The immunosuppressive activity of mesenchymal stromal cells (MSCs) is well documented. However, the therapeutic benefit is completely unpredictable, thus raising concerns about MSC efficacy. One of the affecting factors is the unresolved conundrum that, despite being immunosuppressive, MSCs are undetectable after administration. Therefore, understanding the fate of infused MSCs could help predict clinical responses. Using a murine model of graft-versus-host disease (GvHD), we demonstrate that MSCs are actively induced to undergo perforin-dependent apoptosis by recipient cytotoxic cells and that this process is essential to initiate MSC-induced immunosuppression. When examining patients with GvHD who received MSCs, we found a striking parallel, whereby only those with high cytotoxic activity against MSCs responded to MSC infusion, whereas those with low activity did not. The need for recipient cytotoxic cell activity could be replaced by the infusion of apoptotic MSCs generated ex vivo. After infusion, recipient phagocytes engulf apoptotic MSCs and produce indoleamine 2,3-dioxygenase, which is ultimately necessary for effecting immunosuppression. Therefore, we propose the innovative concept that patients should be stratified for MSC treatment according to their ability to kill MSCs or that all patients could be treated with ex vivo apoptotic MSCs.

Apoptosis in mesenchymal stromal cells induces in vivo recipient-mediated immunomodulation.

The T cell compartment can form a powerful defense against extrinsic (e.g., pathogens) and intrinsic danger (e.g., malignant cells). At the same time, specific subsets of T cells control this process to keep the immune system in check and prevent autoimmunity. A wide variety in T cell functionalities exists, which is dependent on the differentiation and maturation state of the T cells. In this review, we report an overview for the identification of CD4+ T-αβ cells (T-helper (Th)1, Th2, Th9, Th17, Th22, and CD4+ regulatory T cells), CD8+ T-αβ cells (cytotoxic T lymphocyte (Tc)1, Tc2, Tc9, Tc17, and CD8+ regulatory T cells), and their additional effector memory status (naive, stem cell memory, central memory, effector memory, and effector) using flow cytometry. These different subsets can be discriminated based on selective extracellular markers, in combination with intracellular transcription factor and/or cytokine stainings. Additionally, identification of very small subsets, including antigen-specific T cells, and important technical considerations of flow cytometry are discussed. Together, this overview can be used for comprehensive phenotyping of a T cell subset of interest. © 2019 International Society for Advancement of Cytometry.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/cyto.a.23724

Comprehensive Phenotyping of T Cells Using Flow Cytometry.

On antigen encounter, naive CD8+ and CD4+ T cells differentiate into a large number of effector cells that migrate to inflamed tissues to fight infections or tumors. Following elimination of the target, a few cells remain in the long-term, the so-called memory T cells that are capable to reexpand and respond more vigorously on a second encounter with the cognate antigen. While the naive T cell compartment is fairly homogenous, effector and memory T cells are largely diverse, comprising dozens of subsets with diverse functions, molecular characteristics, and localization in the body. In addition, CD4+ and, to some extent, also CD8+ T cells can differentiate into several effector subsets according to their pattern of cytokine expression, including the T helper 1 (Th1), Th2, and Th17 cells. It has become clear that specific subsets of T cells dominate different types of infections and pathological conditions and have different capacities to infiltrate and reject tumors. Their correct phenotypic identification is therefore of foremost importance for their live purification by magnetic or fluorescence-activated cell sorting and subsequent molecular characterization. Here, we present a comprehensive list of the main T cell subpopulations with a major focus on human cells along with their surface phenotypic properties.

