Poster 152 - Tr1 Cells Impact Fibroblast Function to Result in Regenerative Tissue Repair

Saturday, October 26, 2024

10:00 AM - 12:00 PM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

SS

Shreyas Sinha (he/him/his)

Research Intern
Texas Children's Hospital & Baylor College of Medicine, Texas, United States

Co-Author(s)

SK

Sonya Keswani, n/a

Research Assistant
Texas Children's Hospital & Baylor College of Medicine, United States
NN

Navya Nanda (she/her/hers)

Research Assistant
Texas Children's Hospital & Baylor College of Medicine
Houston, Texas, United States
AD

Anjali Degala, n/a

Research Assistant
Texas Children's Hospital & Baylor College of Medicine, United States
PB

Pranav Bommekal

Research Assistant
Texas Children's Hospital & Baylor College of Medicine, United States
JT

Jamie Tellez

Research Technition
Texas Children's Hospital & Baylor College of Medicine, United States
Varun Akondy, n/a

Research Intern
Texas Children's Hospital & Baylor College of Medicine
Cypress, Texas, United States
HL

Hui Li

Research Technician
Texas Children’s Hospital & Baylor College of Medicine, United States
Ling Yu, PhD

Research Assistant
Texas Children's Hospital & Baylor College of Medicine, United States
Mary Elizabeth Guerra, MD (she/her/hers)

Resident Physician
Texas Children's Hospital & Baylor College of Medicine, United States
SK

Sundeep G. Keswani

Surgical Director, Basic Science
Texas Children's Hospital & Baylor College of Medicine, United States

Primary Investigator(s)

Swathi Balaji, PhD (she/her/hers)

Assistant Professor
Texas Children’s Hospital & Baylor College of Medicine
Houston, Texas, United States

Introduction: We have previously reported the critical role of CD4+ T lymphocytes in dermal wound healing and fibrosis, and specifically demonstrated that Tr1 lymphocyte subsets (CD4+, CD44+, CD25+, FOXP3-) can promote regenerative repair in murine wounds. Regulatory T cells (Tr1) are critical for maintaining immune homeostasis and promote wound healing through their anti-inflammatory properties, primarily mediated by interleukin-10 (IL-10) production. However, how Tr1 cells directly affect dermal fibroblasts (Fb)—the main cellular arbiters of extracellular matrix (ECM) deposition, remodeling and fibrosis in the wounds—as part of their regenerative mechanisms remains unknown. We hypothesize that Tr1 cells directly influence fibroblast ECM production and drive distinct Fb subpopulations to promote regenerative wound repair.

Materials and

Methods: CD4+ total lymphocytes were isolated from spleens of C57BL/6J wild type mice (8-10 wk) and stained with antibodies for CD4 (RM4-5), CD44 (IM7), CD25 and CD62L (MEL14) and flow sorted to separate naïve T Lymphocytes, CD25+FoxP3+Treg, and Tr1 precursors. Sorted lymphocytes were cultured for 6 days in RPMI 1640 medium supplemented with 10% FBS, 1% Penicillin/Streptomycin, and 50 µM Beta-mercaptoethanol, activated with a 1:1 bead:cell ratio of anti-CD3/CD28 Dynabeads, and treated with recombinant mouse IL-2 100 U/mL, which is vital for T cell proliferation, survival, and differentiation. T cell conditioned media (CM) from these cultures was transferred to C57BL/6J murine dermal fibroblasts (mDFb) in culture and treated for 24 hours. Rate of proliferation of Tr1 (by cell counting) and IL-10 expression (by ELISA) in Tr1 conditioned medium was assessed at day 3 and day 6. mDFbs were assessed for gene expression of fibrosis-associated markers, Col1a1, Col3a1, TGFb1, and CTGF. To validate the effects of Tr1, we repeated similar experiments using human dermal fibroblasts (hDFb) from our biobank that were isolated from patients stratified into high or low scarring phenotypes based on the assessment of their prior scars. hDFbs were treated with Tr1 CM and assessed for similar endpoints. Data presented is from n=2/group and studies will be statistically powered to reach significance with p values < 0.05 tested by ANOVA indicating significant differences.

Results, Conclusions, and Discussions: Tr1 cell proliferation was assessed which showed cell count increased by 3-fold by day 6 in culture. IL-10 expression by the cells also peaked at day 6 (day 3- 6583 pg/ml, and day 6 – 15937 pg/ml). Tr1 CM-treated mDFb showed a 3.8-fold higher Col3a1/Col1a1 gene expression ratio, an anti-fibrotic pattern, and decreased expression of pro-fibrotic TGFb1 and CTGF genes compared to T naïve treated mDFb, suggesting an anti-fibrotic effect of Tr1 on fibroblasts. Similarly, Tr1 CM-treatment of high scarring hDFb reduced the pro-fibrotic gene expression of all the fibrotic genes tested. Our data showed Tr1 cells impact fibroblasts by downregulating profibrotic genes. These data provide a novel mechanistic basis for the regenerative effects of Tr1 cells that we noted in our murine wounds previously and suggest a role for Tr1 in driving spatial fibroblast heterogeneity in wounds, and support the development of Tr1s as a novel antifibrotic therapy.

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