Immunotherapy-related cognitive impairment after CAR T cell therapy in mice

Abstract

Persistent central nervous system (CNS) immune dysregulation and consequent dysfunction of multiple neural cell types is central to the neurobiological underpinnings of a cognitive impairment syndrome that can occur following traditional cancer therapies or certain infections. Immunotherapies have revolutionized cancer care for many tumor types, but the potential long-term cognitive sequelae are incompletely understood. Here, we demonstrate in mouse models that chimeric antigen receptor (CAR) T cell therapy for both CNS and non-CNS cancers can impair cognitive function and induce a persistent CNS immune response characterized by white matter microglial reactivity and elevated cerebrospinal fluid (CSF) cytokines and chemokines. Consequently, oligodendroglial homeostasis and hippocampal neurogenesis are disrupted. Microglial depletion rescues oligodendroglial deficits and cognitive performance in a behavioral test of attention and short-term memory function. Taken together, these findings illustrate similar mechanisms underlying immunotherapy-related cognitive impairment (IRCI) and cognitive impairment following traditional cancer therapies and other immune challenges.

Figure 1.. Cognitive deficits and neuroinflammation following tumor-clearing CAR T cell therapy

(A) Schematic illustration denoting the murine xenograft models assessed. (B, C, D, E) Schematic illustration of the various xenograft and CAR T cell therapy experimental timelines. (F) Schematic illustration of the Novel Object Recognition Test (NORT) to evaluate attention and short-term memory. Recognition ratio is defined as the percent time spent with the novel object over the total time spent interacting with either object. (G) DIPG xenografted mice treated with either mock T cells, Off-Target CD19-CAR T cells, or On-Target Therapeutic GD2-CAR T cells. NORT performed on day 34 post-CAR T therapy. Mock T cell Control (n=9 mice), CD19-CAR T Control (n=5 mice), GD2-CAR T Therapeutic (n=7 mice). (H) ALL xenografted mice treated with either mock T cells, Off-Target GD2-CAR T cells, or On-Target Therapeutic CD19-CAR T cells. NORT performed on day 27 post-CAR T therapy. Mock T cell Control (n=24 mice), GD2-CAR T Control (n=6 mice), CD19-CAR T Therapeutic (n=18 mice). (I) Aggressive osteosarcoma xenografted mice treated with either mock T cells or On-Target Therapeutic CD19-CAR T cells. NORT performed on day P34 post-CAR T therapy. Mock T cell Control (n=6 mice), CD19-CAR T Therapeutic (n=5 mice). (J) Rapid-clearing osteosarcoma xenografted mice treated with either mock T cells, On-Target Therapeutic GD2-CAR T, or On-Target Therapeutic B7H3-CAR T cells. NORT performed on day 34 post-CAR T therapy. Mock T cell Control (n=3 mice), GD2-CAR T Therapeutic (n=4 mice), B7H3-CAR T Therapeutic (n=4 mice). (K) T Maze Alternation Test to assess spatial working memory and hippocampal-related cognitive dysfunction. Percent Alternation is calculated over 11 trials allowing for 10 decisions. (L) DIPG xenografted mice treated with either mock T cell Control or On-Target Therapeutic GD2-CAR T cells. T maze performed on day 35 post-CAR T therapy. Mock T cell control (n=4 mice), GD2-CAR T therapeutic (n=4 mice). (M) ALL xenografted mice treated with either mock T cell Control or On-Target Therapeutic CD19-CAR T cells. T maze performed on day 28 post-CAR T therapy. Mock T cell control (n=4 mice), CD19-CAR T therapeutic (n=4 mice). (N) Aggressive osteosarcoma xenografted mice treated with either mock T cell Control or On-Target Therapeutic CD19-CAR T cells. T maze performed on day 35 post-CAR T therapy. Mock T cell control (n=7 mice), CD19-CAR T therapeutic (n=5 mice). (O) Rapid-clearing osteosarcoma xenografted mice treated with either mock T cells, On-Target Therapeutic GD2-CAR T, or On-Target Therapeutic B7H3-CAR T cells. T Maze performed on day 35 post-CAR T therapy. Mock T cell Control (n=4 mice), GD2-CAR T Therapeutic (n=4 mice), B7H3-CAR T Therapeutic (n=4 mice). (G-J and L-O) Data shown as mean ± SEM. Each point = one mouse. ns = p > 0.05, *p < 0.05, **p < 0.01, ****p < 0.0001. Analyzed via One Sample T and Wilcoxon Test.

