Low-dose IL-2 for In vivo expansion of CD4+ and CD8+ regulatory T cells in nonhuman primates

Abstract

IL-2 is a known potent T cell growth factor that amplifies lymphocyte responses in vivo. This capacity has led to the use of high-dose IL-2 to enhance T cell immunity in patients with AIDS or cancer. However, more recent studies have indicated that IL-2 is also critical for the development and peripheral expansion of regulatory T cells (Tregs). In the current study, low-dose IL-2 (1 million IU/m(2) BSA/day) was administered to expand Tregs in vivo in naïve nonhuman primates. Our study demonstrated that low-dose IL-2 therapy significantly expanded peripheral blood CD4(+) and CD8(+) Tregs in vivo with limited expansion of non-Treg cells. These expanded Tregs are mainly CD45RA(-) Foxp3(high) activated Tregs and demonstrated potent immunosuppressive function in vitro. The results of this preclinical study can serve as a basis to develop Treg immunotherapy, which has significant therapeutic potential in organ/cellular transplantation.

Figure 1. Treg expansion after low dose IL-2 in cynomolgus monkeys

A: In the peripheral blood, CD4⁺CD25⁺ Foxp3⁺ cells comprise 4.4 ± 2.0% among CD4⁺ cells in adult cynomolgus monkeys (n=7). B: CD4⁺ cells were further divided by staining with CD45RA and Foxp3. Resting Treg (R: rTreg, CD45RA⁺Foxp3^low), activated Treg (A: aTreg, CD45RA^-Foxp3^high) and Foxp3⁺ nonsuppressive T cells (N, CD45RA^-Foxp3^low ) comprise 40 ± 3%, 19 ± 8% and 42 ± 16 % among CD4⁺Foxp3⁺ cells, respectively. C: A representative data of flow cytometric analysis (CD4⁺ gated) before and after daily injection of IL-2 (1 million unit/m²/day). Before IL-2 injection, percentage of CD25⁺Foxp3⁺ cells was 3.4%, but it was increased to 17.3% on day 13. D: Daily treatment with low dose IL-2 significantly expanded absolute counts of Tregs. In contrast, such expansion of Tregs was not observed with lower dose of IL-2 [0.1 (n=2) and 0.6 million IU/m² BSA(n=1)]. E: The majority of expanding Tregs after IL-2 injection were aTregs, which were Foxp3^highCD45RA^-CD62L^-CTLA4^highand KI-67⁺. F: Flow cytometric analysis of a representative monkey. CD8⁺Foxp3⁺ cells were 0.05% before IL-2 injection but increased to 0.77% on day 13. G: absolute counts of Foxp3⁺ cells among CD8⁺ cells significantly increased after low dose IL-2.

Figure 1. Treg expansion after low dose IL-2 in cynomolgus monkeys

A: In the peripheral blood, CD4⁺CD25⁺ Foxp3⁺ cells comprise 4.4 ± 2.0% among CD4⁺ cells in adult cynomolgus monkeys (n=7). B: CD4⁺ cells were further divided by staining with CD45RA and Foxp3. Resting Treg (R: rTreg, CD45RA⁺Foxp3^low), activated Treg (A: aTreg, CD45RA^-Foxp3^high) and Foxp3⁺ nonsuppressive T cells (N, CD45RA^-Foxp3^low ) comprise 40 ± 3%, 19 ± 8% and 42 ± 16 % among CD4⁺Foxp3⁺ cells, respectively. C: A representative data of flow cytometric analysis (CD4⁺ gated) before and after daily injection of IL-2 (1 million unit/m²/day). Before IL-2 injection, percentage of CD25⁺Foxp3⁺ cells was 3.4%, but it was increased to 17.3% on day 13. D: Daily treatment with low dose IL-2 significantly expanded absolute counts of Tregs. In contrast, such expansion of Tregs was not observed with lower dose of IL-2 [0.1 (n=2) and 0.6 million IU/m² BSA(n=1)]. E: The majority of expanding Tregs after IL-2 injection were aTregs, which were Foxp3^highCD45RA^-CD62L^-CTLA4^highand KI-67⁺. F: Flow cytometric analysis of a representative monkey. CD8⁺Foxp3⁺ cells were 0.05% before IL-2 injection but increased to 0.77% on day 13. G: absolute counts of Foxp3⁺ cells among CD8⁺ cells significantly increased after low dose IL-2.

Figure 1. Treg expansion after low dose IL-2 in cynomolgus monkeys

A: In the peripheral blood, CD4⁺CD25⁺ Foxp3⁺ cells comprise 4.4 ± 2.0% among CD4⁺ cells in adult cynomolgus monkeys (n=7). B: CD4⁺ cells were further divided by staining with CD45RA and Foxp3. Resting Treg (R: rTreg, CD45RA⁺Foxp3^low), activated Treg (A: aTreg, CD45RA^-Foxp3^high) and Foxp3⁺ nonsuppressive T cells (N, CD45RA^-Foxp3^low ) comprise 40 ± 3%, 19 ± 8% and 42 ± 16 % among CD4⁺Foxp3⁺ cells, respectively. C: A representative data of flow cytometric analysis (CD4⁺ gated) before and after daily injection of IL-2 (1 million unit/m²/day). Before IL-2 injection, percentage of CD25⁺Foxp3⁺ cells was 3.4%, but it was increased to 17.3% on day 13. D: Daily treatment with low dose IL-2 significantly expanded absolute counts of Tregs. In contrast, such expansion of Tregs was not observed with lower dose of IL-2 [0.1 (n=2) and 0.6 million IU/m² BSA(n=1)]. E: The majority of expanding Tregs after IL-2 injection were aTregs, which were Foxp3^highCD45RA^-CD62L^-CTLA4^highand KI-67⁺. F: Flow cytometric analysis of a representative monkey. CD8⁺Foxp3⁺ cells were 0.05% before IL-2 injection but increased to 0.77% on day 13. G: absolute counts of Foxp3⁺ cells among CD8⁺ cells significantly increased after low dose IL-2.

Figure 2. The effect of low-dose IL-2 on Tregs and non-Treg cell population

To compare the effect of low-dose IL-2 on Tregs and non-Treg cell population, relative increase of each cell population to the pre-treatment level were analyzed (n=5) (Fig.2). There was no significant expansion observed in CD8⁺ memory T cells and NK cells. Most significant expansion was observed in CD4⁺CD25⁺Foxp3⁺ Treg (p<0.0001), followed by CD4⁺CD25⁺Foxp3^- (p<0.0001) and CD8⁺ Tregs (p<0.0001). Significant expansion of CD4⁺ memory T cells (p=0.0002), CD4 (p=0.017) and CD8 (p=0.009) naïve cells were also observed but their expansion was much less significant than those observed in CD4⁺ Tregs

Figure 3. Suppression assays of expanded CD4⁺ and CD8⁺ Tregs

A: Each column represents mean +SE of fold suppression, comparing with PBMC alone from three separate experiments. Addition of CD3⁺CD4⁺CD25^high to PBMCs (20×10³ cells) stimulated with abs anti-CD3/CD28 resulted in dose dependent suppression of T cell activation (black columns). In contrast, addition of CD25^dim cells instead of CD4⁺Tregs, resulted in 1.5 to 2-fold higher responses (dark gray columns) than PBMC alone, which was significantly higher (p<0.002) than PBMC responses with CD4⁺Treg. B: Similar suppressive function was observed with CD3⁺CD8⁺ CD25^high cells. The results were mean +SE of fold suppression, comparing with PBMC alone from two separate experiments.