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The flow cytometry method could support physicians’ decisions in the diagnosis and treatment monitoring of immunodeficient patients. Most clinical recommendations are focused on the search for alterations in T- and B-lymphocyte subsets, less commonly natural killer (NK) cells and granulocytes. While reference values for clinically meaningful lymphocyte subsets have been published ubiquitously among numerous countries, we have not found significant data for a population of adult Polish habitats; thus we determined reference values for T, B, and NK subsets according to sex and age. The female group showed a higher percentage of lymphocytes (CD45 ++ ), T helper lymphocytes with a higher absolute count, as well as CD4/CD8 ratio, marginal zone-like B cells, class-switched B cells, and CD21 low B cells than the male group. The male group was found to have elevated percentages of naïve B lymphocytes, transitional B cells, and plasmablasts. A weak positive correlation with age was found among double positive T lymphocytes, natural killer T cells (NKT) lymphocytes, and CD21 low B cells. A negative correlation with age for double negative T lymphocytes, marginal zone-like B cells, and plasmablasts was noted. The results indicated the importance of creating distinct reference ranges regarding sex and age concerning immunophenotype.

Due to the genetic diversity between the mother and the fetus, heightened control over the immune system during pregnancy is crucial. Immunological parameters determined by clinicians in women with idiopathic recurrent spontaneous abortion (RSA) include the quantity and activity of Natural Killer (NK) and Natural Killer T (NKT) cells, the quantity of regulatory T lymphocytes, and the ratio of pro-inflammatory cytokines, which indicate imbalances in Th1 and Th2 cell response. The processes are controlled by immune checkpoint proteins (ICPs) expressed on the surface of immune cells. We aim to investigate differences in the expression of ICPs on T cells, T regulatory lymphocytes, NK cells, and NKT cells in peripheral blood samples collected from RSA women, pregnant women, and healthy multiparous women. We aim to discover new insights into the role of ICPs involved in recurrent pregnancy loss. Peripheral blood mononuclear cells (PBMCs) were isolated by gradient centrifugation from blood samples obtained from 10 multiparous women, 20 pregnant women (11–14th week of pregnancy), and 20 RSA women, at maximum of 72 h after miscarriage. The PBMCs were stained for flow cytometry analysis. Standard flow cytometry immunophenotyping of PBMCs was performed using antibodies against classical lymphocyte markers, including CD3, CD4, CD8, CD56, CD25, and CD127. Additionally, ICPs were investigated using antibodies against Programmed Death Protein-1 (PD-1, CD279), T cell immunoglobulin and mucin domain-containing protein 3 (TIM-3, CD366), V-domain Ig suppressor of T cell activation (VISTA), T cell immunoglobulin and ITIM domain (TIGIT), and Lymphocyte activation gene 3 (LAG-3). We observed differences in the surface expression of ICPs in the analyzed subpopulations of lymphocytes between early pregnancy and RSA, after miscarriage, and in women. We noted diminished expression of PD-1 on T lymphocytes (p = 0.0046), T helper cells (CD3CD4 positive cells, p = 0.0165), T cytotoxic cells (CD3CD8 positive cells, p = 0.0046), T regulatory lymphocytes (CD3CD4CD25CD127 low positive cells, p = 0.0106), and NKT cells (CD3CD56/CD16 positive cells, p = 0.0438), as well as LAG-3 on lymphocytes T (p = 0.0225) T helper, p = 0.0426), T cytotoxic cells (p = 0.0458) and Treg (p = 0.0293), and cells from RSA women. Impaired expression of TIM-3 (p = 0.0226) and VISTA (p = 0.0039) on CD8 cytotoxic T and NK (TIM3 p = 0.0482; VISTA p = 0.0118) cells was shown, with an accompanying increased expression of TIGIT (p = 0.0211) on NKT cells. The changes in the expression of surface immune checkpoints indicate their involvement in the regulation of pregnancy. The data might be utilized to develop specific therapies for RSA women based on the modulation of ICP expression.

