Inhibitory Effects of Cyclosporine on Human Regulatory T Cells In Vitro

doi:10.1016/j.transproceed.2009.08.043

Transplantation Proceedings

Volume 41, Issue 8, October 2009, Pages 3371-3374

https://doi.org/10.1016/j.transproceed.2009.08.043 Get rights and content

Abstract

Background

Inevitable hepatitis C virus (HCV) recurrence after liver transplantation is a major barrier to the survival of a transplanted liver. It may be promoted by immunosuppression and the emergence of CD4⁺CD25⁺ regulatory T cells (Treg). Treg cells can mediate the induction and maintenance of immunological self-tolerance as well as transplant tolerance. We investigated the effects of cyclosporine (CsA), a widely used immunosuppressive agent, on human CD4⁺CD25⁺ Treg cells.

Methods

Human CD4⁺CD25⁺ cells isolated from healthy donors were cultured in the presence of 40 or 400 ng/mL CsA. The suppressive activity of Treg was assessed in mixed leukocyte reactions (MLR) using CD25⁺ and autologous activated peripheral blood mononuclear cells (PBMC). Phenotype analysis (flow cytometric, Q-PCR) and cytokine production (ELISA) of Treg cells were then performed on cultures.

Results

CsA (40 or 400 ng/mL) inhibited the proliferative capacity of PBMC and CD4⁺CD25⁺ Treg in a dose-dependent manner. Interestingly, addition of 40 ng/mL CsA in MLR impaired the suppressive activity of CD4⁺CD25⁺ cells, whereas a higher dose of CsA had no effect on Treg function. It appears that a therapeutic dose of CsA (40 ng/mL) did not change the phenotype of CD4⁺CD25⁺ T cells, but altered Treg activity by switching the regulatory to an inflammatory cytokine profile.

Conclusion

CsA significantly impaired the function of CD4⁺CD25⁺ Treg cells by inducing interleukin-2 (IL-2) and interferon-γ (IFN-γ) secretion. The present studies suggested that CsA may block the induction of immune tolerance and decrease the risk of hepatitis C recurrence.

Section snippets

Isolation of Human CD4⁺CD25⁺ Cells

CD4⁺ CD25⁺ T cells were isolated from healthy donor peripheral blood mononuclear cells (PBMC) using magnetic beads as recommended by the manufacturer (Miltenyi Biotec, Berlin, Germany).

Flow Cytometric Analysis

Cell purity (>95%) was analyzed using flow cytometry (EPICS XL-MCL, Coulter, Harbor, Calif, USA). Cells washed with phosphate-buffered saline (PBS) were labelled with fluorochrome-conjugated monoclonal antibodies (mAbs). Samples incubated in the dark for 20 minutes were washed twice and analyzed. The antibodies

Results

Freshly isolated CD4⁺CD25⁺ elements were phenotypically characterized by flow cytometry. Based on CD4+ and CD25+ expression, Treg purity was 95%, whereas the CD4+CD25− fraction or the intermediate CD25 expression was not considered to express this marker (Fig 1A). As recently described,⁹ 90% of Treg are CD4+CD25+ and CD127− (Fig 1A). Freshly isolated CD4⁺CD25⁺ were also functionally characterized by mixed leukocyte reaction (MLR), after 48 hours of culture using activated PBMCs plus CD4+CD25+

Discussion

The aim of this study was to elucidate whether CsA affects the induction of immune tolerance, leading to hepatitis recurrence after OLT, by altering Treg activity. Our data suggested that CsA inhibits Treg activity, which fits with other data obtained in both mouse and human studies.7, 10, 11, 12 The main finding of our study was that CsA affects Treg activity only when used at therapeutic doses (40 ng/mL), presumably by affecting inflammatory cytokine production (IL-2 and IFN-γ); whereas

References (12)

M. Charlton
Liver biopsy, viral kinetics, and the impact of viremia on severity of hepatitis C virus reccurence
Liver Transplant
(2003)
S.C. Rayhill et al.
A cyclosporine-based immunosuppression regimen may be better than tacrolimus for long-term liver allograft survival in recipients transplanted for hepatitis C
Transplant Proc
(2006)
F. Villamil et al.
Long-term outcomes in liver transplant patients with hepatic C infection receiving tacrolimus or cyclosporine
Transplant Proc
(2006)
P. Hoffmann et al.
Large-scale in vitro expansion of polyclonal human CD4+CD25 high regulatory T cells
Blood
(2004)
R. Zeiser et al.
Inhibition of CD4+CD25+ regulatory T-cell function by calcineurin-dependent interleukin-2 production
Blood
(2006)
J.P. Lamelin et al.
Lymphotropism oh hepatitis B and C viruses: an update and a newcomer
Int J Clin Lab Res
(1995)

There are more references available in the full text version of this article.

