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, 118 (20), 5671-80

Ex Vivo Expansion of Human Tregs Specific for Alloantigens Presented Directly or Indirectly

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Ex Vivo Expansion of Human Tregs Specific for Alloantigens Presented Directly or Indirectly

Anandharaman Veerapathran et al. Blood.

Abstract

Adoptive transfer of regulatory T cells (Tregs) prevents GVHD in experimental animals. Because antigen activation drives Treg function, we measured the frequency, growth requirements, and function of alloantigen-specific (allospecific) Tregs from human blood. When alloantigen was presented directly, the precursor frequency of allo-specific Tregs in normal individuals was 1.02% (95% confidence interval [95% CI]: 0.65-1.59) and non-Tregs 1.56% (95% CI: 0.94-2.55). When alloantigen was presented indirectly, the frequency of specific Tregs was approximately 100-fold less. Purified Tregs were expanded with APCs, rapamycin, IL-2, and IL-15. In 12 days, allo-specific Tregs expanded 793-fold (95% CI: 480-1107), with duplication approximately every 24 hours. Purified allo-specific Tregs suppressed responses to specific alloantigen selectively and were approximately 100-fold more potent than polyspecific Tregs and nonexpanded Tregs. Allo-specific Tregs maintained high expression of Foxp3, Bcl-2, lymphoid homing receptor CD62L, and chemokine receptor CCR7, predicting sustained function and migration to lymphoid tissues in vivo. Allo-specific Tregs produced TGF-β and IL-10 and expressed more cytoplasmic CTLA-4 compared with non-Tregs. These data provide a platform for the selective expansion of Tregs against major and possibly minor histocompatibility antigens and predict the feasibility of adoptive immunotherapy trials using Tregs with indirect allo-recognition for preventing GVHD while sparing GVL effects.

