Investigating the Role of BMP Receptor Signaling in Function and Stability of Regulatory T Cells
Abstract/Description/Artist Statement
Regulatory T-cells (Tregs) are central to maintaining immune tolerance by suppressing effector T-cell responses and preventing autoimmunity. The T-reg transcriptional and epigenetic 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 define how ALK2 signaling contributes to T-reg stability and plasticity under homeostatic and inflammatory conditions, and whether its loss promotes conversion into pathogenic exT-regs.
To investigate ALK2 function in T-regs, we generated conditional knockout mice using ALK2^fl/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.
Preliminary data indicate successful generation of ALK2 conditional knockout lines and reliable gating strategies for T-reg identification and lineage tracing. 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.
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.
Faculty Advisor/Mentor
Piotr Kraj
Faculty Advisor/Mentor Email
pkraj@odu.edu
Faculty Advisor/Mentor Department
Biological Sciences
College/School Affiliation
Eastern Virginia School of Medicine
Student Level Group
Graduate/Professional
Presentation Type
Oral Presentation
Investigating the Role of BMP Receptor Signaling in Function and Stability of Regulatory T Cells
Regulatory T-cells (Tregs) are central to maintaining immune tolerance by suppressing effector T-cell responses and preventing autoimmunity. The T-reg transcriptional and epigenetic 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 define how ALK2 signaling contributes to T-reg stability and plasticity under homeostatic and inflammatory conditions, and whether its loss promotes conversion into pathogenic exT-regs.
To investigate ALK2 function in T-regs, we generated conditional knockout mice using ALK2^fl/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.
Preliminary data indicate successful generation of ALK2 conditional knockout lines and reliable gating strategies for T-reg identification and lineage tracing. 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.
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.