Cited by (50)

Immunosuppressive drugs modes of action
2021, Best Practice and Research: Clinical Gastroenterology
The innate and adaptive immune systems work as a complex interplay between different cell types, involving cytokines and chemokines mediating extracellular and paracrine effects. At the intracellular level, the inflammatory cascade is mediated by multifaceted processes that have been better described in the last 10 years. Immunosuppressive agents available in clinical practice act at different points of those cascades at the intracellular or extracellular level. Those drugs can mediate their effects on one or more cell types finally limiting inflammation and immune responses to antigens. Every immunosuppressive agent is characterized by intrinsic toxicity and side effects that may be due to the same therapeutic pathways or to off-target secondary effect of each molecule. We will here review the mechanisms of action of the most widely used immunosuppressive agents in the field of solid organ transplantation and autoimmune disorders, describing the mechanisms underlying both the therapeutic and secondary effects.
Modulatory effect of rapamycin and tacrolimus on monocyte-derived dendritic cells phenotype and function
2021, Immunobiology
Immunosuppressive-drugs are needed after solid organ transplantation to prevent allograft rejection but induce severe side effects. Understanding the alloimmune response is critical to modulate it and to achieve graft operational tolerance. The role of regulatory T cells and tolerogenic dendritic cells (Tol-DCs) is undoubtedly essential in tolerance induction. Tacrolimus is considered as the cornerstone of immunosuppression in solid organ transplantation. mTOR inhibitor such as rapamycin are thought to induce tolerance and are used as anticancer drugs in several cancers. The aim of this study was to better understand the effect of these immunosuppressive drugs on the differentiation, maturation and function of human monocyte derived dendritic cells (DCs).
DCs were differentiated from monocytes of healthy donors with either rapamycin (Rapa-DCs) or tacrolimus (Tac-DCs). The phenotype was evaluated by flow cytometry analysis. The production of pro- and anti-inflammatory cytokines was assessed by ELISA. The mRNA expression level of IDO and PD-L1 was assessed by RTqPCR. Mixed leukocytes reactions were performed to analyse suppressive activity of DCs.
Rapa-DC were characterised by a lower expression of the co-stimulatory molecules and CD83 than control-DCs (CTR-DC) (p < 0.05). In contrast, tacrolimus had no effect on the expression of surface markers compared to CTR-DCs. Rapamycin reduced both IL-12 and IL-10 secretions (p < 0.05). Rapa-DCs had a suppressive effect on CD4⁺ allogenic T cells compared to CTR-DCs (p < 0.05). However, neither Rapa-DCs nor Tac-DCs favoured the emergence of a CD4⁺CD25^highFoxp3⁺ population compared to CTR-DCs. Surprisingly, Rapa-DCs had a reduced expression of IDO and PD-L1 compared to Tac-DCs and CTR-DCs.
Rapa-DCs exhibit an incomplete phenotypic tolerogenic profile. To our knowledge this is the first paper showing a reduction of expression of pro-tolerogenic enzyme IDO in DCs. Tacrolimus does not change the phenotypical or functional characteristics of moDCs.
Sirolimus versus cyclosporine for the treatment of pediatric chronic immune thrombocytopenia: A randomized blinded trial
2020, International Immunopharmacology
Chronic immune thrombocytopenia (ITP) of childhood is still a problem. For treating ITP, several immunosuppressive medications can be considered with various response rates. Our goal was to compare effects of sirolimus and cyclosporine on children with chronic ITP.
This randomized and blinded trial was carried out on 67 children over 5 years old with chronic ITP. Patients were assigned 1:1 to cyclosporine and sirolimus for 6 months. Platelet count was assessed and compared between 2 study groups at different intervals. The clinical trial registry number was IRCT20180501039499N1.
Sixty-one children completed the 6-month treatment. Mean age was 9.3 years with an excess of females. Compared to baseline values, both drugs caused a significant increase in number of platelets over the course of treatment; sirolimus group: 15,800/mcL vs 96,566/mcL, (P < 0.001), cyclosporine group: 14,400/mcL vs 111,266/mcL, P < 0.001,). In addition, differences of platelet number were statistically significant at some treatment intervals (3rd and 6th month, P < 0.05). A quicker response was observed in children receiving cyclosporine. Both drugs had similar rate of response which occurred in 50% of included patients. Finally, sirolimus had a better safety profile.
Our study showed that cyclosporine and sirolimus had an equal rate of response in treating chronic ITP of children. At the same time, the two medications showed significant differences in their side effects.
Early reduction of regulatory T cells is associated with acute rejection in liver transplantation under tacrolimus-based immunosuppression with basiliximab induction
2020, American Journal of Transplantation