Figures

Figure 1
Figure 1
Purification of Tregs from human blood. Tregs were isolated using the CD4+CD25+CD127 Treg isolation kit II magnetic MicroBead method (Miltenyi Biotec). The purity of human Tregs and phenotype expression was analyzed as follows: CD4+ cells were gated and analyzed for surface expression IL-7 receptorα chain (CD127) and IL-2 receptorα chain (CD25) and for intracellular expression of Foxp3.
Figure 2
Figure 2
Allo-specific Treg-proliferative response and expansion. (A) Tregs were freshly purified and stimulated with γ-irradiated HLA-mismatched APCs plus rapamycin (100 ng/mL) in the presence or absence of IL-2 (10 U/mL) and IL-15 (10 ng/mL). Each bar represents mean ± SD of 3H-thymidine uptake (CPM) on day 6. Results are one representative of 5 individual experiments. (B) Expansion of allo-specific Tregs (2 × 104/well in triplicate) stimulated with allogeneic DCs (10:1) plus rapamycin (100 ng/mL) in the presence or absence of IL-2 (10 U/mL) or IL-15 (10 ng/mL) for 12 days. Each bar represents mean ± SD of the absolute number of Tregs obtained by timed acquisition after 12 days of expansion. A representative of 5 experiments with similar results is shown. (C) Absolute number of Tregs 12 days after stimulation with allogeneic DCs in the presence or absence of rapamycin, IL-2 (10 U/mL), or IL-15 (10 ng/mL). (D) Bcl-2 expression by expanded Tregs in the presence of IL-2 and IL-15 (bold line) or IL-2 alone (thin line) gated on CD4+CD25+CD127 Tregs. Data shown are representative of 3 experiments. *P < .05; **P < .01.
Figure 3
Figure 3
LDA showing frequency of allo-reactive Treg (CD4+CD25+CD127) and non-Treg (CD4+CD25CD127+) populations in normal human blood. Serially diluted, purified Tregs or non-Tregs were stimulated with allogeneic DCs in the presence or absence of exogenous cytokines. Each LDA was performed with a minimum of 10-12 replicates per cell concentration. (A) Log-fraction plot of the LDA of allo-specific Tregs. The slope represents log-active cell fraction, bold lines represent frequency estimates, and non-bold lines show 95% CIs of Tregs calculated based on the likelihood ratio test of single-hit model. (B) LDA validation by testing CFSE (replicated) or CFSE+ (resting) Tregs. Box plot represents the frequency estimate and the error bar shows 95% CIs calculated by extreme LDA. (C) Box plots show the frequencies of Tregs in the presence or absence of IL-2, IL-2 plus IL-15, and non-Tregs in the presence or absence of IL-2 with 95% CIs. *P < .05; **P < .01; ***P < .001.
Figure 4
Figure 4
Expansion and characterization of allo-specific Tregs. Tregs were seeded at 1 × 105/well in a 48-well plate with irradiated allogeneic DCs (1 × 104), rapamycin (100 ng/mL), IL-2 (10 U/mL), and IL-15 (10 ng/mL). Cells were cultured for 12 days. (A) The total Treg numbers at each time point were calculated by flow cytometry of timed events. The numbers of starting Tregs were normalized to 1 million at time 0. (B) The diagram outlines the numbers of allo-specific Tregs before and after in vitro expansion, normalized to 10 000 Tregs at time 0. The frequency of allo-specific Tregs at time 0 was calculated by LDA. The frequencies of allo-specific Tregs at subsequent times were calculated by flow microfluorometry of CFSE-labeled Tregs (C). (D-F) Flow cytometry plots showed intracellular expression of Foxp3 and cell-surface expression of CD45RA, CCR7, and CD62L on gated CD4+, CD25+, and CD127 Tregs at the end of culture. Dot plot shows the analysis of CFSE dilution versus Ki-67 nuclear protein (G) or Foxp3 (H) staining in expanded Tregs at the end of culture. We observed a dual level of Foxp3 expression with the BD Biosciences kit (panel H), but not with the eBioscience kit (panel D).
Figure 5
Figure 5
Suppressive activity of expanded allo-specific Tregs. (A-C) The suppressive activity of cultured CFSE versus CFSE + (A), CFSE (replicated) versus naive Tregs (B), or allo-specific versus polyspecific Tregs (C) were tested at different ratios to self-CD4+CD25 T-cell responders in the presence of original allogeneic DCs. (D) Allo-specific suppressive function of cultured CFSE Tregs was tested against the original stimulator or a HLA class II–mismatched third party stimulator. Results are shown as average CPM of triplicates measured by the incorporation of 3H-thymidine in cocultures at day 6 after subtracting the CPM of background wells without Tregs. Results shown are representative of 3 individual experiments for each of the panels.
Figure 6
Figure 6
Cytokine release and CTLA-4 expression of expanded allo-specific Tregs. (A) Cytoplasmic staining of cytokines produced by expanded allo-specific CFSE Tregs and non-Tregs. Flow cytometric contour plots show the relative number of cytokine-producing CD4+ T cells. (B) Cytokine secretion of expanded allo-specific Tregs. Supernatants were removed after incubation of expanded Tregs and non-Tregs with PMA and ionomycin in serum-free medium. Each bar represent mean ± SD of cytokine levels of 4 individual experiments. (C) TGF-β/IFN-γ cytokine ratio of Tregs and non-Tregs. *P < .05; **P < .01.
Figure 7
Figure 7
Indirect allo-recognition by Tregs. (A) Tregs were cultured with γ-irradiated autologous monocyte-derived DCs (10:1) pulsed overnight with allogeneic or autologous whole-cell lysate in the presence of cytokines and rapamycin. Results shown are average CPM of triplicates measured by the incorporation of 3H-thymidine in cocultures on day 6. (B) LDA showing frequency of allo-reactive purified CD4+CD25+CD127 Tregs or CD4+CD25CD127+ non-Tregs stimulated with self-DCs and pulsed with allogeneic whole-cell PBMC lysate in the presence of rapamycin, IL-2, and IL-15. Box plot represents the frequency estimate and the error bar shows 95% CI calculated by extreme LDA. Dot plot shows the proliferation of CFSE-labeled Tregs with indirect allospecificity after 14 days in culture (C) and the expression of CD25, Foxp3, and BCL-2 after 27 days in culture (D-E). (F) Expansion kinetics of allo-specific Tregs when antigen was presented indirectly. Tregs were seeded at 7.7 × 105/well in a 48-well plate with irradiated allogeneic cell lysate–loaded self-DCs (7.7 × 104), rapamycin (100 ng/mL), IL-2 (10 U/mL), and IL-15 (10 ng/mL). Cells were cultured for 27 days with APC restimulation on day 14. The total Treg numbers at each time point were calculated by flow cytometry using timed event acquisition. The frequency of allo-specific Tregs at time 0 was calculated by LDA. The frequencies of allo-specific Tregs at subsequent times were calculated by flow microfluorometry of Ki-67+ Tregs. Results shown are from 1 representative experiment of 3. **P < .01; ***P < .001.

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