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. 2008 Oct 1;112(7):2847-57.
doi: 10.1182/blood-2008-01-132951. Epub 2008 Jul 21.

Umbilical cord blood regulatory T-cell expansion and functional effects of tumor necrosis factor receptor family members OX40 and 4-1BB expressed on artificial antigen-presenting cells

Keli L Hippen  1 , Paul Harker-Murray, Stephen B Porter, Sarah C Merkel, Aryel Londer, Dawn K Taylor, Megan Bina, Angela Panoskaltsis-Mortari, Pablo Rubinstein, Nico Van Rooijen, Tatiana N Golovina, Megan M Suhoski, Jeffrey S Miller, John E Wagner, Carl H June, James L Riley, Bruce R Blazar
Affiliations

Umbilical cord blood regulatory T-cell expansion and functional effects of tumor necrosis factor receptor family members OX40 and 4-1BB expressed on artificial antigen-presenting cells

Keli L Hippen et al. Blood. .

Abstract

Previously, we showed that human umbilical cord blood (UCB) regulatory T cells (Tregs) could be expanded approximately 100-fold using anti-CD3/28 monoclonal antibody (mAb)-coated beads to provide T-cell receptor and costimulatory signals. Because Treg numbers from a single UCB unit are limited, we explored the use of cell-based artificial antigen-presenting cells (aAPCs) preloaded with anti-CD3/28 mAbs to achieve higher levels of Treg expansion. Compared with beads, aAPCs had similar expansion properties while significantly increasing transforming growth factor beta (TGF-beta) secretion and the potency of Treg suppressor function. aAPCs modified to coexpress OX40L or 4-1BBL expanded UCB Tregs to a significantly greater extent than bead- or nonmodified aAPC cultures, reaching mean expansion levels exceeding 1250-fold. Despite the high expansion and in contrast to studies using other Treg sources, neither OX40 nor 4-1BB signaling of UCB Tregs reduced in vitro suppression. UCB Tregs expanded with 4-1BBL expressing aAPCs had decreased levels of proapoptotic bim. UCB Tregs expanded with nonmodified or modified aAPCs versus beads resulted in higher survival associated with increased Treg persistence in a xeno-geneic graft-versus-host disease lethality model. These data offer a novel approach for UCB Treg expansion using aAPCs, including those coexpressing OX40L or 4-1BBL.