Figure 2.. Persistent CSF cytokine and chemokine elevation after tumor-clearing CAR T cell therapy

(A) Left, heat map of fold change of cytokine and chemokine analysis of CSF in DIPG+On-Target Therapeutic GD2-CAR T cell treated mice relative to DIPG+mock T cell treated control mice 35-days post-CAR T cell therapy. Right, quantification of raw MFI of CSF levels of cytokines (CCL11, CCL2, CCL7, CXCL1, CXCL10, BAFF). (B) Left, heat map of fold change of cytokine and chemokine analysis of CSF in ALL+On-Target Therapeutic CD19-CAR T cell treated mice relative to ALL+mock T cell treated control mice 28-days post-CAR T cell therapy. Right, quantification of raw MFI of CSF levels of cytokines (CCL11, CCL2, CCL7, CXCL1, CXCL10, BAFF). (C) Left, heat map of fold change of cytokine and chemokine analysis of CSF in aggressive osteosarcoma (OS)+On-Target Therapeutic CD19-CAR T cell treated mice relative to aggressive osteosarcoma (OS)+mock T cell treated control mice 35-days post-CAR T cell therapy. Right, quantification of raw MFI of CSF levels of cytokines (CCL11, CCL2, CCL7, CXCL1, CXCL10, BAFF). (D) Left, heat map of fold change of cytokine and chemokine analysis of CSF in rapid-clearing osteosarcoma (RCOS)+On-Target Therapeutic GD2-CAR T cell treated mice relative to rapid-clearing osteosarcoma (RCOS)+mock T cell treated control mice 35-days post-CAR T cell therapy. Right, quantification of raw MFI of CSF levels of cytokines (CCL11, CCL2, CCL7, CXCL1, CXCL10, BAFF). (A-D) Cytokine and chemokines significantly elevated or depressed, analyzed via Student’s T test of raw MFI values.

Figure 3.. Tumor-clearing CAR T Cell therapy can result in white matter microglial reactivity

(A) Quantification of DIPG xenografted mice microglial reactivity (IBA1+ CD68+) and representative confocal micrographs (white = IBA1; red = CD68) from the corpus callosum 35 days post-CAR T therapy. Mock T cell Control (n=7 mice), CD19-CAR T Control (n=7 mice), GD2-CAR T Therapeutic (n=10 mice). (B) Quantification of DIPG xenografted mice microglial reactivity (IBA1+ CD68+) and representative confocal micrographs (white = IBA1; red = CD68) 35 days post-CAR T cell therapy in the hilar white matter region of the dentate gyrus. Mock T cell Control (n=8 mice), CD19-CAR T Control (n=6 mice), GD2-CAR T Therapeutic (n=10 mice). (C) Quantification of DIPG xenografted mice microglial reactivity (IBA1+ CD68+) 35 days post-CAR T therapy in the cortical grey matter. Mock T cell Control (n=7 mice), CD19-CAR T Control (n=7 mice), GD2-CAR T Therapeutic (n=10 mice). (D) Quantification of ALL xenografted mice microglial reactivity (IBA1+ CD68+) and representative confocal micrographs (white = IBA1; red = CD68) from the corpus callosum 28 days post-CAR T therapy. Mock T cell Control (n=9 mice), GD2-CAR T Control (n=5 mice), CD19-CAR T Therapeutic (n=12 mice). (E) Quantification of ALL xenografted mice microglial reactivity (IBA1+ CD68+) and representative confocal micrographs (white = IBA1; red = CD68;) 28 days post-CAR T cell therapy in the hilar white matter region of the dentate gyrus. Mock T cell Control (n=4 mice), GD2-CAR T Control (n=5 mice), CD19-CAR T Therapeutic (n=4 mice). (F) Quantification of ALL xenografted mice microglial reactivity (IBA1+ CD68+) 28 days post-CAR T therapy in the cortical grey matter. Mock T cell Control (n=9 mice), GD2-CAR T Control (n=5 mice), CD19-CAR T Therapeutic (n=12 mice). (G) Quantification of aggressive osteosarcoma xenografted mice microglial reactivity (IBA1+ CD68+) and representative confocal micrographs (white = IBA1; red = CD68) from the corpus callosum 35 days post-CAR T therapy. Mock T cell Control (n=5 mice), CD19-CAR T Therapeutic (n=5 mice). (H) Quantification of aggressive osteosarcoma xenografted mice microglial reactivity (IBA1+ CD68+) and representative confocal micrographs (white = IBA1; red = CD68) 35 days post-CAR T cell therapy in the hilar white matter region of the dentate gyrus. Mock T cell Control (n=5 mice), CD19-CAR T Therapeutic (n=5 mice). (I) Quantification of aggressive osteosarcoma xenografted mice microglial reactivity (IBA1+ CD68+) 35 days post-CAR T therapy in the cortical grey matter. Mock T cell Control (n=5 mice), CD19-CAR T Therapeutic (n=5 mice). (J) Quantification of rapid-clearing osteosarcoma xenografted mice microglial reactivity (IBA1+ CD68+) and representative confocal micrographs (white = IBA1; red = CD68) from the corpus callosum 35 days post-CAR T therapy. Mock T cell Control (n=4 mice), GD2-CAR Therapeutic (n=4 mice), B7H3-CAR T Therapeutic (n=4 mice). (K) Quantification of rapid-clearing osteosarcoma xenografted mice microglial reactivity (IBA1+ CD68+) and representative confocal micrographs (white = IBA1; magenta = CD68) 35 days post-CAR T cell therapy in the hilar white matter region of the dentate gyrus. Mock T cell Control (n=4 mice), GD2-CAR T Therapeutic (n=4 mice), B7H3-CAR T Therapeutic (n=4 mice). (L) Quantification of rapid-clearing osteosarcoma xenografted mice microglial reactivity (IBA1+ CD68+) 35 days post-CAR T therapy in the cortical grey matter. Mock T cell Control (n=4 mice), GD2-CAR Therapeutic (n=4 mice), B7H3-CAR T Therapeutic (n=4 mice). (A-F, J-L) Data shown as mean ± SEM. Each point = one mouse, ns = p > 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, analyzed via One-way ANOVA. (G-I) Data shown as mean ± SEM. Each point = one mouse, ns = p > 0.05, **p < 0.01, analyzed via Unpaired T Test. Scale bars equal 50um in all confocal images.

