Sequential monitoring and stability of ex vivo-expanded autologous and nonautologous regulatory T cells following infusion in nonhuman primates

Abstract

Ex vivo-expanded cynomolgus monkey CD4(+)CD25(+)CD127(-) regulatory T cells (Treg) maintained Foxp3 demethylation status at the Treg-specific demethylation region, and potently suppressed T cell proliferation through three rounds of expansion. When carboxyfluorescein succinimidyl ester- or violet proliferation dye 450-labeled autologous (auto) and nonautologous (non-auto)-expanded Treg were infused into monkeys, the number of labeled auto-Treg in peripheral blood declined rapidly during the first week, but persisted at low levels in both normal and anti-thymocyte globulin plus rapamycin-treated (immunosuppressed; IS) animals for at least 3 weeks. By contrast, MHC-mismatched non-auto-Treg could not be detected in normal monkey blood or in blood of two out of the three IS monkeys by day 6 postinfusion. They were also more difficult to detect than auto-Treg in peripheral lymphoid tissue. Both auto- and non-auto-Treg maintained Ki67 expression early after infusion. Sequential monitoring revealed that adoptively transferred auto-Treg maintained similarly high levels of Foxp3 and CD25 and low CD127 compared with endogenous Treg, although Foxp3 staining diminished over time in these nontransplanted recipients. Thus, infused ex vivo-expanded auto-Treg persist longer than MHC-mismatched non-auto-Treg in blood of nonhuman primates and can be detected in secondary lymphoid tissue. Host lymphodepletion and rapamycin administration did not consistently prolong the persistence of non-auto-Treg in these sites.

Keywords: Immunosuppressant; T cell biology; animal models: nonhuman primate; basic (laboratory) research / science; immunobiology; immunosuppressant; immunosuppression / immune modulation; mechanistic target of rapamycin (mTOR); polyclonal preparations: rabbit antithymocyte globulin; translational research / science.

Figure 1. Characteristics of expanded cynomolgus monkey Treg and Teff

(A) Expanded Treg (black lines) and Teff (grey shading) were stained for Foxp3, CD25, CD39, CD45RA, CD62L, CD95, CD127, Bcl-2, Helios, CTLA-4, CCR7 and CXCR3 at the end of round 3 of expansion. Compared to expanded Teff, expanded Treg showed much higher expression of Foxp3, Helios, CD39 and CXCR3, and similar high expression of CD25. Data are representative of 3 experiments. (B) Demethylation status of Foxp3 TSDR (Treg-specific demethylation region) in fresh and expanded Treg and Teff, assessed at the end of the 2^nd and 3^rd round of expansion. The % TSDR demethylation in each experiment is shown, as well as the mean % for all experiments in each group (horizontal line). TSDR demethylation was significantly higher (at least 4-fold) in Treg than in Teff under each condition. ***, p<0.001; *, p<0.05.

Figure 2. CFSE / VPD450 labeling does not affect the survival of expanded Treg in vitro or in vivo

Expanded Treg (round 3) were labeled with CFSE or VPD450. (A) They were then cultured in the presence of 300 U/ml of IL-2 for an additional day, and apoptosis analysed by staining with Annexin V and 7-AAD. Unlabeled cells were used as controls. Data are representative of 3 independent experiments. (B, C) Expanded non-auto Treg were labeled with CFSE or VPD450, equal numbers of labeled cells mixed together and infused into an IS monkey (CM118). At the indicated time points, 1ml blood was tested for the presence of infused cells and end Teff and end Treg, as described in Figure 2. (B) dot plots and plot of #/ml blood vs. time post-infusion are shown. (C) MFI of Foxp3, CD25, CD127 in/on infused CFSE-labeled or VPD450-labeled exogenous Treg were compared with those in/on end Teff and end Treg at the indicated time points.

