Intestinal toxicity to CTLA-4 blockade driven by IL-6 and myeloid infiltration YifanZhou 1 , Yusra B. Medik 1 , Bhakti Patel 1 , Daniel B. Zamler 2,3 , Sijie Chen 4 , Thomas Chapman 5 , Sarah Schneider 1,3,6 , Elizabeth M. Park 1,2 , Rachel L. Babcock 1,3 , Taylor T. Chrisikos 1,3 , Laura M. Kahn 1,3 , Allison M. Dyevoich 1 , Josue E. Pineda 1,3 , Matthew C. Wong 7 , Aditya K. Mishra 7 , Samuel H. Cass 5 , Alexandria P. Cogdill 1,2,3 , Daniel H. Johnson 8 , Sarah B. Johnson 5 , Khalida Wani 9 , Debora A. Ledesma 9 , Courtney W. Hudgens 9 , Jingjing Wang 5 , Md Abdul Wadud Khan 5 , Christine B. Peterson 3,10 , Aron Y. Joon 10 , Weiyi Peng 8,11 , Haiyan S. Li 1 , ReetakshiArora 5 , XimingTang 9 , Maria Gabriela Raso 9 , Xuegong Zhang 4 , Wai Chin Foo 12 , Michael T. Tetzlaff 9,13 , Gretchen E. Diehl 14 , Karen Clise-Dwyer 6 , Elizabeth M. Whitley 15 , Matthew M. Gubin 1,3,16 , James P. Allison 1,3,16 , Patrick Hwu 3,8,17 , Nadim J. Ajami 2,7 , Adi Diab 8 , Jennifer A. Wargo 2,3,5,7,16 , Stephanie S. Watowich 1,3,7 * 1. Department of Immunology, The University of Texas MD Anderson Cancer Center, Houston, TX; 2. Department of Genomic Medicine, The University of Texas MD Anderson Cancer Center, Houston, TX; 3. The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences, Houston, TX; 4. MOE Key Lab of Bioinformatics and Bioinformatics Division, BNRIST; Department of Automation, Tsinghua University, Beijing 100084, China; 5. Department of Surgical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX; 6. Department of Hematopoietic Biology and Malignancy, The University of Texas MD Anderson Cancer Center, Houston, TX; 7. Platform for Innovative Microbiome and Translational Research (PRIME-TR), MD Anderson Cancer Center, Houston, TX; 8. Department of Melanoma Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX; 9. Department of Translational Molecular Pathology, The University of Texas MD Anderson Cancer Center, Houston, TX; 10. Department of Biostatistics, The University of Texas MD Anderson Cancer Center, Houston, TX; 11. Current address: Department of Biology and Biochemistry, The University of Houston, Houston, TX; 12. Department of Pathology, The University of Texas MD Anderson Cancer Center, Houston, TX; 13. Current address: Department of Pathology and Dermatology, Dermatopathology and Oral Pathology Unit, University of California, San Francisco; 14. Immunology Program, Memorial Sloan Kettering Cancer Center, New York, NY; 15. Department of Veterinary Medicine and Surgery, The University of Texas MD Anderson Cancer Center, Houston, TX; 16. Parker Institute for Cancer Immunotherapy, The University of Texas MD Anderson Cancer Center, Houston, TX;17. Current address: Moffitt Cancer Center, Tampa, FL
Abstract Immunotherapies such as anti-CTLA-4 ( a CTLA-4) immune checkpoint blockade (ICB) have revolutionized cancer treatment, yet quality of life and continuation of therapy can be constrained by off-target tissue damage or immune-related adverse events (irAEs). At present, there is limited understanding of irAE mechanisms, hampering development of approaches to mitigate their damage. We addressed this problem by generating animal models of intestinal irAE. Our results show that disruption of homeostatic immunity by genetic predisposition to intestinal inflammation or acute gastrointestinal infection sensitizes mice to a CTLA-4-mediated intestinal toxicity. Inflammation-prone mice treated with a CTLA-4 showed neutrophil accumulation, systemic interleukin-6 (IL-6) release, and dysbiosis. Significantly, IL-6 blockade combined with antibiotic treatment improved a CTLA-4 therapeutic efficacy and reduced intestinal irAEs. Immune signatures were validated in biopsies from patients who developed colitis during ICB, supporting the utility of our models. This study provides new pre-clinical models, mechanistic insight into irAEs, and potential approaches to enhance ICB efficacy while mitigating irAEs.
