Katarzyna Bocian

Rapamycin, unlike cyclosporine A, enhances suppressive functions of in vitro-induced CD4+CD25+ Tregs

BACKGROUND A growing body of data shows that CD4(+)CD25(+) regulatory T cells (Tregs) can induce transplantation tolerance by suppressing immune responses to allograft antigens. However, both the generation and the suppressive capacity of CD4(+)CD25(+) Tregs can be substantially affected by different immunosuppressive drugs used in clinical transplantation. The goal of this study was to compare the effects of cyclosporine A and rapamycin on the induction and suppressive functions of human CD4(+)CD25(+) Tregs in vitro. METHODS CD4(+)CD25(+) Tregs were induced in two-way mixed lymphocyte reaction (MLR) in the presence of rapamycin (Treg-Rapa) or cyclosporine A (Treg-CsA). Tregs were identified in MLR cultures by flow cytometry using anti-CD4, anti-CD25, anti-CTLA-4, anti-CD122, anti-GITR mAbs and ant-PE-FOXP3 staining sets. Suppressive capacity of induced Tregs was evaluated by their capability to inhibit anti-CD3 Ab-triggered proliferation of peripheral blood mononuclear cells (PBMCs), as measured by flow cytometry. The concentration of TGF-beta1 in culture supernatants was measured by enzyme-linked immunosorbent assay. RESULTS Although both rapamycin and cyclosporine A suppressed the induction of CD4(+)CD25(+) Tregs during MLRs, this effect was significantly more pronounced in cells cultured with cyclosporine. On the other hand, only rapamycin significantly decreased the percentage of CD4(+)CD25(+) Tregs which expressed GITR, a negative regulator of Tregs suppressive capacity. Importantly, Treg-Rapa, unlike Treg-CsA, displayed significant suppressive activity and were capable of inhibiting the proliferation of anti-CD3 Ab-activated PBMCs. This activity was likely mediated by TGF-beta1. CONCLUSIONS Rapamycin, unlike cyclosporine A, does not inhibit the function of CD4(+)CD25(+) Tregs. This implies that rapamycin could contribute to the development of transplantation tolerance by promoting the induction of functional CD4(+)CD25(+) Tregs. Moreover, our results suggest that rapamycin could be combined with functional Tregs.

CD4+ CD25+ FOXP3+ regulatory T cells in peripheral blood and peritoneal fluid of patients with endometriosis

STUDY QUESTION Is endometriosis associated with changes in CD4⁺ CD25⁺ FOXP3⁺ regulatory T cells (Treg cells)? SUMMARY ANSWER Endometriosis is associated with disturbed compartmentalization of CD25(high) FOXP3⁺ Treg cells. WHAT IS KNOWN ALREADY Endometriosis is associated with an abrogated immune response and displays some features of an autoimmune disorder. Treg cells play a part in the development of autoimmune reactions; however, their role in pathogenesis of endometriosis is still poorly recognized. STUDY DESIGN, SIZE AND DURATION Case-control study comparing 17 women with laparoscopically and histopathologically confirmed ovarian endometriosis with 15 control women without visible endometriosis foci, pelvic inflammation or related pathology who were subjected to laparoscopic surgery between 2010 and 2011. PARTICIPANTS/MATERIALS, SETTING AND METHODS Peripheral blood and peritoneal fluid were collected during laparoscopy and T cell subpopulations were analysed by flow cytometry using specific monoclonal antibodies recognizing CD4⁺, CD25⁺ and FOXP3⁺ markers. MAIN RESULTS The percentage of CD25(high) FOXP3⁺ Treg cells was significantly decreased in the peripheral blood of women with ovarian endometriosis compared with control women. On the other hand, the proportion of these cells was significantly increased in the peritoneal fluid of women with endometriosis. LIMITATIONS, REASONS FOR CAUTION The present study is limited to patients with ovarian endometrioma and further investigations are needed, including patients with lower grade of endometriosis. WIDER IMPLICATIONS OF THE FINDINGS The present results suggest that Treg cells may play a part in immunopathogenesis of endometriosis being responsible for abrogated local cellular immune responses and facilitation and development of autoimmune reactions. Treg cells may be thus a potential target in the treatment of endometriosis. STUDY FUNDING/COMPETING INTEREST(S) This study was supported by 1M15/N/2011 and NK1W grants from the I Faculty of Medicine, Warsaw Medical University. None of the authors has any competing interests to declare.

Immunomodulatory effects of inosine pranobex on cytokine production by human lymphocytes.

