BLIMP-1 and CEACAM1 cooperatively regulate human Treg homeostasis and function to control xenogeneic GVHD
Ying Ding, Aixin Yu, Milos Vujanac, Sabrina N. Copsel, Alejandro Moro, Luis Nivelo, Molly Dalzell, Nicolas Tchitchek, Michelle Rosenzwajg, Alejandro V. Villarino, Robert B. Levy, David Klatzmann, Thomas R. Malek
Ying Ding, Aixin Yu, Milos Vujanac, Sabrina N. Copsel, Alejandro Moro, Luis Nivelo, Molly Dalzell, Nicolas Tchitchek, Michelle Rosenzwajg, Alejandro V. Villarino, Robert B. Levy, David Klatzmann, Thomas R. Malek
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Research Article Immunology

BLIMP-1 and CEACAM1 cooperatively regulate human Treg homeostasis and function to control xenogeneic GVHD

Abstract

Regulatory T cells (Tregs) are essential for peripheral tolerance and depend on TCR and IL-2 receptor (IL-2R) signaling for their homeostasis and function. In mice, IL-2–dependent B-lymphocyte-induced maturation protein 1 (BLIMP-1) contributes to Treg homeostasis. BLIMP-1 is a major transcriptional hub in human Tregs, but its mechanisms of action remain undefined. Here, using CRISPR/Cas9 ablation, we show that BLIMP-1 limits human Treg proliferation but supports IL-10, cytotoxic T lymphocyte-associated protein 4, several immune checkpoints including carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), and Treg functional activity. BLIMP-1 restrains Treg expansion to IL-2 by downregulating CD25 and IL-2R signaling, and by enhancing CEACAM1 expression, which in turn inhibits responsiveness to CD3/CD28 signaling and activation of mTOR. Prolonged IL-2R signaling optimizes BLIMP-1 expression, supporting chromosomal opening of CEACAM1 to increased CEACAM1 expression through STAT5- and BLIMP-1–driven enhancers. Correspondingly, CEACAM1 is highly induced on Tregs from patients with autoimmune disease undergoing low-dose IL-2 therapy, and these Tregs showed reduced proliferation. A humanized mouse model of xenogeneic graft-versus-host disease demonstrates that BLIMP-1 normally promotes, while CEACAM1 restrains, Treg suppressive activity. Collectively, our findings reveal that BLIMP-1 and CEACAM1 function in an IL-2–dependent feedback loop to restrain Treg proliferation and affect suppressive function. CEACAM1 also acts as a highly selective biomarker of IL-2R signaling in human T cells.

Authors

Ying Ding, Aixin Yu, Milos Vujanac, Sabrina N. Copsel, Alejandro Moro, Luis Nivelo, Molly Dalzell, Nicolas Tchitchek, Michelle Rosenzwajg, Alejandro V. Villarino, Robert B. Levy, David Klatzmann, Thomas R. Malek

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Figure 4

CEACAM1 expression is IL-2 dependent and more robust in human Tregs than Teff cells.

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CEACAM1 expression is IL-2 dependent and more robust in human Tregs than...
(A) BLIMP-1–dependent DEGs identified in human Tregs (Figure 1D) were compared with Blimp-1–regulated DEGs in mouse Tregs. Venn diagram shows overlapping genes (≥1.5-fold change, P < 0.05); BLIMP-1–activated genes in red, repressed in blue. (B) Human CD4+ T cells were cultured for 2 days with IL-2; CEACAM1 expression was measured in Tregs (CD4+Foxp3+) and TEM cells (CD4+Foxp3–CD25med) by flow cytometry; representative histograms (100 U/mL) and quantification (n = 3, mean ± SEM) are shown. (C) CD4+ T cells were cultured with IL-2 or anti-CD3/CD28 ± anti-IL-2; CEACAM1 expression was examined on gated Tregs and Teff cells (CD4+Foxp3–) (n = 4, mean ± SEM; paired 2-sided t test). (D and E) FACS-sorted CD4+CD25hiCD127lo Tregs were cultured in media (n = 6), IL-2 (100 U/mL) (n = 4), or anti-CD3/CD28 with anti–IL-2 (TCR/CD28) (n = 6) for 4 or 16 hours. (D) RNA-Seq identified IL-2–induced DEGs (≥3-fold, FDR < 0.01) at 4 hours compared with basal expression in media. (E) Comparison of IL-2–dependent DEGs in D in relationship to the fold-change observed after culture with IL-2 versus TCR/CD28 at 4 and 16 hours. (F) FACS-sorted Tregs and CD4+CD45RA–CD25medCD127hi TEM cells were cultured for 5–6 days with IL-2 ± anti-CD3/CD28. CEACAM1 expression was determined by flow cytometry; representative histograms and quantification (n = 2, mean ± SEM). (G and H) Purified Tregs and TEM cells were activated by anti-CD3/CD28 and IL-2 on day 0 and subcultured with only IL-2 on days 3 and 6. CEACAM1 and CD25 RNA (G) or surface protein (H) were determined by RNA-Seq or flow cytometry (n = 4, mean ± SEM; 2-way ANOVA). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.