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Theranostics, Published 2025-2-25; 15(8):3610-3626 | doi:10.7150/thno.105069
Interleukin 35-producing B cells prolong the survival of GVHD mice by secreting exosomes with membrane-bound IL-35 and upregulating PD-1/LAG-3 checkpoint proteins
Jin Kyeong Choi1,2,#, Evaristus C. Mbanefo1,#, Manoj Kumar Yadav1, Sahar A. Alhakeem1,3, Vijayaraj Nagarajan1, Natalia S. Nunes4, Christopher G. Kanakry4, Charles E. Egwuagu1
1Molecular Immunology Section, Laboratory of Immunology, National Eye Institute (NEI), National Institutes of Health (NIH), Bethesda, MD 20892, United States of America (USA).
2Department of Immunology, Jeonbuk National University Medical School, Jeonju, Jeonbuk, 54907, Republic of Korea.
3Department of Biomedical Sciences, College of Health Sciences, University of Wisconsin-Milwaukee, Milwaukee, WI, United States
4Center for Immuno-Oncology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda MD 20892, United States of America.
#These authors contributed equally to this work.
Corresponding author: Charles E. Egwuagu
1Molecular Immunology Section, Laboratory of Immunology, National Eye Institute (NEI), National Institutes of Health (NIH), Bethesda, MD 20892, United States of America (USA).
2Department of Immunology, Jeonbuk National University Medical School, Jeonju, Jeonbuk, 54907, Republic of Korea.
3Department of Biomedical Sciences, College of Health Sciences, University of Wisconsin-Milwaukee, Milwaukee, WI, United States
4Center for Immuno-Oncology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda MD 20892, United States of America.
#These authors contributed equally to this work.
Corresponding author: Charles E. Egwuagu
Abstract
Background: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is an effective treatment for aggressive hematologic malignancies. However, the risk of developing graft-versus-host disease (GVHD) is a significant barrier to allo-HSCT. GVHD is a debilitating condition with high mortality rates and current therapeutic options for GVHD are limited, with corticosteroids being the standard treatment. However, the adverse effects of steroids make prolonged use difficult, necessitating the development of safer therapies. IL-35-producing B-cells (i35-Bregs) have emerged as critical regulators of immunity during autoimmune diseases. In this study, we investigated whether i35-Bregs immunotherapy can suppress and mitigate GVHD.
Methods: We administered a single dose of i35-Bregs (1.5×106) to mice undergoing allo-HSCT and monitored disease severity and survival of GVHD mice over 90 days post-transplantation. We discovered that i35-Bregs secrete exosomes containing membrane-bound IL-35 (i35-Exosomes) and investigated whether ex-vivo generated i35-exosomes can be used as stand-alone immunotherapy for GVHD. i35-Breg-induced expression of cytokines or checkpoint proteins (PD-1, LAG-3, CTLA-4) was analyzed by Flow cytometry, ELISA, and RNA-seq analysis. Characterization of membrane-bound IL-35 was by Proximity ligation assay (PLA), immunohistochemistry/Confocal microscopy and Alpha Fold-Multimer modeling.
Results: A single dose of 1.5×106 i35-Breg reduced severity of GVHD and prolonged GVHD survival, with more than 70% i35-Breg-treated mice surviving beyond day-90 post-transplantation while observing 100% mortality among untreated mice by day-45. Contrary to the view that IL-35 is secreted cytokine, we show here that i35-Bregs mitigate GVHD via membrane-bound IL-35 and by secreting i35-exosomes. Furthermore, i35-Bregs or ex-vivo generated i35-exosomes induce alloreactive T-cells to upregulate checkpoint proteins associated with T-cell exhaustion and anergy, inhibiting alloreactive responses and propagating infectious-tolerance mechanisms that suppress GVHD. Importantly, i35-Bregs or i35-exosomes suppresses GVHD by increasing bystander lymphocytes coated with immunosuppressive i35-exosomes.
Conclusions: This study demonstrates that i35-Bregs and i35-exosomes play a critical role in mitigating GVHD. The combination of i35-Breg and i35-exosome immunotherapy may be an effective strategy for treating GVHD and other inflammatory diseases.
Methods: We administered a single dose of i35-Bregs (1.5×106) to mice undergoing allo-HSCT and monitored disease severity and survival of GVHD mice over 90 days post-transplantation. We discovered that i35-Bregs secrete exosomes containing membrane-bound IL-35 (i35-Exosomes) and investigated whether ex-vivo generated i35-exosomes can be used as stand-alone immunotherapy for GVHD. i35-Breg-induced expression of cytokines or checkpoint proteins (PD-1, LAG-3, CTLA-4) was analyzed by Flow cytometry, ELISA, and RNA-seq analysis. Characterization of membrane-bound IL-35 was by Proximity ligation assay (PLA), immunohistochemistry/Confocal microscopy and Alpha Fold-Multimer modeling.
Results: A single dose of 1.5×106 i35-Breg reduced severity of GVHD and prolonged GVHD survival, with more than 70% i35-Breg-treated mice surviving beyond day-90 post-transplantation while observing 100% mortality among untreated mice by day-45. Contrary to the view that IL-35 is secreted cytokine, we show here that i35-Bregs mitigate GVHD via membrane-bound IL-35 and by secreting i35-exosomes. Furthermore, i35-Bregs or ex-vivo generated i35-exosomes induce alloreactive T-cells to upregulate checkpoint proteins associated with T-cell exhaustion and anergy, inhibiting alloreactive responses and propagating infectious-tolerance mechanisms that suppress GVHD. Importantly, i35-Bregs or i35-exosomes suppresses GVHD by increasing bystander lymphocytes coated with immunosuppressive i35-exosomes.
Conclusions: This study demonstrates that i35-Bregs and i35-exosomes play a critical role in mitigating GVHD. The combination of i35-Breg and i35-exosome immunotherapy may be an effective strategy for treating GVHD and other inflammatory diseases.
논문정보
- Research article
- 2025년 02월 (BRIC 등록일 2025-03-04)
- 국내+국외 연구진
- 의약학 > 면역의학
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