Figure 3. Persistence of ex vivo-expanded Treg in peripheral blood of control monkeys

Auto- or non-auto-Treg were labeled with either CFSE or VPD450, respectively, then infused i.v. into healthy control monkeys. At the days indicated post-Treg infusion, 1ml blood was tested for the presence of the infused cells and endogenous (end) Teff and Treg by flow cytometry. Counting beads were added before running flow analysis. Cell numbers per ml blood were calculated as the number of cell events/number of bead events × total number of beads added. Duplicate samples were tested at each time-point. (A) Flow cytometric profiles of CD4-gated PBMC from 3 monkeys showing the percentages of infused, labeled auto-and non-auto-Treg. Endogenous (unlabeled) CD4⁺ T cells are also evident (lower left quadrant). (B) Kinetics of the number of infused Treg (auto- and non-auto-Treg) and endogenous (end) Teff and Treg numbers in the peripheral blood after labeled Treg infusion in the 3 control monkeys. (C) Kinetics of the ratio of infused (ex) Treg to endogenous (end) Treg in the peripheral blood after labeled Treg infusion in these monkeys (n=3). *, The ratio of auto-Treg/end Treg was significantly greater than that of non-auto-Treg/end Treg, p<0.05.

Figure 4. Protocol for infusion of Treg in monkeys treated with ATG, tacrolimus and rapamycin

(A) Immunosuppressive (IS) drug regimen and Treg infusion time points (days after last ATG infusion) in 3 monkeys. (B) Kinetics of host T cell, B cell and NK cell depletion and recovery.

Figure 5. Persistence of ex vivo-expanded Treg in peripheral blood of immunosuppressed (IS) monkeys

Auto- or non-auto-Treg were labeled with either CFSE or VPD450, respectively and infused (3.10⁶ to 3.10⁷/kg) i.v. into 3 IS monkeys. At the days indicated post-Treg infusion, 1ml blood was tested for the presence of the infused cells and endogenous (end) Teff and Treg by flow cytometry. Counting beads were added before running flow analysis. Cell numbers per ml blood were calculated as the number of cell events/number of bead events × total number of beads added. Duplicate samples were tested at each time-point. (A) Flow cytometric profiles of CD4-gated PBMC from 3 IS monkeys, showing the percentages of infused, labeled auto-and non-auto-Treg. Endogenous (unlabeled) CD4⁺ T cells are also evident (lower left quadrant). Two IS monkeys were monitored beyond 21 days. (B) Kinetics of the number of infused Treg (auto- and non-auto-Treg) and endogenous (end) Teff and Treg numbers in the peripheral blood after labeled Treg infusion in 3 IS monkeys for 30 min to day 6 and in 2 of these monkeys beyond day 21. (C) Kinetics of the ratio of infused (ex) Treg to endogenous (end) Treg in the peripheral blood after labeled Treg infusion in the IS monkeys (n=3).

Figure 6. Detection of ex vivo-expanded Treg in host lymph nodes after infusion

Ex vivo-expanded auto-and non-auto-Treg were labeled with CFSE or VPD450 before infusion. On day 1 post-infusion, inguinal LN were dissected and assessed for the presence of infused Treg. (A) Presence of exogenous Treg shown as dot plots. Expression of CCR7, CXCR3 and CD25 (offset histogram) by endogenous (end) Teff, end Treg, and exogenous auto-Treg in all 4 monkeys examined and non-auto-Treg in 2 of these monkeys. (B) Ratio of infused (ex) Treg and endogenous (end) Teff (left) or end Treg (right) in 2 control and 2 IS monkeys. *, P<0.05.

Figure 7. Expression of Treg signature and activation and tissue-homing markers by ex vivo-expanded auto-Treg after their infusion

Ex vivo-expanded auto-Treg were labeled with CFSE or VPD450 before infusion. Expression (mean fluorescence intensity; MFI) of Foxp3, CD25, CD127, CXCR3, CCR7 and CD62L at the times indicated post-Treg infusion is shown as flow histogram overlays for endogenous (end) Teff (grey solid), end Treg (black line), and exogenous auto-Treg (red line). Representative histogram overlays and pooled MFI from (A) 3 control monkeys and (B) 3 IS-monkeys, with 2 IS monkeys beyond day 21, respectively are shown.

Figure 7. Expression of Treg signature and activation and tissue-homing markers by ex vivo-expanded auto-Treg after their infusion

Ex vivo-expanded auto-Treg were labeled with CFSE or VPD450 before infusion. Expression (mean fluorescence intensity; MFI) of Foxp3, CD25, CD127, CXCR3, CCR7 and CD62L at the times indicated post-Treg infusion is shown as flow histogram overlays for endogenous (end) Teff (grey solid), end Treg (black line), and exogenous auto-Treg (red line). Representative histogram overlays and pooled MFI from (A) 3 control monkeys and (B) 3 IS-monkeys, with 2 IS monkeys beyond day 21, respectively are shown.