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 6

IL-2R–dependent CEACAM1 expression depends on chromatin opening and BLIMP-1.

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IL-2R–dependent CEACAM1 expression depends on chromatin opening and BLIM...
(A) Purified human Tregs were stimulated with anti-CD3/CD28 and IL-2, then subcultured with IL-2 alone on days 3 and 6. ATAC-Seq and RNA-Seq were performed at the indicated times, and Genome browser tracks were plotted for the CEACAM1 locus. Peaks with STAT and PRDM1 motifs were identified using Meme suite 5.4.1. Peak IDs refer to regions with substantial changes in sequence reads, showing peaks with STAT and PRDM1 motifs. (B) Variance in accessibility across peaks over time is shown. (C and D) Purified Tregs stimulated for 3 days with anti-CD3/CD28 plus IL-2 were transfected with scramble or Cas9 RNPs targeting accessible regions (regions 3–5) and cultured for 7 more days with IL-2. (C) T7EI assay confirmed editing at the targeted region. Expected PCR product sizes are 928 bp (region 3), 663 bp (region 4), and 1,036 bp (region 5). (D) CEACAM1 and Foxp3 expression was analyzed by flow cytometry; representative and quantitative data (n = 3). (E) HEK-Blue IL-2 cells were cotransfected with luciferase reporters driven by the indicated wild-type or mutated CEACAM1 regions and either control or PRDM1 vectors. Cells were treated ± IL-2 (500 U/mL) for 24 hours, and luciferase activity was measured and normalized to baseline. (F) CEACAM1 expression was assessed by flow cytometry; representative histograms and quantitative data (n = 5). (G and H) Purified Tregs were initially stimulated with anti-CD3/CD28 and IL-2 and subcultured with IL-2 on days 3 and 6. (G) Time course of PRDM1 and CEACAM1 mRNA expression by RNA-Seq. (H) Expression of BLIMP-1 during expansion was examined by Western blotting analysis. Data were analyzed by 1-way ANOVA (D and E) or paired 2-sided t test (F). *P < 0.05, ***P < 0.001, ****P < 0.0001.