Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2009 May 21;113(21):5125-33.
doi: 10.1182/blood-2009-01-199950. Epub 2009 Mar 18.

Selective expression of latency-associated peptide (LAP) and IL-1 receptor type I/II (CD121a/CD121b) on activated human FOXP3+ regulatory T cells allows for their purification from expansion cultures

Dat Q Tran  1 , John Andersson, Donna Hardwick, Lolita Bebris, Gabor G Illei, Ethan M Shevach
Affiliations

Selective expression of latency-associated peptide (LAP) and IL-1 receptor type I/II (CD121a/CD121b) on activated human FOXP3+ regulatory T cells allows for their purification from expansion cultures

Dat Q Tran et al. Blood. .

Abstract

Although adoptive transfer of regulatory T cells (Foxp3(+) Tregs) has proven to be efficacious in the prevention and treatment of autoimmune diseases and graft-versus-host disease in rodents, a major obstacle for the use of Treg immunotherapy in humans is the difficulty of obtaining a highly purified preparation after ex vivo expansion. We have identified latency-associated peptide (LAP) and IL-1 receptor type I and II (CD121a/CD121b) as unique cell-surface markers that distinguish activated Tregs from activated FOXP3(-) and FOXP3(+) non-Tregs. We show that it is feasible to sort expanded FOXP3(+) Tregs from non-Tregs with the use of techniques for magnetic bead cell separation based on expression of these 3 markers. After separation, the final product contains greater than 90% fully functional FOXP3(+) Tregs. This novel protocol should facilitate the purification of Tregs for both cell-based therapies as well as detailed studies of human Treg function in health and disease.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Treg expansion cultures contain cytokine-producing FOXP3− and FOXP3+ non-Tregs. (A) Fold expansion of magnetic bead–purified CD4+CD127lowCD25+ T cells from 6 donors after stimulation with anti-CD3/CD28 and IL-2 in the absence (none) or presence of rapamycin. Error bars represent SEM. (B) Percentage of CD4+FOXP3+ T cells in the starting population and after expansion of CD4+CD127lowCD25+ T cells as described in panel A. Horizontal lines represent the mean of each group. (C) Day 14 expansion cultures generated in the absence (none) or presence of rapamycin were restimulated for 5 hours with PMA/ionomycin. IFN-γ and IL-2 production was evaluated by intracellular staining. Data are representative of 6 independent experiments. The number in each quadrant represents the percentage of total population.
Figure 2
Figure 2
Selective expression of LAP, CD121a, and CD121b on activated Tregs. (A) Flow cytometric analysis of surface LAP, CD121a, and CD121b and intracellular FOXP3 expression on day 14 and day 21 Treg expansion cultures after restimulation for 48 hours with anti-CD3/CD28 (data are from 1 representative donor of 6). (B) Kinetics of LAP, CD121a, and CD121b expression on fresh Tregs stimulated with anti-CD3/CD28 and 100 U/mL IL-2. (C) Costaining of LAP, CD121a, and CD121b on 48-hour restimulated day 14 cultures (data are from 1 donor representative of 6). (D) Expression of LAP, CD121a, and CD121b on 48-hour restimulated day 14 CD4+CD25−CD127+CD45RA+ and CD45RO+ T cells previously stimulated on day 0 with anti-CD3/CD28 and IL-2 for 5 days in the absence (−) or presence (+) of TGFβ1 and rested in IL-2 until day 12. Data are representative of 3 independent experiments. Number in each quadrant represents the percentage of total population.
Figure 3
Figure 3
Selective expression of LAP and CD121b allows for separation of Tregs from non-Tregs in ex vivo expansion cultures. (A) Percentage of CD4+LAP+ cells after 48 hours of restimulation of day 14 and 21 expansion cultures. (B) Day 14 expansion cultures generated in the absence (CD25+/NONE) or presence (CD25+/RAPA) of rapamycin were restimulated for 48 hours with anti-CD3/CD28. LAP+/LAP− and CD121b+/CD121b− fractions were then purified with magnetic beads and analyzed by flow cytometry for FOXP3 expression. Number in each quadrant represents the percentage of total population. (C) Percentage of CD4+FOXP3+ and (D) cell yield after purification of LAP+ cells from restimulated 14-day expansion cultures. Horizontal lines in panels A, C, and D represent the mean of each group.