The Single-Cell Phenotypic Identity of Human CD8+ and CD4+ T Cells

Granzyme B (GrzB) is a serine proteinase expressed by memory T cells and NK cells. Methods to measure GrzB protein usually involve intracellular (flow cytometry) and extracellular (ELISA and ELISpot) assays. CD8 T cells are the main source of GrzB during immunological reactions, but activated CD4 T cells deploy GrzB as well. Because GrzB is an important mediator of cell death, tissue pathology and disease, clarification of differences of GrzB expression and secretion between CD4 and CD8 T cells is important for understanding effector functions of these cells. Memory CD4 and memory CD8 T cells were purified from human peripheral blood of healthy donors, and production of GrzB was directly compared between memory CD4 and memory CD8 T cells from the same donors using parallel measurements of flow cytometry (intracellular GrzB), ELISpot (single cell secretion of GrzB), and ELISA (bulk extracellular GrzB). Memory CD8 T cells constitutively stored significantly more GrzB protein (~25%) compared to memory CD4 T cells as determined by flow cytometry (~3%), and this difference remained stable after 24 hrs of activation. However, measurement of extracellular GrzB by ELISA revealed that activated memory CD4 T cells secrete similar amounts of GrzB (~1,000 pg/ml by 1x105 cells/200 μl medium) compared to memory CD8 T cells (~600 pg/ml). Measurement of individual GrzB-secreting cells by ELISpot also indicated that similar numbers of activated memory CD4 (~170/1×105) and memory CD8 (~200/1×105) T cells secreted GrzB. Expression of CD107a further indicated that Grzb is secreted similarly by activated CD4 and CD8 T cells, consistent with the ELISA and ELISpot results. However, memory CD8 T cells expressed and secreted more perforin compared to memory CD4 T cells, suggesting that perforin may be less associated with GrzB function for memory CD4 T cells. Although measurement of intracellular GrzB by flow cytometry suggests that a larger proportion of CD8 T cells have higher capacity for GrzB production compared to CD4 T cells, ELISpot and ELISA show that similar numbers of activated CD4 and CD8 T cells secrete similar amounts of GrzB. Secretion of GrzB by activated CD8 T cells may be more tightly controlled compared to CD4 T cells.

/pdf/granzyme-b-secretion-by-human-memory-cd4-t-cells-is-less-1nq5r9s18z.pdf

Granzyme B secretion by human memory CD4 T cells is less strictly regulated compared to memory CD8 T cells

Objective Adult drug-resistant epilepsy (DRE) is associated with significant morbidity. Infiltration of immune cells is observed in DRE epileptic foci; however, the relation between DRE and the peripheral immune cell compartment remains only partially understood. We aimed to investigate differences in immune cell populations, cytokines, and neurodegenerative biomarkers in the peripheral blood of subjects with epilepsy versus healthy controls, and in DRE compared to well-controlled epilepsy (WCE). Methods Peripheral blood mononuclear cells and serum from >120 age- and sex-matched adults suffering from focal onset epilepsy and controls were analyzed by multipanel flow cytometry, multiplex immunoassays, and ultrasensitive single molecule array. Results Using a data-driven analytical approach, we identified that CD4 T cells in the peripheral blood are present in a higher proportion in DRE patients. Moreover, we observed that the frequency of CD4 T cells expressing proinflammatory cytokines interleukin (IL)-17A, IL-22, tumor necrosis factor, interferon-γ, and granulocyte-macrophage colony-stimulating factor, but not anti-inflammatory cytokines IL-10 and IL-4, is elevated in the peripheral blood of DRE subjects compared to WCE. In parallel, we found that Th17-related circulating proinflammatory cytokines are elevated, but Th2-related cytokine IL-4 is reduced, in the serum of epilepsy and DRE subjects. As Th17 cells can exert neurotoxicity, we measured levels of serum neurofilament light chain (sNfL), a marker of neuronal injury. We found significantly elevated levels of sNfL in DRE compared to controls, especially among older individuals. Significance Our data support that DRE is associated with an expansion of the CD4 Tcell subset in the peripheral blood and with a shift toward a proinflammatory Th17/Th1 CD4 Tcell immune profile. Our results further show that pathological levels of sNfL are more frequent in DRE, supporting a potential neurodegenerative component in adult DRE. With this work, we provide evidence for novel potential inflammatory and degenerative biomarkers in DRE.

Increased frequency of proinflammatory CD4 T cells and pathological levels of serum neurofilament light chain in adult drug-resistant epilepsy.

https://works.bepress.com/claudia-kozinetz/4/download/

HIV replication in conjunction with granzyme B production by CCR5+ memory CD4 T cells: implications for bystander cell and tissue pathologies

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Granzyme B secretion by human memory CD4 T cells is less strictly regulated compared to memory CD8 T cells

HIV replication in conjunction with granzyme B production by CCR5+ memory CD4 T cells: implications for bystander cell and tissue pathologies