Abstract Inosine pranobex (inosine dimepranol acedoben, isoprinosine) (Inos) is an immunomodulatory and antiviral drug used in some viral infections, especially in patients with weakened immunity. In the present study, effects of Inos on the production of cytokines attributable to Th1 (IL-2, IFN-g, and TNF-a) or Th2 cells (IL-4, IL-5, and IL-10) were tested in human peripheral blood lymphocyte cultures stimulated with phytohemagglutinin (PHA). Inos enhanced TNF-a secretion significantly (in short-term - 24-hour, and prolonged term - 72-hour cultures) and IFN-g (in 72-hour cultures). Surprisingly, production of IL-10 by PHA-stimulated lymphocytes was suppressed by Inos in a dose-dependent manner in both 24-hour and 72-hour cultures. These results shed some light on immunomodulatory properties of Inos and suggest applicability of this agent in patients with a depressed function of the immune system.

Expanding Diversity and Common Goal of Regulatory T and B Cells. I: Origin, Phenotype, Mechanisms

Immunosuppressive activity of regulatory T and B cells is critical to limit autoimmunity, excessive inflammation, and pathological immune response to conventional antigens or allergens. Both types of regulatory cells are intensively investigated, however, their development and mechanisms of action are still not completely understood. Both T and B regulatory cells represent highly differentiated populations in terms of phenotypes and origin, however, they use similar mechanisms of action. The most investigated CD4+CD25+ regulatory T cells are characterized by the expression of Foxp3+ transcription factor, which is not sufficient to maintain their lineage stability and suppressive function. Currently, it is considered that specific epigenetic changes are critical for defining regulatory T cell stability in the context of their suppressive function. It is not yet known if similar epigenetic regulation determines development, lineage stability, and function of regulatory B cells. Phenotype diversity, confirmed or hypothetical developmental pathways, multiple mechanisms of action, and role of epigenetic changes in these processes are the subject of this review.

Inflammatory response during slow- and fast-twitch muscle regeneration.

Introduction: Skeletal muscles are characterized by their unique ability to regenerate. Injury of a so‐called fast‐twitch muscle, extensor digitorum longus (EDL), results in efficient regeneration and reconstruction of the functional tissue. In contrast, slow‐twitch muscle (soleus) fails to properly reconstruct and develops fibrosis. This study focuses on soleus and EDL muscle regeneration and associated inflammation. Methods: We determined differences in the activity of neutrophils and M1 and M2 macrophages using flow cytometry and differences in the levels of proinflammatory cytokines using Western blotting and immunolocalization at different times after muscle injury. Results: Soleus muscle repair is accompanied by increased and prolonged inflammation, as compared to EDL. The proinflammatory cytokine profile is different in the soleus and ED muscles. Conclusions: Muscle repair efficiency differs by muscle fiber type. The inflammatory response affects the repair efficiency of slow‐ and fast‐twitch muscles. Muscle Nerve 55: 400–409, 2017

The Effects of T4 and A3/R Bacteriophages on Differentiation of Human Myeloid Dendritic Cells

Bacteriophages (phages) are viruses of bacteria. Here we evaluated the effects of T4 and A3/R bacteriophages, as well as phage-generated bacterial lysates, on differentiation of human myeloid dendritic cells (DCs) from monocytes. Neither of the phages significantly reduced the expression of markers associated with differentiation of DCs and their role in the activation of T cells (CD40, CD80, CD83, CD86, CD1c, CD11c, MHC II, PD-L1, PD-L2, TLR2, TLR4, and CCR7) and phagocytosis receptors (CD64 and DEC-205). By contrast, bacterial lysate of T4 phage significantly decreased the percentages of DEC-205- and CD1c-positive cells. The percentage of DEC-205-positive cells was also significantly reduced in DCs differentiated in the presence of lysate of A3/R phage. Thus while bacteriophages do not substantially affect differentiation of DCs, some products of phage-induced lysis of bacterial cells may influence the differentiation and potentially also some functions of DCs. Our results have important implications for phage therapy of bacterial infections because during infections monocytes recruited to the site of inflammation are an important source of inflammatory DCs.

LPS-Activated Monocytes Are Unresponsive to T4 Phage and T4-Generated Escherichia coli Lysate

A growing body of data shows that bacteriophages can interact with different kinds of immune cells. The objective of this study was to investigate whether T4 bacteriophage and T4-generated Escherichia coli lysate affect functions of monocytes, the key population of immune cells involved in antibacterial immunity. To that end, we evaluated how T4 and E. coli lysate influence the expression of main costimulatory molecules including CD40, CD80 and CD86, TLR2, TLR4 on monocytes, as well as the production of IL-6 and IL-12 in cultures of peripheral blood mononuclear cells (PBMCs). Separate experiments were performed on unactivated and LPS-activated PBMCs cultures. Both studied preparations significantly increased the percentage of CD14+CD16-CD40+ and CD14+CD16-CD80+ monocytes in unactivated PBMCs cultures, as well as the concentration of IL-6 and IL-12 in culture supernates. However, neither purified T4 nor E. coli lysate had any significant effect on monocytes in LPS-activated PBMCs cultures. We conclude that LPS-activated monocytes are unresponsive to phages and products of phage-induced lysis of bacteria. This study is highly relevant to phage therapy because it suggests that in patients with infections caused by Gram-negative bacteria the administration of phage preparations to patients and lysis of bacteria by phages are not likely to overly stimulate monocytes.