Results
Fig. 1 Inflammation-prone mice show exaggerated intestinal inflammation upon a CTLA-4 therapy.
Fig. 5 Therapeutic interventions to enhance a CTLA-4 efficacy and suppress irAE.
Fig. 3 a CTLA-4 remodels the intestinal immune repertoire in inflammation-prone conditions.
B
A
C
D
Fig. 3 (A-C) Stat3 D / D and Stat3 +/+ mice bearing B16-OVA tumors were treated biweekly for 2 weeks with IgG or a CTLA-4, as indicated in Fig. 1. Colonic LP immune cells were subjected to scRNAseq. (A) UMAP plot showing distinct clusters. (B) Proportion of individual clusters in each experimental group. (C) Expression module scores of Gene Ontology terms.
Fig. 1 (A) Stat3 D / D and Stat3 +/+ mice bearing B16-OVA melanoma tumors were treated biweekly for 2 weeks with IgG or a CTLA-4, beginning 4 d after tumor establishment. (B) Body weight change over time. (C, D) Colon histology by H&E staining. (E) Colon cytokine levels determined by multiplex assay.
Fig. 5 Stat3 D / D and Stat3 +/+ mice bearing B16-OVA tumors were treated biweekly for 2 weeks with IgG or a CTLA-4, as indicated in Fig. 1. (A, B) Feces were collected prior to or following a CTLA-4 treatment; fecal microbiota composition was determined by 16S ribosomal RNA profiling. (C, D) B16-OVA bearing Stat3 D / D mice on a CTLA-4 were treated with or without broad-spectrum antibiotics (Abx) and an IL6 blocking antibody ( a IL-6), as indicated. Colon histopathological scores and immune profiles were analyzed in mice 4 days following conclusion of therapy.
Fig. 4 a CTLA-4 drives systemic cytokine release and myelopoiesis in inflammation-prone mice.
Fig. 2 Immune signatures in human intestinal irAE.
Summary of irAE-driving mechanisms
Introduction
Microbiome dysbiosis & infection
Most clinically important irAEs with a CTLA-4/combination blockade or a PD-1 blockade
Macrophage
α -CTLA-4
IL-6 G-CSF
CTLA-4
Cytokines
B7
CD28
MHC
TCR
Hypothyroidism Pneumonitis Myocarditis Arthralgia Autoimmune Diabetes
Dendritic cell
Hypophysitis
T cell
Neutrophil infiltration
IFN γ
Myeloid inflammation
Treg
Gzmb + T
Rash/Dermatitis
Th1
Acknowledgements
Fig. 2 (A-D) Colitis regions and normal intestinal biopsies were evaluated by NanoString. (A) Principal component analysis of all biopsies. (B) Estimation of immune subset abundance using expression of cell type-specific marker genes. Data represent relative abundance scores. (C) Heatmap of pathway z-scores, summarized from data representing pathway-related gene expression with a single score. (D) Volcano plot showing differential gene expression.
Fig. 4 (A-E) Stat3 D / D and Stat3 +/+ mice bearing B16-OVA tumors were treated biweekly for 2 weeks with IgG or a CTLA-4, as indicated in Fig. 1. (A) Mean concentration of differently expressed cytokines in serum. (B) tSNE plot showing prospective clustering of live spleen cells. (C) Neutrophil and monocyte amounts in spleen.
Enteritis
Colitis
MD Anderson Center for Inflammation and Cancer MD Anderson South Campus Flow Core
NIH NIAID (R01AI109294, 3R01AI109294-04S1, R56AI109294, R01AI133822) (to SSW) Research Training Award from the Cancer Prevention and Research Institute of Texas (CPRIT RP170067) (to RLB, TTC, APC) MD Anderson NIH NCI P30CA016672 grant
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