Figure 4.. Tumor-clearing CAR T Cell therapy can result in oligodendroglial and myelin deficits

(A) Quantification and representative confocal micrographs of PDGFRa+ oligodendrocyte precursor cells from the corpus callosum in DIPG xenografted mice 35 days post-CAR T therapy. Mock T cell Control (n=11 mice), CD19-CAR T Control (n=10 mice), GD2-CAR T Therapeutic (n=10 mice). (B) Quantification and representative confocal micrographs of PDGFRa+ oligodendrocyte precursor cells from the corpus callosum in ALL xenografted mice 28 days post-CAR T therapy. Mock T cell Control (n=6 mice), GD2-CAR T Control (n=5 mice), CD19-CAR T Therapeutic (n=11 mice). (C) Quantification and representative confocal micrographs of PDGFRa+ oligodendrocyte precursor cells from the corpus callosum in aggressive osteosarcoma xenografted mice 35 days post-CAR T therapy. Mock T cell Control (n=5 mice), CD19-CAR T Therapeutic (n=5 mice). (D) Quantification and representative confocal micrographs of ASPA+ mature oligodendrocytes from corpus callosum in DIPG xenografted mice 35 days post-CAR T therapy. Mock T cell Control (n=11 mice), CD19-CAR T Control (n=10 mice), GD2-CAR T Therapeutic (n=10 mice). (E) Quantification and representative confocal micrographs of ASPA+ mature oligodendrocytes from the corpus callosum in ALL xenografted mice 28 days post-CAR T therapy. Mock T cell Control (n=6 mice), CD19-CAR T Control (n=5 mice), GD2-CAR T Therapeutic (n=10 mice). (F) Quantification and representative confocal micrographs of ASPA+ mature oligodendrocytes from the corpus callosum in aggressive osteosarcoma xenografted mice 35 days post-CAR T therapy. Mock T cell Control (n=5 mice), CD19-CAR T Therapeutic (n=5 mice). (G) Quantification and representative confocal micrographs of PDGFRa+ oligodendrocyte precursor cells from the corpus callosum in rapid-clearing osteosarcoma xenografted mice 35 days post-CAR T therapy. Mock T cell Control (n=4 mice), GD2-CAR T Therapeutic l (n=4 mice), B7H3-CAR T Therapeutic (n=4 mice). (H) Quantification and representative confocal micrographs of ASPA+ mature oligodendrocytes from the corpus callosum in rapid-clearing osteosarcoma xenografted mice 35 days post-CAR T therapy. Mock T cell Control (n=4 mice), GD2-CAR T Therapeutic (n=4 mice), B7H3-CAR T Therapeutic (n=4 mice). (I) Schematic illustration denoting the cingulum region of the corpus callosum used for the transmission electron microscopy (TEM) DIPG xenografted mice 35 days post-CAR T therapy. (J) Quantification and Representative TEM images of white matter myelinated axons per area at day P35 post-CAR T therapy. Myelinated axonal projections are in cross section and visible as electron-dense sheaths encircling axons. Scale bar = 2 um. Mock T cell control (n=6 mice), GD2-CAR T therapeutic (n=6 mice). (A, B, D, E, G and H) Data shown as mean ± SEM. Each point = one mouse. ns = p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, analyzed via One-way ANOVA. (C, F and J) Data shown as mean ± SEM. Each point = one mouse, *p < 0.05, analyzed via Unpaired T Test. Scale bars equal 50um in all confocal micrographs (A-H), and 2um in electron microscopy images (J).

