Relative resistance of human CD4(+) memory T cells to suppression by CD4(+) CD25(+) regulatory T cells

Abstract

Successful expansion of functional CD4(+) CD25(+) regulatory T cells (T(reg)) ex vivo under good manufacturing practice conditions has made T(reg) -cell therapy in clinical transplant tolerance induction a feasible possibility. In animals, T(reg) cells home to both transplanted tissues and local lymph nodes and are optimally suppressive if active at both sites. Therefore, they have the opportunity to suppress both naïve and memory CD4(+) CD25(-) T cells (Tresp). Clinical transplantation commonly involves depleting therapy at induction (e.g. anti-CD25), which favors homeostatic expansion of memory T cells. Animal models suggest that T(reg) cells are less suppressive on memory, compared with naïve Tresp that mediate allograft rejection. As a result, in the context of human T(reg) -cell therapy, it is important to define the effectiveness of T(reg) cells in regulating naïve and memory Tresp. Therefore, we compared suppression of peripheral blood naïve and memory Tresp by fresh and ex vivo expanded T(reg) cells using proliferation, cytokine production and activation marker expression (CD154) as readouts. With all readouts, naïve human Tresp were more suppressible by approximately 30% than their memory counterparts. This suggests that T(reg) cells may be more efficacious if administered before or at the time of transplantation and that depleting therapy should be avoided in clinical trials of T(reg) cells.

Figure 1. T_regcells suppress naïve more than memory Tresp

(A–C) Suppression of ³H-Thymidine incorporation in naïve and memory Tresp by T_reg cells at 1:1 ratio. (A), plot showing pooled data from seven independent experiments taken from day 7 with a bead:Tresp ratio of 0.2. Mean percentage suppression against increasing stimulus for days 5 (B) and seven (C). Solid lines show regression for naïve while dashed lines indicate those for memory Tresp; *p < 0.05. (D and E) Suppression of CFSE dilution in Tresp by T_reg cells at 1:1 ratio. (D and –E) Representative CFSE profiles of Tresp in the presence (E) and absence (D) of 1:1 T_reg cells. (F) Suppression, expressed as the increase in the number of Tresp not dividing. (D and E) are representative examples, from day 5 of culture, from two independent experiments. Due to differences in absolute numbers between the two experiments, representative, rather than pooled data, have been shown.

Figure 2. Suppression of naïve and memory Tresp by T_reg cells in dose–response

(A) Representative example from three independent experiments of suppression of ³H-Thymidine incorporation by naïve and memory Tresp at different T_reg cell:Tresp ratios (but constant bead:Tresp ratio of 0.2) for 5 days. Solid and dashed arrows show the IC₅₀ for naïve and memory Tresp respectively. (B and C) cumulative IC₅₀ (B) and S_max (C) for the three experiments. Lines join paired data.

Figure 3. T/_reg–cell-mediated suppression of early activation markers on naïve Tresp is greater than on memory Tresp

(A) CD45RA and CD45RO gating strategy to identify naïve and memory Tresp respectively in cocultures of FACS-sorted Tresp and T_reg cells. (B), CD154 expression on naïve and memory Tresp after 7 h of stimulation (bead:Tresp ratio of 0.2) in the presence and absence of T_reg cells. (C), percentage suppression of CD154 expression on Tresp by T_reg cells at increasing T_reg cell:Tresp ratios. (A–C) are representative examples from three independent experiments. (D), cumulative IC₅₀ (left panel) and S_max from the three experiments.

Figure 4. IL-2 production from naïve Tresp is more regulated by T_reg cells than those from memory Tresp

(A) IL-2 concentrations in supernatants of naïve and memory Tresp polyclonally stimulated with anti-CD3/CD28 beads (bead:Tresp ratio of 0.2) in vitro at days 3, 5 and 7. (B) Percentage suppression of IL-2 production from naïve and memory Tresp by T_reg cells at 1:1 ratio on days 3, 5 and 7. All graphs show pooled (mean ± s.d.) results from three independent experiments; *p < 0.05.

Figure 5. Rapamycin-expanded T_reg-cell lines suppress naïve Tresp more than memory Tresp

(A) Percentage suppression of CFSE dilution in memory (A) and naïve (B) Tresp by fresh T_reg cells and a T_reg-cell line generated in vitro in the presence of Rapamycin. (C) IC₅₀ (left panel) and S_max (right panel).