The human "Treg MLR": immune monitoring for FOXP3+ T regulatory cell generation.
BACKGROUND
Controversy exists about the conditions effecting the development of forkhead/winghead helix transcription factor P3 (FOXP3) expressing T cells and their relevance in transplant recipients.


METHODS
We generated carboxy-fluorescein diacetate succinimidyl ester-labeled CD4+CD25 high FOXP3+ cells in mixed lymphocyte reactions (MLRs) ("the Treg MLR"), with varying human leukocyte antigen (HLA) disparities and cell components. Five color flow cytometry and H-thymidine uptakes were the readouts.


RESULTS
(1) Despite lower stimulation indices (SIs) than two DR-mismatched MLRs, 2 DR-matched MLRs generated more than twofold higher percentages when gating on proliferating CD4+CD25 high FOXP3+ cells; (2) Even with low numbers of proliferating cells, autologous and HLA identical MLRs generated the highest FOXP3+:FOXP3- cell ratios; (3) Elimination of either non-CD3+ responding cells (resulting in "direct presentation" only) or responding CD25+ (Treg generating) cells increased the SI but inhibited proliferating CD4+CD25 high FOXP3+ cell development; (4) MLR-generated CD4+CD25 high FOXP3+ cells added as third components specifically inhibited the same freshly set MLR SI and caused recruitment of new CD4+CD25 high FOXP3+ cells. As an example of the "Treg MLR" immune monitoring potential, addition of third component peripheral blood mononuclear cell containing high percentages of CD4+CD25 high FOXP3+ cells from an HLA identical kidney transplant recipient (in a tolerance protocol) caused donor-specific Treg MLR inhibition or recruitment. This was similar to the third component MLR Tregs generated entirely in vitro.


CONCLUSIONS
In the Treg MLR, the generation of CD4+CD25 high FOXP3+ cells is more pronounced in the context of self-recognition (HLA matching, indirect presentation). These cells can be assayed for MLR inhibitory and Treg recruitment functions, so as to immunologically monitor the allospecific regulation after transplantation.
Levitsky
Josh
J
Division of Hepatology, Department of Medicine, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA.
Miller
Joshua
J
Leventhal
Joseph
J
Huang
Xuemei
X
Flaa
Cathy
C
Wang
Edward
E
Tambur
Anat
A
Burt
Richard K
RK
Gallon
Lorenzo
L
Mathew
James M
JM
D017951:Antigen Presentation; D017252:CD3 Complex / Q000276:immunology; D049109:Cell Proliferation; D002478:Cells, Cultured; D005434:Flow Cytometry; D051858:Forkhead Transcription Factors / Q000276:immunology*; D006680:HLA Antigens / Q000276:immunology; D006648:Histocompatibility; D006801:Humans; D053645:Interleukin-2 Receptor alpha Subunit / Q000276:immunology; D016030:Kidney Transplantation / Q000276:immunology*; D007959:Lymphocyte Culture Test, Mixed; D011237:Predictive Value of Tests; D050378:T-Lymphocytes, Regulatory / Q000276:immunology*; D013997:Time Factors; D023001:Transplantation Tolerance
D017252:CD3 Complex; C418974:FOXP3 protein, human; D051858:Forkhead Transcription Factors; D006680:HLA Antigens; C508017:IL2RA protein, human; D053645:Interleukin-2 Receptor alpha Subunit