Although therapies based on mesenchymal stromal cells (MSCs) are being implemented in clinical settings, many aspects regarding these procedures require further optimization. Domestic dogs suffer from numerous immune-mediated diseases similar to those found in humans. This study aimed to assess the immunomodulatory activity of canine (c) Wharton jelly (WJ)-derived MSCs and refer them to human (h) MSCs isolated from the same tissue. Canine MSC(WJ)s appeared to be more prone to in vitro aging than their human counterparts. Both canine and human MSC(WJ)s significantly inhibited the activation as well as proliferation of CD4+ and CD8+ T cells. The treatment with IFNγ significantly upregulated indoleamine-2,3-dioxygenase 1 (IDO1) synthesis in human and canine MSC(WJ)s, and the addition of poly(I:C), TLR3 ligand, synergized this effect in cells from both species. Unstimulated human and canine MSC(WJ)s released TGFβ at the same level (p > 0.05). IFNγ significantly increased the secretion of TGFβ in cells from both species (p < 0.05); however, this response was significantly stronger in human cells than in canine cells. Although the properties of canine and human MSC(WJ)s differ in detail, cells from both species inhibit the proliferation of activated T cells to a very similar degree and respond to pro-inflammatory stimulation by enhancing their anti-inflammatory activity. These results suggest that testing MSC transplantation in naturally occurring immune-mediated diseases in dogs may have high translational value for human clinical trials.

Immune checkpoints (ICPs) serve as regulatory switches on immune-competent cells. Soluble ICPs consist of fragments derived from ICP molecules typically located on cell membranes. Research has demonstrated that they perform similar functions to their membrane-bound counterparts but are directly present in the bloodstream. Effective control of the maternal immune system is vital for a successful pregnancy due to genetic differences between the mother and fetus. Abnormalities in the immune response are widely acknowledged as the primary cause of spontaneous abortions. In our research, we introduce a novel approach to understanding the immune-mediated mechanisms underlying recurrent miscarriages and explore new possibilities for diagnosing and preventing pregnancy loss. The female participants in the study were divided into three groups: RSA (recurrent spontaneous abortion), pregnant, and non-pregnant women. The analysis of soluble forms of immune checkpoints and their ligands in the serum of the study groups was conducted using the Luminex method Statistically significant differences in the concentrations of (ICPs) were observed between physiological pregnancies and the RSA group. Among patients with RSA, we noted reduced concentrations of sGalectin-9, sTIM-3, and sCD155, along with elevated concentrations of LAG-3, sCD80, and sCD86 ICPs, in comparison to physiological pregnancies. Our study indicates that sGalectin-9, TIM-3, sLAG-3, sCD80, sCD86, sVISTA, sNectin-2, and sCD155 could potentially serve as biological markers of a healthy, physiological pregnancy. These findings suggest that changes in the concentrations of soluble immune checkpoints may have the potential to act as markers for early pregnancy loss.

Mycelial cultures of Lentinula edodes, an edible and medicinal mushroom, have been used in our previous research to obtain selenium-containing immunomodulatory preparations. Our current attempts to obtain a new preparation containing both selenium and zinc, two micronutrients necessary for the functioning of the immune system, extended our interest in the simultaneous accumulation of these elements by mycelia growing in media enriched with selenite and zinc(II) ions. Subsequently, we have studied the effects of new L. edodes mycelium water extracts with different concentrations of selenium and zinc on the activation of T cell fraction in human peripheral blood mononuclear cells (PBMCs). Flow cytometry analysis was used to measure the expression of activation markers on human CD4+ and CD8+ T cells stimulated by anti-CD3 and anti-CD3/CD28 antibodies (Abs). It was demonstrated that statistically significant changes were observed for PD-1 and CD25 antigens on CD8+ T cells. The selenium and zinc content in the examined preparations modified the immunomodulatory activity of mycelial polysaccharides; however, the mechanisms of action of various active ingredients in the mycelial extracts seem to be different.

Polysaccharides isolated from Lentinula edodes are bioactive compounds with immunomodulatory properties. In our previous studies from L. edodes mycelium, we have isolated a selenium(Se)-enriched fraction (named Se-Le-30), a mixture of linear 1,4-α-glucan and linear 1,3-β- and 1,6-β-glucans. In this study, we analyzed the effects of Se-Le-30 on the activation and proliferation of human T lymphocytes stimulated by anti-CD3 and anti-CD3/CD28 antibodies (Abs) and on the production of cytokines by peripheral blood mononuclear cells (PBMCs). Se-Le-30 had effects on T cell proliferation induced by Abs against CD3 and CD28. It significantly inhibited the proliferation of CD3-stimulated CD4+ and CD8+ T cells and enhanced the proliferation of CD4+ T cells stimulated with anti-CD3/CD28 Ab. Moreover, Se-Le-30 downregulated the number of CD3-stimulated CD4+CD69+ cells, CD4+CD25+ cells, as well as CD8+CD25+ cells, and upregulated the expression of CD25 marker on CD4+ and CD8+ T cells activated with anti-CD3/CD28 Abs. Furthermore, Se-Le-30 enhanced the synthesis of IFN-γ by the unstimulated and anti-CD3/CD28-stimulated PBMCs, inhibited synthesis of IL-2 and IL-4 by CD3-stimulated cells, and augmented the synthesis of IL-6 and IL-10 by unstimulated, CD3-stimulated, and CD3/CD28-stimulated PBMCs. Together, we demonstrated that Se-Le-30 exerts immunomodulatory effects on human T lymphocytes. These observations are of importance for the prospective use of Se-Le-30 in research or as a therapeutic compound.

