IL-2 receptor engineering enhances regulatory T cell function suppressed by calcineurin inhibitor

Abstract

Clinical trials utilizing regulatory T cell (Treg) therapy in organ transplantation have shown promising results, however, the choice of a standard immunosuppressive regimen is still controversial. Calcineurin inhibitors (CNIs) are one of the most common immunosuppressants for organ transplantation, although they may negatively affect Tregs by inhibiting IL-2 production by conventional T cells. As a strategy to replace IL-2 signaling selectively in Tregs, we have introduced an engineered orthogonal IL-2 (ortho IL-2) cytokine/cytokine receptor (R) pair that specifically binds with each other but does not bind with their wild-type counterparts. Murine Tregs were isolated from recipients and retrovirally transduced with ortho IL-2Rβ during ex vivo expansion. Transduced Tregs (ortho Tregs) were transferred into recipient mice in a mixed hematopoietic chimerism model with tacrolimus administration. Ortho IL-2 treatment significantly increased the ortho IL-2Rβ(+) Treg population in the presence of tacrolimus without stimulating other T cell subsets. All the mice treated with tacrolimus plus ortho IL-2 achieved heart allograft tolerance, even after tacrolimus cessation, whereas those receiving tacrolimus treatment alone did not. These data demonstrate that Treg therapy can be adopted into a CNI-based regimen by utilizing cytokine receptor engineering.

Keywords: animal models: murine; basic (laboratory) research/science; bioengineering; cellular biology; cytokines/cytokine receptors; immunobiology; immunosuppressant - calcineurin inhibitor (CNI); immunosuppression/immune modulation; tolerance: chimerism.

FIGURE 1

Ortho IL-2 and tacrolimus synergistically increase %ortho Tregs. Foxp3^GFP+ Tregs transduced with ortho IL-2Rβ (ortho Tregs) and naïve Tcons were incubated with CD3/CD28 beads. Wt IL-2 (1000 IU/ml), ortho IL-2 (100 000 IU/ml), and tacrolimus (100 ng/ml) were added as indicated and replenished every 2 days. (A) Representative pseudocolor plots of co-cultured cells. (B-D) Box plots indicating the number of Tcons (B), ortho Tregs (C), and the proportion of ortho Tregs among total cells (D) on day 4. Quantification of triplicate wells in 1 representative experiment of 2 independent experiments. *<0.05, **<0.01, ***<0.001; p-values calculated by Dunnett test comparing each column to PBS-control (gray bar) among tacrolimus (−) or tacrolimus (+) group. ns, not significant.

FIGURE 2

Ortho IL-2 increase ortho IL-2Rβ hi Tregs. (A) Representative pseudo color plot for tRFP (ortho IL-2Rβ) hi and low gate. The absolute number of ortho IL-2Rβ hi (B) and low (C) fractions in each treatment group are shown. Quantification of triplicate wells in 1 representative experiment of 2 independent experiments. *<0.05, **<0.01, ***<0.001; p-values calculated by Dunnett test comparing each column to PBS-control (gray bar) among tacrolimus (−) or tacrolimus (+) group.

FIGURE 3

Ortho IL-2 injection together with tacrolimus facilitates ortho Treg expansion and the establishment of heart transplant tolerance. (A) Schema of in vivo experiment. Anti-CD40L (MR1); 0.5 mg i.p. on d0. ortho IL-2; 25 000 IU, tacrolimus; 5 mg/kg, both i.p. from d0 to d14. (B) Left; representative pseudocolor plots for CD4⁺ gated cells on d14. Right; mean ± SD of %Foxp3^GFP+/CD4⁺ cells at indicated time points. (C) Mean ± SD of %donor (CD45.1+ cells/CD45⁺ PBMCs) at indicated time points, dashed line: 1%. Pooled data from two independent experiments with at least three mice per group. *p-values <0.05 calculated by unpaired t-test between indicated two groups.

FIGURE 4

Ortho IL-2 increased the expression of ortho IL-2Rβ. (A) Representative pseudocolor plot of CD4⁺Foxp3^GFP+ gated cells in the peripheral blood on d14 showing gating strategy for ortho IL-2Rβ (tRFP) hi and low fraction. Mean ± SD with individual plots showing the proportion of ortho IL-2Rβ hi in Foxp3^GFP+ cells (B) and the proportion of ortho IL-2Rβ hi and low cells among CD45.2 (C). Pooled data from two independent experiments with at least three mice per group. *p-values <0.05 calculated by unpaired t-test between indicated two groups.

FIGURE 5

The proportion of ortho Treg and donor cell chimerism shows positive correlation. Pearson's correlation for %Foxp3^GFP+/CD4⁺ cells (x-axis) and %donor (y-axis) on d14. Each dots represent individual mouse in ortho IL-2-treated (red) and PBS-treated mouse (blue). R = 0.6, p = 0.023.

FIGURE 6

Combination of ortho IL-2 plus tacrolimus treatment promotes heart allograft tolerance. Heart allograft survival curve. **p-value <0.01 calculated by Log-rank test. Red, ortho IL-2-treated, N = 7; blue, PBS-treated, N = 7. Pooled data from two independent experiments.

FIGURE 7

Ortho IL-2 replaces IL-2 signaling in ortho IL-2Rβ⁺ Tregs that was deprived by calcineurin inhibitor treatment. (Left) TCR stimulation activates NFAT and promotes transcription of IL-2 in Teffs. Wt IL-2 secreted from activated Teffs stimulate both Teffs and Tregs. (Mid) CNIs inhibit NFAT activation and result in IL-2 deprivation for both Teffs and Tregs. (Right) ortho IL-2 restores IL-2 signaling through transduced ortho IL-2R in Tregs but does not affect wt IL-2R on Teffs. Created with BioRender.com.