TABLE 1.
Timeline of reports of Treg isolation, expansion and suppressive activity in NHP species.
| Species | Treg selection (purity) | Cell yield | Expansion method | Expansion rate | Suppressive activity | Reference(s) |
| Rhesus macaque | T cells from spleen | 200 × 106 cells per recipient (splenectomized) | Donor splenocytes + anti-CD80/CD86; 13 days | 2–4-fold | In vivo infusion 13 days post allogeneic kidney transplant; donor-specific inhibition of rejection | Bashuda et al., 2005 |
| Rhesus | MACS (90%) or FACS (98%) CD4+ CD25+ | Not indicated | Anti-CD3/CD28 beads + IL-2; 4 weeks | 300–2,000-fold | Up to 1:8a ratio, suppression of autologous PBMC proliferation | Gansuvd et al., 2007 |
| Rhesus | Anti-CD8 and anti-CD20 Dynabeads, or CD4+ MACS, followed by anti-CD25 MACS (82%) | 10% of CD3+CD4+ T cells | Fresh cells used; no expansion | n/a | Proliferation of Teff in response anti-CD3 or irradiated PBMC decreased at 1:1a ratio, however variation between animals | Haanstra et al., 2008 |
| Rhesus | FACS CD4+CD25hi or CD4+CD25+CD127– | 105 cells/mL blood | Anti-CD3/CD28 beads + IL-2; 4 weeks | Up to 450-fold | CFSE-MLR, up to 1:100a ratio; suppression of alloreactive response by responder-specific or third-party Treg | Anderson et al., 2008 |
| Rhesus | MACS CD4+CD127–/lo | 7% of CD4+/1.3% of total PBMC; 3.7 × 104 cells/ml blood | Immature Mo-DC+ IL-2+ IL-15; 14 days, or 10 days followed by 2 days without DC | No expansion | Suppression up to 1:40a ratio; donor-specific | Zahorchak et al., 2009 |
| Rhesus | FACS CD4+ CD25- CD127–/lo cells (> 80%) | >or = 106 | Stimulation with anti-CD3 and anti-CD28 microbeads (1 cell: 2 beads) + IL-2 (2,000 U/ml) ± rapamycin (1,000 nM); re-stimulation at 7 and 14 days; harvested on day 21 | 210–760-fold | Strong suppression of CD4+ and CD8+ T cell proliferation in CFSE-MLR | Singh K. et al., 2012 |
| Cynomolgus macaque | FACS CD4+ CD25+CD127– (> 98%) | 0.4% of PBMC | Allo DC (BM-DC/Mo-DC) + IL-2; 7 days | 12–25-fold | CFSE-MLR, PBMC+ anti-CD3/CD28+ Treg: 30% inhibition at 1:3a ratio. Treg suppressive function stimulated by BM-DC > Mo-DC | Moreau et al., 2008 |
| Cynomolgus (MS model) | MACS CD4+ CD25+ (>90%) | 6.4% of T cells | Fresh cells used; no expansion | n/a | Proliferation to anti-CD3/CD28 or CD3/CD46 stimulation impaired during active MS | Ma et al., 2009 |
| Cynomolgus | FACS CD4+CD25hiCD127– from PBMC | 5% total CD4+ cells | NHP-specific anti-CD2/3/28 microbeads (cell: bead ratio 1:2), rhu IL-2 (1,000 U/ml) and rhu TGF-b; 5 ng/mL); 20 day culture | >80-fold | Strong suppressive effect at ratios up to 1:4 (Treg:Teff) on CD4+ and CD8+ T cell proliferation | Dons et al., 2013 |
| Cynomolgus | FACS CD4+CD25+CD127– (>95%) | 4.7 (2–6.9)% of blood CD4+ cells | Artificial (a)APCs (L32) loaded with anti-CD3 (1:1 ratio) for 7–8 days with IL-2 + rapamycin; restimulation with aAPCs on days 7 and 14 without rapamycin; harvested on day 21 | 1,000-fold | Potent suppression of anti-CD3/CD28-indiced CD4 and CD8 T cell proliferation | Guo et al., 2015; Zhang et al., 2015 |
| Cynomolgus | FACS CD4+CD25hiCD127lo | 0.5–1% of total PBMC | Donor Ag alloreactive Tregs stimulated with donor CD40L-activated B cells + IL-2; harvest on day 9 and co-culture with aAPCs (L32 cells) + IL-2 for 2 subsequent rounds of 7 days | 10,000-fold | Strong suppression (>polyclonal Tregs) of autologous T cell proliferation in MLR in response to donor APCs. | Ezzelarab et al., 2020 |
| Cynomolgus | FACS CD4 + CD8–CD25hiCD127– | Top 1% CD25+ cells in the CD4+ CD8–CD127– gate | Polyspecific Tregs expanded with a panel of CD40L-stimulated B cells (CD40L−sBc) | 10,000-fold after 28 days (4 rounds) | Expanded Tregs suppressive after activation by any APCs whose MHC was shared by CD40L−sBc used during expansion | Alonso-Guallart et al., 2021 |
| Baboon | MACSFACS sorting (> 95%) CD4+CD25hi | 1.7% spleen, 3.1% lymph node, 1.9% blood T cells; 10 × 104 cells/ml blood | Pig PBMC+ IL-2; 3–4 week + 7–10 days without PBMC | ≤ 2,000-fold, depending on IL-2 concentration | 1:1a, strong xenogeneic suppression, donor-specific: 4–10 x less efficient to third-party | Porter et al., 2007 |
| Baboon | CD4+CD25hiFACS-sorted from PBMC (> 95% purity) | 1–2% of CD4+ cells | Stimulation with irradiated pig PBMC or anti-CD3/CD28 in cultures supplemented with IL-2 and rapamycin for 4 weeks | 200-fold | Suppression of autologous anti-pig T effector cell and B cell proliferation | Singh A. K. et al., 2012 |
| Baboon | FACS CD4+CD25hi (top 1% of CD25+ cells) from PBMC | 105 Treg isolated from 20 ml blood | Culture for 26 days with IL-2, anti-CD3 Ab, aAPCs (transfected with human CD58, CD32, and CD80) and rapamycin, with weekly restimulation | >10,000-fold | Excellent suppressive function,. preserved or even enhanced by increasing amounts of restimulation after thawing | Weiner et al., 2015 |
| Baboon | CD4+ T cells | n/a | Stimulation with tolerogenic, baboon Mo-derived DCs loaded with porcine-specific in vitro-transcribed RNA in cocultures supplemented with IL-2 and rapamycin for 10 days | Fivefold within 10 days | Highly suppressive effects on porcine-specific T effector cell proliferation; reduced IFNg/enhanced IL-10/TGFb production | Li et al., 2018 |
(a)APC, (artificial) antigen-presenting cell; BM-DC, bone-marrow derived dendritic cells; CD40L-sBc, CD40 ligand-stimulated B cells; CFSE-MLR, carboxyfluorescein succinimidyl ester-mixed leukocyte reaction; FACS, fluorescence-activated cell sorting; LN, lymph node; MACS, magnetic-activated cell sorting; Mo-derived DC, monocyte-derived dendritic cells; MS, multiple sclerosis; TGFb, transforming growth factor b.