Regulatory T (Treg) cells are important in preventing acute rejection (AR) in solid organ transplantation, but the clinical relevance of the different kinetics early after liver transplantation (LT) in acute rejectors and non-rejectors is unclear. We analyzed peripheral blood samples of 128 LT recipients receiving basiliximab induction plus tacrolimus immunosuppression. Samples were obtained at pretransplant, D7, and D30 after LT. Frequency and phenotype of Tregs were analyzed by flow cytometry. The predictive value of Treg frequency at D7 was assessed for suspected acute rejection (SAR) and was validated for biopsy-proven AR (BPAR). We found that the frequencies of total and activated Tregs at D7 were significantly lower in recipients with SAR and BPAR. Treg was more reduced in BPARs by in vitro tacrolimus treatment in the presence of basiliximab. Moreover, an early reduction of Treg frequency in rejectors was associated with a greater increase in Treg apoptosis and further attenuated IL-2 signaling. D7 Treg frequency was an independent risk factor for SAR, which was also validated for BPAR. In conclusion, first-week peripheral blood Treg frequency correlates with AR after LT under tacrolimus-based immunosuppression, which needs to be proven in larger, geographically and clinically diverse populations.
NFAT-Specific Inhibition by dNP2-VIVITAmeliorates Autoimmune Encephalomyelitisby Regulation of Th1 and Th17
2020, Molecular Therapy Methods and Clinical Development
Citation Excerpt :
Treatment with calcineurin inhibitors upregulated the expression of transforming growth factor β (TGF-β) and endothelin, which induced nephrotoxic effects such as endothelial dysfunction, impaired glomerular filtration, systemic hypertension, and tubular fibrosis.49–52 Moreover, recent studies have reported that calcineurin inhibitors negatively affected Treg cell proliferation and function, which have a pivotal role in immune tolerance.31,32 To overcome the limitations associated with the currently used calcineurin inhibitors, we developed the VIVIT peptide.
Nuclear factor of activated T cells (NFATs) is an important transcription factor for T cell activation and proliferation. Recent studies have highlighted the role of NFATs in regulating the differentiation of effector CD4 T helper (Th) subsets including Th1 and Th17 cells. Because controlling the effector T cell function is important for the treatment of autoimmune diseases, regulation of NFAT functions in T cells would be an important strategy to control the pathogenesis of autoimmune diseases. Here, we demonstrated that an NFAT inhibitory peptide, VIVIT conjugated to dNP2 (dNP2-VIVIT), a blood-brain barrier-permeable peptide, ameliorated experimental autoimmune encephalomyelitis (EAE) by inhibiting Th1 and Th17 cells, but not regulatory T (T_reg) cells. dNP2-VIVIT negatively regulated spinal cord-infiltrating interleukin-17A (IL-17A) and interferon (IFN)-γ-producing CD4⁺ T cells without affecting the number of Foxp3⁺ CD4⁺ T_reg cells, whereas dNP2-VEET or 11R-VIVIT could not significantly inhibit EAE. In comparison with cyclosporin A (CsA), dNP2-VIVIT selectively inhibited Th1 and Th17 differentiation, whereas CsA inhibited the differentiation of all T cell subsets including that of Th2 and T_reg cells. Collectively, this study demonstrated the role of dNP2-VIVIT as a novel agent for the treatment of autoimmune diseases such as multiple sclerosis by regulating the functions of Th1 and Th17 cells.
The Immune Response to the Allograft
2017, Kidney Transplantation, Bioengineering, and Regeneration: Kidney Transplantation in the Regenerative Medicine Era
Allotransplantation is the most common treatment for end stage kidney, liver, lung, and heart disease. In order to accommodate the allograft, the recipient must be immune-suppressed, to prevent rejection of the graft by the innate and adaptive immune system. Acute rejection has been largely avoided through improved methods of organ procurement, surgical techniques, and immunosuppressive regimens. However, long-term immunosuppression has many drawbacks, leaving patients susceptible to nephrotoxicity, malignancies, and infection. Tolerance is the absolute goal in clinical transplantation, where a patient can be weaned from immunosuppression with no subsequent rejection; however, this is rarely achieved. Withdrawal from these therapies usually results in allograft destruction by the recipient immune system. Here, we outline current knowledge on the immune response to the allograft at the innate and adaptive levels, the impact of withdrawal from immunosuppression and subsequent allograft rejection mediated by a highly activated T cell compartment, and the role of homeostatic proliferation in this setting. Finally, we describe current investigations into novel therapies for the induction of tolerance.

View all citing articles on Scopus

: V.P. and N.D. equally contributed to the work.

View full text

Experimental transplantationInhibitory Effects of Cyclosporine on Human Regulatory T Cells In Vitro

Abstract

Background

Methods

Results

Conclusion

Section snippets

Isolation of Human CD4+CD25+ Cells

Flow Cytometric Analysis

Results

Discussion

Liver Transplant

Transplant Proc

Transplant Proc

Blood

Blood

Lymphotropism oh hepatitis B and C viruses: an update and a newcomer

Int J Clin Lab Res

Experimental transplantation
Inhibitory Effects of Cyclosporine on Human Regulatory T Cells In Vitro

Isolation of Human CD4⁺CD25⁺ Cells