Immunoregulatory effects of everolimus on in vitro alloimmune responses

Josh Levitsky; Joshua Miller; Xuemei Huang; Lorenzo Gallon; Joseph R. Leventhal; James M. Mathew

doi:10.1371/journal.pone.0156535

Immunoregulatory effects of everolimus on in vitro alloimmune responses

Josh Levitsky, Joshua Miller, Xuemei Huang, Lorenzo Gallon, Joseph R. Leventhal, James M. Mathew

Research output: Contribution to journal › Article › peer-review

12 Scopus citations

Abstract

Everolimus (EVL) is a novel mTOR-inhibitor similar to sirolimus (SRL) that is used in organ transplant recipients, often in combination with tacrolimus (TAC) or mycophenolate (MPA). The current study aims to determine its effects on regulatory T cells. Increasing concentrations of EVL, MPA and TAC alone or in combination were added to MLRs of healthy volunteers. Lymphoproliferation by ³H-TdR incorporation and the percentage of newly generated CD4⁺CD127^-CD25⁺FOXP3⁺ (total Treg) and CD4⁺CD127^-CD25^HighFOXP3⁺ (natural Treg) in CFSE labeled responder cells were assessed by flow cytometry. In comparison to medium controls, EVL and other agents dose-dependently inhibited ³H-TdR incorporation in HLA-2DR-matched and HLA-mismatched MLRs (n = 3-10). However, EVL significantly amplified newly generated total and natural Tregs in CFSE labeled responder cells (p<0.05) at all concentrations, while MPA and SRL did this only at sub-therapeutic concentrations and inhibited at therapeutic levels. In contrast, TAC inhibited newly generated Tregs at all concentrations. When tested in combination with TAC, EVL failed to reverse TAC inhibition of Treg generation. Combinations of EVL and low concentrations of MPA inhibited proliferation and amplified Treg generation in an additive manner when compared to medium controls or each drug tested alone (p<0.05). The relative tolerogenic effect from high to low was EVL > SRL> MPA > TAC. If the results from these in vitro studies are extrapolated to clinical transplantation, it would suggest EVL plus low concentrations of MPA may be the most tolerogenic combination.

Original language	English (US)
Article number	e0156535
Journal	PloS one
Volume	11
Issue number	6
DOIs	https://doi.org/10.1371/journal.pone.0156535
State	Published - Jun 1 2016

Funding

This work was supported by Novartis (CRAD001AUSNC10T; https://www.novartis.com/; JL; JM and JMM). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. However, the funders approved the publication of the results. This work was supported by the National Institutes of Health (2R01DK25243-25A2; http://www.niddk.nih.gov; JM). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors declare that they have received funding from a commercial source: "Novartis" in the form of a research grant (CRAD001AUSNC10T) consisting of funds for supplies and salary support. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. However, the funders approved the publication of the results. This does not alter the authors' adherence to PLOS ONE policies on sharing data and materials.

ASJC Scopus subject areas

General
General Biochemistry, Genetics and Molecular Biology
General Agricultural and Biological Sciences

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title = "Immunoregulatory effects of everolimus on in vitro alloimmune responses",

abstract = "Everolimus (EVL) is a novel mTOR-inhibitor similar to sirolimus (SRL) that is used in organ transplant recipients, often in combination with tacrolimus (TAC) or mycophenolate (MPA). The current study aims to determine its effects on regulatory T cells. Increasing concentrations of EVL, MPA and TAC alone or in combination were added to MLRs of healthy volunteers. Lymphoproliferation by 3H-TdR incorporation and the percentage of newly generated CD4+CD127-CD25+FOXP3+ (total Treg) and CD4+CD127-CD25HighFOXP3+ (natural Treg) in CFSE labeled responder cells were assessed by flow cytometry. In comparison to medium controls, EVL and other agents dose-dependently inhibited 3H-TdR incorporation in HLA-2DR-matched and HLA-mismatched MLRs (n = 3-10). However, EVL significantly amplified newly generated total and natural Tregs in CFSE labeled responder cells (p<0.05) at all concentrations, while MPA and SRL did this only at sub-therapeutic concentrations and inhibited at therapeutic levels. In contrast, TAC inhibited newly generated Tregs at all concentrations. When tested in combination with TAC, EVL failed to reverse TAC inhibition of Treg generation. Combinations of EVL and low concentrations of MPA inhibited proliferation and amplified Treg generation in an additive manner when compared to medium controls or each drug tested alone (p<0.05). The relative tolerogenic effect from high to low was EVL > SRL> MPA > TAC. If the results from these in vitro studies are extrapolated to clinical transplantation, it would suggest EVL plus low concentrations of MPA may be the most tolerogenic combination.",

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N2 - Everolimus (EVL) is a novel mTOR-inhibitor similar to sirolimus (SRL) that is used in organ transplant recipients, often in combination with tacrolimus (TAC) or mycophenolate (MPA). The current study aims to determine its effects on regulatory T cells. Increasing concentrations of EVL, MPA and TAC alone or in combination were added to MLRs of healthy volunteers. Lymphoproliferation by 3H-TdR incorporation and the percentage of newly generated CD4+CD127-CD25+FOXP3+ (total Treg) and CD4+CD127-CD25HighFOXP3+ (natural Treg) in CFSE labeled responder cells were assessed by flow cytometry. In comparison to medium controls, EVL and other agents dose-dependently inhibited 3H-TdR incorporation in HLA-2DR-matched and HLA-mismatched MLRs (n = 3-10). However, EVL significantly amplified newly generated total and natural Tregs in CFSE labeled responder cells (p<0.05) at all concentrations, while MPA and SRL did this only at sub-therapeutic concentrations and inhibited at therapeutic levels. In contrast, TAC inhibited newly generated Tregs at all concentrations. When tested in combination with TAC, EVL failed to reverse TAC inhibition of Treg generation. Combinations of EVL and low concentrations of MPA inhibited proliferation and amplified Treg generation in an additive manner when compared to medium controls or each drug tested alone (p<0.05). The relative tolerogenic effect from high to low was EVL > SRL> MPA > TAC. If the results from these in vitro studies are extrapolated to clinical transplantation, it would suggest EVL plus low concentrations of MPA may be the most tolerogenic combination.

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Immunoregulatory effects of everolimus on in vitro alloimmune responses

Abstract

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