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Figures

Figure 1
Figure 1
Treg lines expanded with cell-based aAPCs have equivalent purity and expansion, with increased suppressive function. (A) Representative example (i) and summary (ii) of the CD4, CD25 versus Foxp3 (CD4-gated), and CD127 versus Foxp3 (CD4-gated) profiles for purified cord blood cells expanded 21 days in vitro with bead- or cell-based aAPCs. (B) Representative example (i) and summary (ii) of in vitro expansion of total cells or Tregs (CD4+, CD127−, Foxp3+). Representative example (C) and summary (D) demonstrating that cord blood Tregs expanded with aAPCs potently suppress an in vitro MLR assay (average values for Treg-to-Tresponder ratio of 1:4), and cell-based aAPCs are even more effective than bead-based aAPCs. For each summary, the data are presented as the mean plus or minus the standard error of the mean (SEM), with n = 7. *P ≤ .05.
Figure 2
Figure 2
Rapamycin inhibits, rather than aids, the in vitro expansion of UCB Tregs. Data from a series of 3 experiments showing that rapamycin does not significantly increase the purity (A) or suppressive function (B) of UCB Treg cultures. (C) Rapamycin significantly inhibits the expansion of UCB Treg cultures (P ≤ .05).
Figure 3
Figure 3
Treg lines expanded with cell-based aAPCs have equivalent purity and expansion, with increased suppressive function. (A) Representative example (i) and summary (ii) of the CD4, CD25 versus Foxp3 (CD4-gated), and CD127 versus Foxp3 (CD4-gated) profiles for purified cord blood cells expanded 21 days in vitro with bead- or cell-based aAPCs. (B) Representative example (i) and summary (ii) of in vitro Treg expansion demonstrating the increased effectiveness of 4-1BBL or OX40L costimulation. (C) Representative example (i) and summary (ii; average values for Treg-to-Tresponder ratio of 1:4) demonstrating cord blood Tregs expanded with aAPCs potently suppress an in vitro MLR assay, and cell-based aAPCs are even more effective than bead-based aAPCs. Tregs were cultured for 18 to 21 days, and each summary is presented as the mean plus or minus SEM, with n = 6. TGF-β (D) or IFN-γ (E) present in day 6 or 17 supernatants from UCB Treg cultures expanded with anti-CD3/28 beads or KT32/4.1BBL. Note that the limit of detection for IFN-γ was 25 ng/mL. *P ≤ .05 for comparison with CD3/28 beads; #P ≤ .05 for comparison with KT32.
Figure 4
Figure 4
Tregs expanded with cell-based aAPCs have increased survival and accumulation in in vitro suppression assays. Cord blood Tregs were expanded in vitro using bead-or cell-based aAPCs with or without 4.1BBL or OX40L costimulation, then CFSE-labeled and incubated with allogeneic PBMNCs and anti-CD3 beads for 4 days and Treg cell division monitored by CFSE-dye dilution. (A) Representative example of CFSE dilution in Tregs during bead-based suppression assays. (B) Quantitation of Treg cell number (CD4+, CFSE+) on day 4 of suppression assay at Treg-to-PBMNC ratio of 1:2 as assessed by flow cytometry using counting beads and CFSE-dilution. (C) Pro- and antiapoptotic gene expression in UCB Tregs expanded with CD3/28 beads versus KT32/4.1BBL. Data are mean plus or minus SEM for 5 independent cultures. (E) Data in panel A is representative of a single experiment, while data in panels B and C represent the mean plus or minus SEM for 3 and 5 independent experiments, respectively. *P < .05.
Figure 5
Figure 5
In vitro–expanded cord blood Tregs decrease mortality in a xenogeneic model of GVHD. Tregs were purified from frozen cord blood and expanded with a single stimulation of bead- or cell-based aAPCs for 18 days, then were cotransferred with allogeneic PBMNCs into clodronate-treated, irradiated Rag2−/−, γc−/− mice. (A-C) Day-18 flow phenotype showing percent CD4+ and percent CD25+Foxp3+ (CD4-gated), fold expansion, and suppression index (respectively) of the cell lines used. (D) Kaplan-Meyer survival curve showing increased survival of clodronate-treated, irradiated Rag2−/−, γc−/− mice receiving human PBMNCs with or without groups of Tregs in a 1:1 ratio (ie, 30 × 106 each). For groups PBMNCs, CD3/28 beads, KT32/4.1BBL, KT32/OX40L, and PBS, n = 8, 9, 8, 8, and 4, respectively. P ≤ .05 for each Treg-treated group compared with PBMNCs. (E) Average weight (% of initial) for mice surviving on given day for different groups of mice. *P ≤ .05 from days 8 to 73 for 4.1BBL and days 10 to 31 and 49 to 80 for OX40L. Data are representative of 4 experiments ameliorating XGVHD with Tregs.
Figure 6
Figure 6
Cotransfer of Tregs leads to amelioration of GVHD pathology. (A) Differential persistence of Tregs in blood of animals on the indicated days after transfer. (B) Comparison of Treg (A2+) numbers in circulation on day 7 versus day of death due to disease (open symbols) or conclusion of the experiment (closed symbols). (C,D,F) Disease severity was assessed at time of death (open symbols) or at the conclusion of the experiment (closed symbols). (C) Decreased percentage of human CD4+ cells was observed in blood from Treg-treated animals that survived to day 80. (D) Moribund animals were found to have decreased hematocrit readings, except for the one peri-transplant, non–GVHD-related death observed in the 4.1BBL group. Representative example (E) and disease severity scores (F) for H&E staining of liver, lung, and ileum from animals with or without PBMNCs and Tregs. (G) Representative examples of anti-hCD4 peroxidase staining of liver and lung from animals with or without PBMNCs and Tregs expanded with KT32/OX40L. Pathology data from same experiment as Figure 5, and the pathology is representative of 3 experiments analyzed. Images were acquired at room temperature with an Olympus BX51 microscope, Olympus U-plan Apo and 10× objective lens (aperture = 0.40; Olympus, Hamburg, Germany) with an RTT Spot camera and Spot Advanced software (Diagnostic Instruments, Sterling Heights, MI).
Figure 7
Figure 7
Tregs expanded with cell-based aAPCs offer increased protection from GVHD mortality and pathology over Tregs expanded with bead-based aAPCs. Cord blood Tregs were purified, expanded in vitro using bead- or cell-based aAPCs, and injected along with allogeneic PBMNCs into clodronate-treated, irradiated Rag2−/−, γc−/− mice as in Figure 5. (A) Kaplan-Meyer survival curve showing increased survival of mice receiving human PBMNCs with or without groups of Tregs in a 3:1 ratio (ie, 30 × 106 PBMNCs and 10 × 106 Tregs). For groups PBMNCs, CD3/28 beads, and KT32, n = 8, 10, and 10, respectively. P ≤ .05 for each Treg-treated group compared with PBMNCs. (B) Enumeration of Tregs in blood of animals on the indicated days after transfer, showing increased persistence for KT32-expanded Tregs. P < .04 on day 10. (C) Decreased percentage of human CD4+ cells were observed in blood and spleen from Treg-treated animals that survived to day 67.
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