Immune-Effector-Cell-Associated-Neurotoxicity-Syndrome (ICANS) Pathophysiology Is Mediated By Microglia TGF-β-Activated Kinase-1 Signaling
Authors: Vinnakota Janaki Manoja , Biavasco Francesca , Schwabenland Marius , Chhatbar Chintan , Adams Rachael C , Erny Daniel , Duquesne Sandra , El Khawanky Nadia , Salié Henrike , Athanassopoulos Dimitrios , Braun Lukas , Schmidt Dominik , Fetsch Viktor , Javorniczky Rebeka N. , Ho Jenny N.H.G , Marks Reinhard , Wäsch Ralph , Simonetta Federico , Andrieux Geoffroy , Pfeifer Dietmar , Monaco Gianni , Capitini Christian , Fry Terry J. , Blank Thomas , Blazar Bruce R , Wagner Drouet Eva-Maria , Theobald Matthias , Sommer Clemens , Stelljes Matthias , Reicherts Christian , Jeibmann Astrid , Schittenhelm Jens , Monoranu Camelia-Maria , Rosenwald Andreas , Kortüm K. Martin , Rasche Leo , Einsele Hermann , Meyer Philipp Tobias , Brumberg Joachim , Mackensen Andreas , von Bergwelt-Baildon Michael , Coras Roland , Albert Nathalie L. , Bartos Laura M. , Brendel Matthias , Holzgreve Adrien , Boerries Melanie , Mackall Crystal L. , Duyster Justus , Koehler Natalie , Strübing Felix , Bengsch Bertram , Ruella Marco , Subklewe Marion , Baumgarten Louisa von , Gill Saar , Prinz Marco , Zeiser Robert 
Published: December 2023

Chimeric antigen receptor (CAR)-T cell therapy has revolutionized the field of immune therapy in treating relapsed and refractory B cell malignancies. Despite remarkable progress, CD19 CAR-T cell therapy is associated with major side effects, most significantly are cytokine-release syndrome and neurotoxicity. Of note, 40-60% of patients receiving CD19 CAR-T cells suffer from severe neurotoxicity termed immune cell-associated-neurotoxicity-syndrome (ICANS), which remains a major obstacle limiting their therapeutic efficacy. Several pre-clinical reports using xenograft mouse models have been published to address ICANS, however, they do not fully recapitulate the clinical scenario. Identifying and targeting the molecular mediators of neurotoxicity could help ameliorate ICANS induced by CD19 CAR-T cells.
In order to understand the molecular mechanisms leading to CD19 CAR-T cell-induced ICANS, we established syngeneic ICANS mouse models for B-cell malignancies including non-Hodgkin's Lymphoma (B-NHL) and Acute Lymphoblastic Leukemia (B-ALL). Micro-array and single-nuclei RNA sequencing analyses were performed to unveil key signaling pathways in microglia leading to ICANS pathophysiology. Cognitive impairment resulting from the transfer of CD19 CAR-T cells was assessed with behavior studies in murine models. Imaging mass cytometry was performed on autopsied ICANS and control patient cohorts to study the relevance of microglial activation in patients.
We previously demonstrated that microglial upregulation of tumor necrosis factor-alpha (TNFα) results in graft versus host disease of the central nervous system (Mathew and Vinnakota et al. JCI 2020). Similarly, our ICANS mouse models exhibit morphological and functional signs of microglial activation with increased expression of TNFα and GM-CSF along with endothelial damage in lymphoma-bearing mice that received CD19 CAR-T cells when compared to non-transduced controls. Behavioral studies revealed that mice receiving CD19 CAR-T cells exhibit cognitive deficits and increased anxiety. The behavior deficits were however rescued by microglia depletion indicating an important role for microglia in mediating CD19 CAR-T cells induced ICANS. Mechanistically, we identified the activation of p-38 mitogen-activated protein kinase (MAPK) in microglia isolated from mice receiving CD19 CAR-T cells. Furthermore, pharmacological inhibition of TGF-β-activated kinase-1 (TAK1), an upstream target for p-38 MAPK significantly reduced microglial activation, lowered microglia production of TNF and GM-CSF, and improved cognitive function in these mice. In line with these results, microglia-specific knockdown of TAK1 using Cx3cr1 creER:Tak1 fl/fl mice revealed reduced microglial activation and improved cognitive function when compared to littermate controls. Moreover, TAK1 inhibition combined with CD19 CAR-T cell therapy synergistically improved anti-lymphoma/leukemia effects leading to better survival in B-cell malignancy-bearing mice. Translocator-protein-positron-emission-tomography (TSPO-PET) on ICANS patients revealed signs of microglial activation. In addition, Imaging-mass cytometry analysis on post-mortem ICANS patient cohort revealed upregulation of myeloid cell markers further confirming the in vivo findings.
In summary, our results identify TAK1/p38 MAPK-pathway as a key mediator of CAR19 CAR-T cell-induced neurotoxicity. Targeting this axis diminished the neurotoxicity associated with this therapy. This study provides a rationale for testing TAK1-inhibition in a clinical trial for treating CD19 CAR-T cell-induced neurotoxicity.