"Cerberus" T Cells: A Glucocorticoid-Resistant, Multi-Pathogen Specific T Cell Product to Fight Infections in Severely Immunocompromised Patients.

Kiriakos Koukoulias,Achilles Anagnostopoulos,Maria Alvanou,Antonios Makris,Anastasios Kouimtzidis,Penelope-Georgia Papayanni,Timoleon-Achilleas Vyzantiadis,Anastasia Chatzidimitriou,Nikoletta Psatha,Alexandros Spyridonidis,Evangelia Yannaki,Stamatia Laidou,Glykeria Gkoliou,Anastasia Papadopoulou,Chrysoula Pantazi,Aphrodite Georgakopoulou

doi:10.3389/fimmu.2020.608701

Abstract

Adoptive immunotherapy (AI) with pathogen-specific T cells is a promising alternative to pharmacotherapy for the treatment of opportunistic infections after allogeneic hematopoietic cell transplantation or solid organ transplantation. However, clinical implementation of AI is limited to patients not receiving high-dose steroids, a prerequisite for optimal T-cell function, practically excluding the most susceptible to infections patients from the benefits of AI. To address this issue, we here rapidly generated, clinical doses of a steroid-resistant T-cell product, simultaneously targeting four viruses (adenovirus, cytomegalovirus, Epstein Barr virus, and BK virus) and the fungus Aspergillus fumigatus, by genetic disruption of the glucocorticoid receptor (GR) gene using CRISPR/CAS9 ribonucleoprotein delivery. The product, “Cerberus” T cells (Cb-STs), was called after the monstrous three-headed dog of Greek mythology, due to its triple potential; specificity against viruses, specificity against fungi and resistance to glucocorticoids. Following efficient on-target GR disruption and minimal off-target editing, the generated Cb-STs maintained the characteristics of pentavalent-STs, their unedited counterparts, including polyclonality, memory immunophenotype, specificity, and cytotoxicity while they presented functional resistance to dexamethasone. Cb-STs may become a powerful, one-time treatment for severely immunosuppressed patients under glucocorticoids who suffer from multiple, life-threatening infections post-transplant, and for whom therapeutic choices are limited.

Highlights

The outcome of allogeneic hematopoietic cell transplantation and solid organ transplantation (SOT) is severely impaired by the development of opportunistic infections from viruses and/or fungi [1,2,3]
Flow cytometry analysis revealed no effect in glucocorticoid receptor (GR) expression after targeting exon 1, which was not unexpected since it represents the 5’-untranslated region (5’-UTR) of the gene [38, 39]
Targeting the exon 2 proximally to methionine 1 (Met1), the first initiator codon resulting in the GR-A isoform, failed to functionally disrupt the GR, indicating that translation might start from an alternative translational initiation site, such as Met27, resulting in the translational isoform GR-B, which is more transcriptionally active [39, 40] Targeting all the downstream loci in exons 2, 4, and 5 resulted in significant reductions in the percentage of cells expressing the GR, in approximately 32–42% of the cells (Figures 1C, D)

Summary

Introduction

The outcome of allogeneic hematopoietic cell transplantation (allo-HCT) and solid organ transplantation (SOT) is severely impaired by the development of opportunistic infections from viruses and/or fungi [1,2,3]. Immunosuppressive drugs significantly impair T-cell functionality [24,25,26,27,28], confining the use of antigen-specific T cells only to patients in whom immunosuppression has been tapered or withdrawn. The latter, creates the paradox of precluding from the potential benefits of AI, the most vulnerable to life-threatening infections patients; those receiving high-dose glucocorticoids, the first-line treatment of graft-versus-host disease (GvHD) post HCT or rejection post SOT

Methods

Results

Conclusion