Figure 5.. Tumor-clearing CAR T Cell therapy can impair hippocampal neurogenesis

(A) Quantification of neuroblasts (DCX+) in DIPG model and representative confocal micrographs (magenta = DCX; cyan = DAPI) from the dentate gyrus 35 days after CAR T cell therapy. Mock T cell control (n=8 mice), CD19-CAR T control (n=8 mice), GD2-CAR T Therapeutic (n=8 mice). (B) Quantification of neuroblasts (DCX+) in ALL model and representative confocal micrographs (magenta = DCX; cyan = DAPI) from the dentate gyrus 28 days after CAR T cell therapy. Mock T cell control (n=4 mice), GD2-CAR T control (n=4 mice), CD19-CAR T Therapeutic (n=5 mice). (C) Quantification of neuroblasts (DCX+) in aggressive osteosarcoma model and representative confocal micrographs (magenta = DCX; cyan = DAPI) from the dentate gyrus 35 days after CAR T cell therapy. Mock T cell control (n=5 mice), CD19-CAR T Therapeutic (n=5 mice). (D) Quantification of neuroblasts (DCX+) in rapid-clearing osteosarcoma model and representative confocal micrographs (magenta = DCX; cyan = DAPI) from the dentate gyrus 35 days after CAR T cell therapy. Mock T cell control (n=3 mice), GD2-CAR T Therapeutic (n=3 mice), B7H3-CAR T Therapeutic (n=4 mice). (A, B, and D) Data shown as mean ± SEM. Each point = one mouse. ns = p > 0.05, *p < 0.05, **p < 0.01, ****p < 0.0001, analyzed via One-way ANOVA. (C) Data shown as mean ± SEM. Each point = one mouse, **p < 0.01, analyzed via Unpaired T Test. Scale bars equal 50um in all confocal images.

Figure 6.. Microglial depletion rescues oligodendroglial dysregulation cognitive performance in novel object recognition testing

