Selective expansion of regulatory T cells using an orthogonal IL-2/IL-2 receptor system facilitates transplantation tolerance
Toshihito Hirai, Teresa L. Ramos, Po-Yu Lin, Federico Simonetta, Leon L. Su, Lora K. Picton, Jeanette Baker, Jian-Xin Lin, Peng Li, Kinya Seo, Juliane K. Lohmeyer, Sara Bolivar-Wagers, Melissa Mavers, Warren J. Leonard, Bruce R. Blazar, K. Christopher Garcia, Robert S. Negrin
Toshihito Hirai, Teresa L. Ramos, Po-Yu Lin, Federico Simonetta, Leon L. Su, Lora K. Picton, Jeanette Baker, Jian-Xin Lin, Peng Li, Kinya Seo, Juliane K. Lohmeyer, Sara Bolivar-Wagers, Melissa Mavers, Warren J. Leonard, Bruce R. Blazar, K. Christopher Garcia, Robert S. Negrin
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Research Article

Selective expansion of regulatory T cells using an orthogonal IL-2/IL-2 receptor system facilitates transplantation tolerance

Abstract

Adoptive transfer of Tregs has been shown to improve alloengraftment in animal models. However, it is technically challenging to expand Tregs ex vivo for the purpose of infusing large numbers of cells in the clinic. We demonstrate an innovative approach to engineering an orthogonal IL-2/IL-2 receptor (IL-2R) pair, the parts of which selectively interact with each other, transmitting native IL-2 signals, but do not interact with the natural IL-2 or IL-2R counterparts, thereby enabling selective stimulation of target cells in vivo. Here, we introduced this orthogonal IL-2R into Tregs. Upon adoptive transfer in a murine mixed hematopoietic chimerism model, orthogonal IL-2 injection significantly promoted orthogonal IL-2R+Foxp3GFP+CD4+ cell proliferation without increasing other T cell subsets and facilitated donor hematopoietic cell engraftment followed by acceptance of heart allografts. Our data indicate that selective target cell stimulation enabled by the engineered orthogonal cytokine receptor improves Treg potential for the induction of organ transplantation tolerance.

Authors

Toshihito Hirai, Teresa L. Ramos, Po-Yu Lin, Federico Simonetta, Leon L. Su, Lora K. Picton, Jeanette Baker, Jian-Xin Lin, Peng Li, Kinya Seo, Juliane K. Lohmeyer, Sara Bolivar-Wagers, Melissa Mavers, Warren J. Leonard, Bruce R. Blazar, K. Christopher Garcia, Robert S. Negrin

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Figure 3

RNA-Seq reveals upregulation of transcripts involved in IL-2 signaling after ortho IL-2 stimulation.

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RNA-Seq reveals upregulation of transcripts involved in IL-2 signaling a...
Foxp3GFP+ Tregs were transduced with hEGFR/oIL-2Rβ. hEGFR+ cells were enriched by magnetic cell isolation using an anti-hEGFR mAb. Enriched cells were restimulated with WT or ortho IL-2 for 4 hours after overnight IL-2 starvation. (A) Principal component analysis revealed clustering of WT- (orange) and ortho IL-2–stimulated (red) samples away from unstimulated control cells (no IL-2, gray). (B) Scatterplot of mean RPKM values (log2) in no IL-2 vs. WT IL-2 (left), no IL-2 vs. ortho IL-2 (middle), and WT IL-2 vs. ortho IL-2 (right). (C) Venn diagram showing overlap of DEGs in WT and ortho IL-2–stimulated samples. (D) Volcano plots reveal DEGs in WT- (left) or ortho IL-2–stimulated (right) cells compared with no IL-2. Vertical dashed lines on volcano plots indicate a fold change of ± 1.5. The top 20 most differentially expressed genes are indicated. (E) Heatmap displaying DEGs in IL-2 signaling signature genes among no IL-2–, WT IL-2–, and ortho IL-2–stimulated samples. (F) The top 13 pathways with the smallest adjusted P values in WT IL-2–stimulated cells shown in the BioPlanet 2019 pathway analysis. Pathway analysis of DEGs shown in ortho IL-2–treated cells reveals the same pattern as shown with WT IL-2–treated cells. Plots show the adjusted P values, circle area represents the number of DEGs overlapping with genes in a given pathway, and circle color represents the rich factor reflecting the proportion of DEGs in a given pathway. Data shown are from 1 representative experiment from 3 independent experiments with biological triplicates.