Lentinula edodes (Berk.) Pegler, also known as shiitake mushroom, is a popular edible macrofungus and a source of numerous bioactive substances with multiple beneficial health effects. L. edodes-derived polysaccharides are the most valuable compounds, with anticancer, antioxidant, antimicrobial, and immunomodulatory properties. It has been demonstrated that their biological activity depends on the extraction method, which affects monosaccharide composition, molecular weight, branching degrees, and helical conformation. In this review, we discuss the immunomodulatory properties of various polysaccharides from L. edodes in animal models and in humans.

A common edible mushroom Lentinula edodes, is an important source of numerous biologically active substances, including polysaccharides, with immunomodulatory and antitumor properties. In the present work, the biological activity of the crude, homogenous (Se)-enriched fraction (named Se-Le-30), which has been isolated from L. edodes mycelium by a modified Chihara method towards human peripheral blood mononuclear cells (PBMCs) and peripheral granulocytes, was investigated. The Se-Le-30 fraction, an analog of lentinan, significantly inhibited the proliferation of human PBMCs stimulated with anti-CD3 antibodies or allostimulated, and down-regulated the production of tumor necrosis factor (TNF)-α by CD3+ T cells. Moreover, it was found that Se-Le-30 significantly reduced the cytotoxic activity of human natural killer (NK) cells. The results suggested the selective immunosuppressive activity of this fraction, which is non-typical for mushroom derived polysaccharides.

Despite intensive clinical research on the use of mesenchymal stromal cells (MSCs), further basic research in this field is still required. Herein, we compared human bone marrow MSCs (BM-MSCs, n = 6) and Wharton’s jelly MSCs (WJ-MSCs, n = 6) in their ability to interact with human primary macrophages. Evaluation of secretory potential revealed that under pro-inflammatory stimulation, WJ-MSCs secreted significantly more IL-6 than BM-MSCs (2-fold). This difference did not translate into the effect of MSCs on macrophages: both types of MSCs significantly directed M1-like macrophages toward the M2 phenotype (based on CD206 expression) to a similar extent. This observation was consistent both in flow cytometry analysis and immunocytochemical assessment. The effect of MSCs on macrophages was sustained when IL-6 signaling was blocked with Tocilizumab. Macrophages, regardless of polarization status, enhanced chemotaxis of both BM-MSCs and WJ-MSCs (p < 0.01; trans-well assay), with WJ-MSCs being significantly more responsive to M1-derived chemotactic signals than BM-MSCs. Furthermore, WJ-MSCs increased their motility (scratch assay) when exposed to macrophage-conditioned medium while BM-MSCs did not. These results indicate that although both BM-MSCs and WJ-MSCs have the ability to reciprocally interact with macrophages, the source of MSCs could slightly but significantly modify the response under clinical settings.

The aim of our study was to determine whether sildenafil citrate influences the production of Th1- (TNF-α, INF-γ) or Th2-type (TGF-β, IL-10) cytokines by lymphocytes of healthy men. Sildenafil citrate (SC) is a selective blocker of phosphodiesterase 5, by competing for the binding site with cGMP. It was reported that a higher risk of sexually transmitted diseases (STD) could be correlated with a recreational use of sildenafil, especially when combined with another drug. While behavioral causes of these findings are understood, it is worth considering other causes of that phenomenon that might rely on the influence of sildenafil on the immune system. Material and Methods. Peripheral blood mononuclear cells (PBMCs) were isolated from 27 healthy men donors and cultured in the presence of SC at a concentration of 400 ng/ml. The first set of research was performed on cells stimulated, for at least 4 hours, by incubation with phorbol myristate acetate (PMA), ionomycin, and Golgi-Stop. Subsequently, we determined cytokine production in cells stimulated with phytohemagglutinin (PHA) for 12 hours in the presence of Golgi-Stop. Flow cytometry immunophenotyping of PBMC was performed towards the surface marker of T cells: CD3 and intracellular cytokine expression: TNF-α, IFN-γ, TGF-β, and IL-10. Our findings show that SC significantly decreased the percentage of T cells producing TNF-α and displayed tendency to decrease IFN-γ, when stimulated with PMA. Frequent usage of SC might strengthen this effect. That could partially explain the impaired immune response to the pathogens of men using the drug.