Investigating the Role of ALK2 Signaling in Function and Stability of Regulatory T Cells
Poster #: 121
Session/Time: B
Author:
Afia Salsabil Alam, BS
Mentor:
Piotr Kraj, PhD DVM
Research Type: Clinical Research
Abstract
INTRODUCTION:
Regulatory T-cells (Tregs) are central to maintaining immune tolerance by suppressing effector T-cell responses and preventing autoimmunity. The T-reg transcriptional landscape is programmed by the transcription factor Foxp3 on which their lineage stability depends. However, T-regs can lose Foxp3 expression and convert into pro-inflammatory "exT-regs," under inflammatory conditions, produce cytokines such as IFN-γ and IL-17 that exacerbate pathology. Signaling pathways including TGF-β and bone morphogenetic proteins (BMPs) control this phenomenon known as Treg plasticity. Our laboratory has made progress studying BMPR1α which is a type-I BMP receptor that safeguards T-reg stability and restrains Th-17 conversion. In contrast, the role of ALK2 (ACVR1), another BMP type-I receptor with distinct ligand preferences and signaling properties, is yet unventured. As ALK2 is widely expressed in CD4⁺ T cells, we hypothesize that it serves as a critical regulator of T-reg identity and function. Our objective is to identify the genes and regulatory networks involved in Treg plasticity and stability and define how ALK2 signaling contributes to these processes under homeostatic and inflammatory conditions, and whether its loss promotes conversion into pathogenic exT-regs.
METHODS:
To investigate ALK2 function in T-regs, we generated conditional knockout mice using ALK2fl/fl alleles with reporters. Genotyping was performed using DNA extracted from toe biopsies, followed by PCR analysis of floxed alleles and reporter constructs. Blood was stained for CD4/CD8, Foxp3-GFP, and Ly5.1/Ly5.2 congenic markers to confirm construct integrity and allow donor/recipient tracking. T-regs were flow-sorted from wild-type or ALK2-deficient mice and transferred into recipients injected with diphtheria toxin (DTR) to model T-reg instability in inflammatory settings. Spleens and lymph nodes were collected for flow cytometry analysis of T-reg frequency, Foxp3 stability, and cytokine expression. Parallel in vitro cultures assessed T-reg proliferation and conversion under activating conditions. RNA was extracted from sorted T-regs for downstream transcriptomic and epigenomic profiling, including single-cell RNA-seq (scRNA-seq) and ATAC-seq.
RESULTS:
Preliminary data indicate successful generation of ALK2 conditional knockout lines and reliable gating strategies for T-reg identification. In adoptive transfer experiments, ALK2-deficient T-regs exhibited reduced Foxp3 stability compared to wild-type controls when placed in lymphopenic DTR hosts, with increased proportions of Foxp3^low and cytokine-producing exT-regs. Ongoing RNA-seq and ATAC-seq analyses will define transcriptional modules and chromatin accessibility changes specific to ALK2 deficiency, These experiments will establish whether ALK2 deletion promotes epigenetic remodeling that destabilizes Foxp3 expression and drives T-reg to exT-reg conversion.
CONCLUSION:
Our study investigates the previously unexplored role of ALK2 in T-reg biology. Early results suggest that ALK2 signaling is required for maintaining T-reg stability under inflammatory stress, with its loss leading to Foxp3 downregulation, acquisition of effector functions, and impaired immune homeostasis. By integrating adoptive transfer models with high-resolution transcriptomic and epigenomic profiling, this work will provide mechanistic insight into how ALK2 controls T-reg plasticity. Understanding ALK2-mediated regulation of T-regs may identify new therapeutic targets to modulate immune tolerance in autoimmunity and inflammation.
Regulatory T-cells (Tregs) are central to maintaining immune tolerance by suppressing effector T-cell responses and preventing autoimmunity. The T-reg transcriptional landscape is programmed by the transcription factor Foxp3 on which their lineage stability depends. However, T-regs can lose Foxp3 expression and convert into pro-inflammatory "exT-regs," under inflammatory conditions, produce cytokines such as IFN-γ and IL-17 that exacerbate pathology. Signaling pathways including TGF-β and bone morphogenetic proteins (BMPs) control this phenomenon known as Treg plasticity. Our laboratory has made progress studying BMPR1α which is a type-I BMP receptor that safeguards T-reg stability and restrains Th-17 conversion. In contrast, the role of ALK2 (ACVR1), another BMP type-I receptor with distinct ligand preferences and signaling properties, is yet unventured. As ALK2 is widely expressed in CD4⁺ T cells, we hypothesize that it serves as a critical regulator of T-reg identity and function. Our objective is to identify the genes and regulatory networks involved in Treg plasticity and stability and define how ALK2 signaling contributes to these processes under homeostatic and inflammatory conditions, and whether its loss promotes conversion into pathogenic exT-regs.
METHODS:
To investigate ALK2 function in T-regs, we generated conditional knockout mice using ALK2fl/fl alleles with reporters. Genotyping was performed using DNA extracted from toe biopsies, followed by PCR analysis of floxed alleles and reporter constructs. Blood was stained for CD4/CD8, Foxp3-GFP, and Ly5.1/Ly5.2 congenic markers to confirm construct integrity and allow donor/recipient tracking. T-regs were flow-sorted from wild-type or ALK2-deficient mice and transferred into recipients injected with diphtheria toxin (DTR) to model T-reg instability in inflammatory settings. Spleens and lymph nodes were collected for flow cytometry analysis of T-reg frequency, Foxp3 stability, and cytokine expression. Parallel in vitro cultures assessed T-reg proliferation and conversion under activating conditions. RNA was extracted from sorted T-regs for downstream transcriptomic and epigenomic profiling, including single-cell RNA-seq (scRNA-seq) and ATAC-seq.
RESULTS:
Preliminary data indicate successful generation of ALK2 conditional knockout lines and reliable gating strategies for T-reg identification. In adoptive transfer experiments, ALK2-deficient T-regs exhibited reduced Foxp3 stability compared to wild-type controls when placed in lymphopenic DTR hosts, with increased proportions of Foxp3^low and cytokine-producing exT-regs. Ongoing RNA-seq and ATAC-seq analyses will define transcriptional modules and chromatin accessibility changes specific to ALK2 deficiency, These experiments will establish whether ALK2 deletion promotes epigenetic remodeling that destabilizes Foxp3 expression and drives T-reg to exT-reg conversion.
CONCLUSION:
Our study investigates the previously unexplored role of ALK2 in T-reg biology. Early results suggest that ALK2 signaling is required for maintaining T-reg stability under inflammatory stress, with its loss leading to Foxp3 downregulation, acquisition of effector functions, and impaired immune homeostasis. By integrating adoptive transfer models with high-resolution transcriptomic and epigenomic profiling, this work will provide mechanistic insight into how ALK2 controls T-reg plasticity. Understanding ALK2-mediated regulation of T-regs may identify new therapeutic targets to modulate immune tolerance in autoimmunity and inflammation.