(A) Schematic illustrations of experimental timeline of the DIPG model including administration of the CSF1R inhibitor (PLX5622). (B) Schematic illustrations of experimental timeline of the ALL model including administration of the CSF1R inhibitor (PLX5622). (C) Quantification of DIPG xenografted mice corpus callosum white matter microglia (IBA1+) 35 days post-CAR T therapy with or without 14 days of CSF1R inhibition. Mock T cell+Vehicle Chow (n=4 mice), mock T cell+CSF1Ri Chow (n=4 mice), GD2-CAR T Therapeutic+Vehicle Chow (n=4 mice), GD2-CAR T Therapeutic+CSF1Ri Chow (n=4 mice). Representative confocal micrographs of microglia (white = IBA1) in the corpus callosum of DIPG xenografted mice 35 days after CAR T cell therapy with or without 14 days of CSF1R inhibitor (PLX5622). (D) Quantification of ALL xenografted mice corpus callosum white matter microglia (IBA1+) 28 days post-CAR T therapy with or without 14 days of CSF1R inhibition. Mock T cell+Vehicle Chow (n=6 mice), CD19-CAR T Therapeutic+Vehicle Chow (n=5 mice), CD19-CAR T Therapeutic+CSF1Ri Chow (n=6 mice). Representative confocal micrographs of microglia (white = IBA1) in the corpus callosum of ALL xenografted mice 28 days after CAR T cell therapy with or without 14 days of CSF1R inhibitor (PLX5622). (E) Quantification of DIPG xenografted mice dentate gyrus microglia (IBA1+) 35 days post-CAR T therapy with or without 14 days of CSF1R inhibition. Mock T cell+Vehicle Chow (n=4 mice), mock T cell+CSF1Ri Chow (n=4 mice), GD2-CAR T Therapeutic+Vehicle Chow (n=4 mice), GD2-CAR T Therapeutic+CSF1Ri Chow (n=4 mice). Representative confocal micrographs of microglia (white = IBA1) in the dentate gyrus of DIPG xenografted mice 35 days after CAR T cell therapy with or without 14 days of CSF1R inhibitor (PLX5622). (F) Quantification of ALL xenografted mice dentate gyrus microglia (IBA1+) 28 days post-CAR T therapy with or without 14 days of CSF1R inhibition. Mock T cell+Vehicle Chow (n=6 mice), CD19-CAR T Therapeutic+Vehicle Chow (n=5), CD19-CAR T Therapeutic+CSF1Ri Chow (n=6 mice). Representative confocal micrographs of microglia (white = IBA1) in the dentate gyrus of ALL xenografted mice 28 days after CAR T cell therapy with or without 14 days of CSF1R inhibitor (PLX5622). (G) Quantification of DIPG xenografted mice oligodendrocyte precursor cells (PDGFRa+) 35 days post-CAR T therapy with or without 14 days of CSF1R inhibition. Mock T cell+Vehicle Chow (n=7 mice), mock T cell+CSF1Ri Chow (n=5 mice), GD2-CAR T Therapeutic+Vehicle Chow (n=10 mice), GD2-CAR T Therapeutic+CSF1Ri Chow (n=10 mice). Representative confocal micrographs of PDGFRa+ oligodendrocyte precursor cells (white = PDGFRa) in the corpus callosum of DIPG xenografted mice 35 days after CAR T cell therapy with or without 14 days of CSF1R inhibitor (PLX5622). (H) Quantification of ALL xenografted mice oligodendrocyte precursor cells (PDGFRa+) 28 days post-CAR T therapy with or without 14 days of CSF1R inhibition. Mock T cell+Vehicle Chow (n=6 mice), CD19-CAR T Therapeutic+Vehicle Chow (n=5 mice), CD19-CAR T Therapeutic+CSF1Ri Chow (n=4 mice). Representative confocal micrographs of PDGFRa+ oligodendrocyte precursor cells (white = PDGFRa) in the corpus callosum of ALL xenografted mice 28 days after CAR T cell therapy with or without 14 days of CSF1R inhibitor (PLX5622). (I) Quantification of DIPG xenografted mice mature oligodendrocytes (ASPA+) 35 days post-CAR T therapy with or without 14 days of CSF1R inhibition. Mock T cell+Vehicle Chow (n=4 mice), mock T cell+CSF1Ri Chow (n=5 mice), GD2-CAR T Therapeutic+Vehicle Chow (n=4 mice), GD2-CAR T Therapeutic+CSF1Ri Chow (n=4 mice). Representative confocal micrographs of ASPA+ oligodendrocytes (white = ASPA) in the corpus callosum of DIPG xenografted mice 35 days after CAR T cell therapy with or without 14 days of CSF1R inhibitor (PLX5622). (J) Quantification of ALL xenografted mice mature oligodendrocytes (ASPA+) 28 days post-CAR T therapy with or without 14 days of CSF1R inhibition. Mock T cell+Vehicle Chow (n=6 mice), CD19-CAR T Therapeutic+Vehicle Chow (n=5 mice), CD19-CAR T Therapeutic+CSF1Ri Chow (n=4 mice). Representative confocal micrographs of ASPA+ oligodendrocytes (white = ASPA) in the corpus callosum of ALL xenografted mice 28 days after CAR T cell therapy with or without 14 days of CSF1R inhibitor (PLX5622). (K) NORT results from DIPG xenografted mice 34 days post-CAR T therapy with or without 14 days of CSF1R inhibition. Mock T cell+Vehicle Chow (n=9 mice), mock T cell+CSF1Ri Chow (n=8 mice), GD2-CAR T Therapeutic+Vehicle Chow (n=9 mice), GD2-CAR T Therapeutic+CSF1Ri Chow (n=10 mice). (L) NORT results from ALL xenografted mice 27 days post-CAR T therapy with or without 14 days of CSF1R inhibition. Mock T cell+Vehicle Chow (n=14 mice), CD19-CAR T Therapeutic+Vehicle Chow (n=12 mice), CD19-CAR T Therapeutic+CSF1Ri Chow (n=14 mice). Data shown as mean ± SEM (C-J). Each point = one mouse. ns = p > 0.05, * p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, analyzed via 2-way ANOVA. (K and L) Data shown as mean ± SEM. Each point = one mouse. ns = p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Analyzed via One Sample T and Wilcoxon Test. Scale bars equal 50um in all confocal images (A-J).