bims-hummad 2025-02-09 papers

Mol Ther. 2025 Feb 03. pii: S1525-0016(25)00083-8. [Epub ahead of print]

Gene editing of CD3 epsilon gene to redirect regulatory T cells for adoptive T cell transfer.

Adoptive transfer of antigen-specific regulatory T cells (Tregs) is a promising strategy to combat immunopathologies in transplantation and autoimmune diseases. However, their low frequency in peripheral blood poses challenges for both manufacturing and clinical application. Chimeric antigen receptors (CARs) have been used to redirect the specificity of Tregs, employing retroviral vectors. However, retroviral gene transfer is costly, time consuming, and raises safety issues. Here, we explored non-viral CRISPR-Cas12a gene editing to redirect Tregs, using HLA-A2-specific constructs for proof-of-concept studies in transplantation models. Knock-in of an antigen-binding domain into the N terminus of CD3 epsilon (CD3ε) gene generates Tregs expressing a chimeric CD3ε-T cell receptor fusion construct (TRuC) protein which integrates into the endogenous TCR/CD3 complex. These CD3ε-TRuC Tregs exhibit potent antigen-dependent activation while maintaining responsiveness to TCR/CD3 stimulation. This enables preferential enrichment of TRuC-redirected Tregs over CD3ε KO Tregs via repetitive CD3/CD28-stimulation in a GMP-compatible expansion system. CD3ε-TRuC Tregs retained their phenotypic, epigenetic, and functional identity. In a humanized mouse model, HLA-A2-specific CD3ε-TRuC Tregs demonstrate superior protection of allogeneic HLA-A2+ skin grafts from rejection compared to polyclonal Tregs. This approach provides a pathway for developing clinical-grade CD3ε-TRuC-based Treg cell products for transplantation immunotherapy and other immunopathologies.

DOI: https://doi.org/10.1016/j.ymthe.2025.01.045

J Transl Med. 2025 Feb 04. 23(1): 150

Non-invasive imaging with ICOS-targeting monoclonal antibody for preclinical diagnosis of rheumatoid arthritis in a humanized mouse model.

Shao Duan, Chao Li, Feng Yan, Yifei Xia, Shuaiming Shao, Weiyu Chen, Zunyu Xiao, Gongping Xu.

BACKGROUND: Activated T cells play a pivotal role in rheumatoid arthritis (RA) pathogenesis, and imaging of activated T cells may provide a non-invasive tool for RA detection. Here, we first developed an optical probe targeting human inducible T cell co-stimulator (ICOS) and tested its capacity in RA diagnosis by capturing ICOS+ activated T cells in vivo in a humanized mouse model.
METHODS: The humanized arthritis model, Human peripheral blood mononuclear cells- adjuvant induced arthritis (HuPBMC-AIA) was established, and flow cytometry and immunofluorescence were employed to determine ICOS expression in huPBMC-AIA model. Anti-human ICOS monoclonal antibody (mAb) was conjugated to Cy7 via NHS ester amine reaction. A cell uptake study was used to confirm the specificity of Cy7-ICOS mAb to activated T cells. 4-view near-infrared fluorescence (NIRF) imaging study was performed to test Cy7-ICOS mAb in detecting RA in vivo.
FINDINGS: ICOS was confirmed as an indicator of RA pathogenesis via RNA-seq, flow cytometry and immunofluorescence data. An in-vitro cellular uptake study validated the specificity of Cy7-ICOS mAb to activated T cells. Cy7-ICOS mAb could detect ICOS+ activated T cells in vivo through 4-view NIRF imaging. The receiver operating characteristic (ROC) curve created based on NIRF imaging quantification could distinguish the huPBMC-AIA group from the control group at all time points imaged.
CONCLUSION: In this study, we first developed an optical imaging probe targeting human ICOS, Cy7-ICOS mAb. The 4-view NIRF imaging with Cy7-ICOS mAb could detect pathogenic ICOS+ activated T cells with high sensitivity and specificity in vivo, which indicated the great potential of this imaging probe in RA early diagnosis.

Keywords: Activated T cell; Humanized model; ICOS; NIRF imaging; Rheumatoid arthritis

DOI: https://doi.org/10.1186/s12967-024-05899-w

Arch Iran Med. 2024 Dec 01. 27(12): 683-692

Clinicopathologic Effects of Xenogeneic GvHD Induced by Adoptively Transferred Human-Derived T Cells in Severely Immunodeficient Mice.

Hami Ashraf, Farid Kosari, Amir Arsalan Khorsand, Samad Muhammadnejad, Vahid Mansouri, Ahad Muhammadnejad, Naser Ahmadbeigi, Seyed Mostafa Monzavi.

BACKGROUND: Xenogeneic graft-versus-host disease (xGvHD) is an inevitable confounder of preclinical evaluation of adoptive immunotherapies on tumor-bearing immunodeficient mouse models. This study was designed to appraise the clinical and histopathological effects caused by xGvHD in severely immunodeficient mice considering the T cell dosage.
METHODS: Fifty NOG mice underwent intraperitoneal injection of three different doses of human-derived total T cells, a high dose of CD8+T cells, or vehicle (as control). Clinical and histopathological status of the study subjects were evaluated and compared according to scoring systems.
RESULTS: In mice receiving higher doses of total T cells, the clinical severity of xGvHD was greater. However, recipients of CD8+T cells developed none to mild xGvHD manifestations. Higher doses of T cells were associated with poorer outcomes including premature death and more severe histopathologic damages. Greater CD3+T cell tissue engraftment (immunohistochemical CD3 positivity) was associated with more severe xGvHD-induced histopathological damages. Clinical xGvHD scores were significantly correlated with histopathological xGvHD scores in total and in each tissue. Mice with severe cutaneous symptoms had higher scores of xGvHD-induced histopathologic changes in the skin. Lethargy was associated with higher histopathological scores in the lungs, liver and spleen.
CONCLUSION: In preclinical evaluations, lower doses of T cell-based therapies are associated with milder xGvHD. Development of xGvHD may be averted by the use of CD4+T cell-depleted grafts. Histopathological and clinical scoring systems for evaluating xGvHD are significantly correlated. The lungs and liver are reliable organs for histopathological assessment and scoring of xGvHD.

Keywords: Adoptive immunotherapy; Evaluation; Graft; Host disease; Immunodeficient mice; Preclinical drug; Xenotransplantation

DOI: https://doi.org/10.34172/aim.28597