and self-antigens. METHODS: We analysed the profile of immunoregulatory (REG) and pro-inflammatory (INFLAMMA) cytokines for the antigen-specific response directed to these three antigen groups, by Luminex. RESULTS: We showed that, in contrast to chronic rejection and healthy individuals, OT gives rise to an immunoregulatory deviation in the cellular response to donor human leucocyte antigen DR isotype peptides, while preserving the pro-inflammatory response to pathogenic peptides. Cellular autoreactivity to the N6 heat shock protein 60 (Hsp60) peptide also showed a REG profile in OT, increasing IL4, IL-5, IL-10 and IL-13. CONCLUSIONS: The REG shift of donor indirect alloreactivity in OT, with inhibition of interleukin (IL)-1B, IL-8, IL-12, IL-17, granulocyte colony-stimulating factor, Interferon-γ and monocyte chemoattractant protein-1, indicates that this may be an important mechanism in OT. In addition, the differential REG profile of cellular response to the Hsp60 peptide in OT suggests that REG autoimmunity may also play a role in human transplantation tolerance. Despite cross-reactivity of antigen-specific T cell responses, a systemic functional antigen-specific discrimination takes place in OT.

Generation of T. cruzi-Specific Primary CD4+ T Cell Lines from Peripheral Blood Mononuclear Cells Isolated from Chagas Disease Patients.

Human CD8+ and CD4+ T cell lines and clones are valuable tools to explore the role of these cells in the context of diseases, especially in cases in which the main underlying actor is the immune response, like Chagas disease. These cell lines and clones provide a good experimental system to address the phenotypic and functional features of specific T cell subpopulations and furthermore settle the framework necessary for analyzing their antigen/peptide specificity.This chapter details a culture method for the establishment of T. cruzi-specific memory T cell lines from mononuclear cells isolated from Chagas disease patients' peripheral blood. The presented protocol comprises (1) enrichment of memory CD4+ T cells, (2) stimulation with parasite lysate, (3) evaluation of specificity, and (4) expansion and maintenance of specific T cell lines.

Pluripotent Nontumorigenic Adipose Tissue-Derived Muse Cells have Immunomodulatory Capacity Mediated by Transforming Growth Factor-ß1.

Adult mesenchymal stromal cell-based interventions have shown promising results in a broad range of diseases. However, their use has faced limited effectiveness owing to the low survival rates and susceptibility to environmental stress on transplantation. We describe the cellular and molecular characteristics of multilineage-differentiating stress-enduring (Muse) cells derived from adipose tissue (AT), a subpopulation of pluripotent stem cells isolated from human lipoaspirates. Muse-AT cells were efficiently obtained using a simple, fast, and affordable procedure, avoiding cell sorting and genetic manipulation methods. Muse-AT cells isolated under severe cellular stress, expressed pluripotency stem cell markers and spontaneously differentiated into the three germ lineages. Muse-AT cells grown as spheroids have a limited proliferation rate, a diameter of ∼15 µm, and ultrastructural organization similar to that of embryonic stem cells. Muse-AT cells evidenced high stage-specific embryonic antigen-3 (SSEA-3) expression (∼60% of cells) after 7-10 days growing in suspension and did not form teratomas when injected into immunodeficient mice. SSEA-3+ -Muse-AT cells expressed CD105, CD29, CD73, human leukocyte antigen (HLA) class I, CD44, and CD90 and low levels of HLA class II, CD45, and CD34. Using lipopolysaccharide-stimulated macrophages and antigen-challenged T-cell assays, we have shown that Muse-AT cells have anti-inflammatory activities downregulating the secretion of proinflammatory cytokines, such as interferon-γ and tumor necrosis factor-α. Muse-AT cells spontaneously gained transforming growth factor-ß1 expression that, in a phosphorylated SMAD2-dependent manner, might prove pivotal in their observed immunoregulatory activity through decreased expression of T-box transcription factor in T cells. Collectively, the present study has demonstrated the feasibility and efficiency of obtaining Muse-AT cells that can potentially be harnessed as immunoregulators to treat immune-related disorders. Stem Cells Translational Medicine 2017;6:161-173.