Allospecific regulatory effects of sirolimus and tacrolimus in the human mixed lymphocyte reaction.
BACKGROUND
Tacrolimus (TAC) and sirolimus (SRL), two commonly used immunosuppressive agents, have demonstrated contrasting immunoregulatory effects. We recently described factors affecting the generation of allospecific CD4CD25 forkhead/winged helix transcription factor P3 (FOXP3) T-regulatory (Treg) cells in mixed lymphocyte reaction (Treg MLR) and now report additional findings on the effects of TAC and SRL.


METHODS
TAC, SRL, or media without agents were added separately to MLRs using human leukocyte antigen two DR-matched and -mismatched healthy volunteers and prekidney transplant donor/recipient pairs. Concentrations correlated with subtherapeutic and therapeutic blood levels. Stimulation indices of H-TDR uptake, cell proliferation, and the generation of carboxy-fluorescein diacetate succinimidyl ester (CFSE) labeled CD4CD25FOXP3 cells by flow cytometry were initially compared. Each group of (non-CFSE labeled) MLR-generated cells were then added as third components to CFSE-labeled responding cells in freshly prepared primary MLRs, to determine allospecific and nonspecific inhibitory and Treg recruitment effects.


RESULTS
TAC inhibited stimulation indices and CD4CD25 FOXP3 cell generation in both human leukocyte antigen DR-matched and -mismatched pairs, particularly at therapeutic levels (≥5 ng/mL). SRL had an equivalent effect in matched pairs but was associated with a significantly higher %generation of CD4CD25FOXP3 cells than TAC. SRL-MLR-generated Tregs added as third components allospecifically inhibited MLR proliferation and recruited additional CFSE-labeled autologous Tregs compared with addition of TAC- or media-MLR-generated Tregs.


CONCLUSIONS
Calcineurin and mammalian target of rapamycin inhibitors have disparate effects on allospecific Treg generation using the Treg MLR. This assay can thereby be helpful in assessing allospecific regulatory effects of diverse immunosuppressive agents.
Division of Hepatology, Department of Medicine, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA. j-levitsky@northwestern.edu
Josh
J
Levitsky
Lorenzo
L
Gallon
Joshua
J
Miller
Anat R
AR
Tambur
Joseph
J
Leventhal
Catherine
C
Flaa
Xuemei
X
Huang
Bara
B
Sarraj
Edward
E
Wang
James M
JM
Mathew
D049109:Cell Proliferation / Q000187:drug effects; D006684:HLA-DR Antigens / Q000276:immunology; D006801:Humans; D007166:Immunosuppressive Agents / Q000494:pharmacology*; D066298:In Vitro Techniques; D016030:Kidney Transplantation / Q000276:immunology; D008213:Lymphocyte Activation / Q000187:drug effects; D007959:Lymphocyte Culture Test, Mixed; D020123:Sirolimus / Q000494:pharmacology*; D050378:T-Lymphocytes, Regulatory / Q000166:cytology / Q000187:drug effects* / Q000276:immunology*; D016559:Tacrolimus / Q000494:pharmacology*; D014019:Tissue Donors
D006684:HLA-DR Antigens; D007166:Immunosuppressive Agents; D020123:Sirolimus; D016559:Tacrolimus
Transplantation. 1998 Apr 15;65(7):947-55
9565100
Eur J Immunol. 2000 Jul;30(7):1807-12
10940869
Transplantation. 2006 Feb 15;81(3):445-54
16477233
Science. 1964 Feb 21;143(3608):815-6
14088079
J Clin Invest. 1997 Dec 15;100(12):3173-83
9399965
Diabetes. 2006 Jan;55(1):40-9
16380475
Transplantation. 2005 Jul 15;80(1):110-7
16003241
Transplantation. 2007 Jun 27;83(12):1611-9
17589345
Transpl Immunol. 2003 Jul-Sep;11(3-4):307-21
12967784
J Investig Med. 2001 Sep;49(5):442-9
11523700
Am J Transplant. 2010 Jun;10(6):1375-84
20486921
Blood. 2008 Jan 1;111(1):453-62
17967941
Hum Immunol. 2009 Mar;70(3):146-50
19141306
Transplantation. 2008 Mar 27;85(6):783-9
18360256
J Immunol. 2007 Jan 1;178(1):320-9
17182569
Blood. 2005 Jun 15;105(12):4743-8
15746082
Am J Transplant. 2007 Jul;7(7):1722-32
17511761
Hepatology. 2013 Jan;57(1):239-48
22234876
Transplantation. 2009 Dec 15;88(11):1303-11
19996930
J Immunol. 2007 Jun 1;178(11):7018-31
17513751
Transplantation. 2010 Apr 27;89(8):928-36
20305583
Transplantation. 2000 Oct 15;70(7):1104-6
11045651
Blood. 2006 Jul 1;108(1):390-9
16522809
Cytotherapy. 2007;9(2):144-57
17453966
J Immunol. 1977 Apr;118(4):1232-8
66282