Suppressor Properties of Human CD8(+)CD28(-) T Cells in Mixed Leukocyte Reaction are not Affected by CsA and RAPA.

Human CD8+CD28− T suppressor cells were previously shown to be involved in the control of the immune response to transplanted allografts. It seems essential to examine how immunosuppressive drugs influence these cells. However, the CD8+CD28− population contains both suppressor (Ts) and cytotoxic (Tc) T cells, and the phenotype of the Ts subpopulation has not been identified explicitly. It is proposed that the transcription factor FOXP3 may be helpful in distinguishing the Ts and Tc subpopulations. The aim of this study was to evaluate the influence of the immunosuppressive drugs cyclosporine A (CsA) and rapamycin (RAPA) on the level, suppressor properties, and phenotype of human CD8+CD28− T cells in vitro. The model used was the mixed leukocyte reaction performed with peripheral blood mononuclear cells from healthy volunteers. It was observed that CD8+CD28− T cells from cultures with CsA or RAPA had similar suppressor properties to cells from control cultures, although the drugs influenced the expression of FOXP3. CsA and RAPA did not interfere with the suppressor properties of human CD8+CD28− T cells in vitro, although they affected the expression of the FOXP3 molecule.

Human peripheral blood CD8+ CD28- T cells of renal allograft recipients do not express FOXP3 protein.

INTRODUCTION In recent studies, the FOXP3 molecule has been suggested to be a marker of a suppressor subset of human CD8+ CD28- T cells based on correlations between the level of its mRNA and allograft function. Because this transcriptional factor produces a protein, we suggest that these correlations should focus on the FOXP3 protein. The aim of our study was to evaluate whether FOXP3 protein was present in cells of the CD8+ CD28- population in the peripheral blood of renal allograft recipients and whether the level of CD8+ CD28- FOXP3+ cells correlated with allograft function. METHODS The study was performed on 30 renal allograft recipients with uneventful stable courses (n=18) or biopsy-proven chronic rejection (n=12). The immunosuppression was based on cyclosporine (n=12) or rapamycin (n=9). Peripheral blood mononuclear cells isolated from recipient blood samples were labeled with anti-CD8 and anti-CD28 MAbs conjugated with fluorochromes. After incubation, washing, and labeling using a PE anti-human FOXP3 Kit, we determined the percentage of cells by flow cytometry. RESULTS FOXP3 protein expression was not observed either in the CD8+ CD28- population, or the whole populations of CD8+ or CD28- cells among patient groups. CONCLUSIONS The expression of FOXP3 protein in CD8+ CD28- cells seems to be of a questionable value as a diagnostic tool for allograft function, it is probably not a marker for the CD8+ CD28- T cell subset.

Expanding Diversity and Common Goal of Regulatory T and B Cells. II: In Allergy, Malignancy, and Transplantation

Regulation of immune response was found to play an important role in the course of many diseases such as autoimmune diseases, allergy, malignancy, organ transplantation. The studies on immune regulation focus on the role of regulatory cells (Tregs, Bregs, regulatory myeloid cells) in these disorders. The number and function of Tregs may serve as a marker of disease activity. As in allergy, the depletion of Tregs is observed and the results of allergen-specific immunotherapy could be measured by an increase in the population of IL-10+ regulatory cells. On the basis of the knowledge of anti-cancer immune response regulation, new directions in therapy of tumors are introduced. As the proportion of regulatory cells is increased in the course of neoplasm, the therapeutic action is directed at their inhibition. The depletion of Tregs may be also achieved by an anti-check-point blockade, anti-CD25 agents, and inhibition of regulatory cell recruitment to the tumor site by affecting chemokine pathways. However, the possible favorable role of Tregs in cancer development is considered and the plasticity of immune regulation should be taken into account. The new promising direction of the treatment based on regulatory cells is the prevention of transplant rejection. A different way of production and implementation of classic Tregs as well as other cell types such as double-negative cells, Bregs, CD4+ Tr1 cells are tested in ongoing trials. On the basis of the results of current studies, we could show in this review the significance of therapies based on regulatory cells in different disorders.