Figure 4
Figure 4
LAP+ and CD121b+ Tregs are anergic and manifest potent T-suppressor activity. CD4+CD25− T cells were stimulated with anti-CD3 and APCs alone (■) or in the presence of various numbers of unseparated CD25+ or purified LAP+, LAP−, CD121b+, and CD121b− cells from 48 hours of restimulated day 14 cultures generated in the absence (A) or presence (B) of rapamycin. Error bars represent SEM. 3H-TdR incorporation was determined after 72 hours of stimulation. CFSE-labeled CD4+CD25− T cells were stimulated with anti-CD3 and APCs alone or in the presence of unseparated CD25+ or purified LAP+, LAP−, CD121b+, and CD121b− cells from 48-hour restimulated day 14 cultures generated in the absence (C) or presence (D) of rapamycin. Cocultures were performed at responder to suppressor ratios of 4:1 and 8:1. CFSE dilution was measured by FACS analysis after 72 hours of culture. Number in each quadrant represents percentage of dividing cells from the total population. Data are from 1 donor representative of 6.
Figure 5
Figure 5
LAP+, CD121a+, and CD121b+ Tregs maintain purity, suppressive function, and phenotype after expansion. (A) Expression and purification based on LAP, CD121a, and CD121b on 48-hour restimulated CD25+ cells initially obtained with one-step CD25 selection method and expanded for 21 days. (B) Analysis of FOXP3 during an additional 21 more days of expansion for LAP+, CD121a+, and CD121b+ Tregs with anti-CD3/CD28 Dynabeads and IL-2 and (C) their typical Treg surface markers at the end of the 21-day expansion (42 total days in culture). Data are representative of CD121a+ and CD121b+ Tregs as well. Numbers in quadrants in panels A and B represent percentage of total population. (D) In vitro suppression assay of day 21 unseparated CD25+ cells (CD25+) and postpurified LAP+, CD121a+, and CD121b+ Tregs from panel A. Error bars represent SEM. (E) In vitro suppression assay of fresh Tregs (CD25hi) and 21-day expanded LAP+, CD121a+, and CD121b+ Tregs from panel B. Error bars represent SEM.
Figure 6
Figure 6
Expansion and purification of Tregs from patients with Sjögren syndrome and SLE with 5 to 10 mL peripheral blood. Expression of LAP, CD121a, CD121b, and FOXP3 on CD4+ T cells from a patient with (A) Sjögren syndrome or (B) SLE (top). Correlation of LAP, CD121a, and CD121b with FOXP3 on 48-hour restimulated CD25+ cells from one-step CD25 selection method expanded for 14 days (bottom). Numbers in each quadrant represent percentage of total population. Right panel represents the FOXP3 purity after reisolation with anti-LAP from day 14–expanded CD25+ cells of patients with Sjögren syndrome and SLE. In vitro suppression assay of LAP+ Tregs from patient with (C) Sjögren syndrome or (D) SLE, comparing with LAP+ Tregs from healthy control donors (HD). Data represent 1 of 3 patients with Sjögren syndrome and SLE. Error bars represent SEM.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Sakaguchi S, Ono M, Setoguchi R, et al. Foxp3+ CD25+ CD4+ natural regulatory T cells in dominant self-tolerance and autoimmune disease. Immunol Rev. 2006;212:8–27. - PubMed
    1. Valencia X, Yarboro C, Illei G, Lipsky PE. Deficient CD4+CD25high T regulatory cell function in patients with active systemic lupus erythematosus. J Immunol. 2007;178:2579–2588. - PubMed
    1. Lindley S, Dayan CM, Bishop A, Roep BO, Peakman M, Tree TI. Defective suppressor function in CD4(+)CD25(+) T-cells from patients with type 1 diabetes. Diabetes. 2005;54:92–99. - PubMed
    1. Hafler DA, Slavik JM, Anderson DE, O'Connor KC, De Jager P, Baecher-Allan C. Multiple sclerosis. Immunol Rev. 2005;204:208–231. - PubMed
    1. Solomou EE, Rezvani K, Mielke S, et al. Deficient CD4+ CD25+ FOXP3+ T regulatory cells in acquired aplastic anemia. Blood. 2007;110:1603–1606. - PMC - PubMed

Publication types

MeSH terms