Special Poster Session 50th International Society for the Study of the Lumbar Spine Annual Meeting 2024

INTRODUCTION: Modic type 1 changes (MC1) are among the most specific features for chronic low back pain. Yet, the pathobiology of the affected marrow remains largely unknown. Endplate damage with MC1 expose the immune-privileged disc to immune cells of the bone marrow, potentially triggering an autoimmune response of the marrow against disc tissue. T-cell infiltration has been reported in a MC1 rat model, and plasma cell infiltration in human MC1 biopsies suggest B-cell involvement. However, a comprehensive analysis of immune system remodeling in human MC1 lesions is missing. This would identify the underlying immune mechanisms and pave ways for targeted treatments of MC1. The aim of this study was to discover changes in the bone marrow immune cell composition in MC1.

METHODS: Vertebral bone marrow aspirates from patients undergoing lumbar spinal fusion surgery (n=17) were taken before screw insertion through the pedicle screw trajectory. In patients with MC1 (n=8), one aspirate was taken from the MC lesion, and one other from visually unaffected bone marrow adjacent to MC1. From patients without MC1 (n=9), a control bone marrow aspirate was processed. Mononuclear cells were isolated and analyzed with a novel broad spectral flow cytometry-based immunophenotyping panel using CD45 multiplexing to avoid batch effects. CD45 was barcoded with three out of five antibodies against CD45 but conjugated with different fluorochromes. Barcoded samples were mixed and co-stained with 36 antibodies against surface markers, allowing us to distinguish major immune cell lineages and to follow the activation and functional maturation of these cell subsets. Samples were analyzed on Cytek Aurora spectral flow cytometer using SpectroFlo software. Demultiplexing and gating were performed in FlowJo. Prism software was used for statistical analysis. 

RESULTS: T-cells were the only major immune cell subset increased in MC1 patients (Figure_1A). This was associated with changes in functional maturation: decreased proportion of naïve T-cells and an increase in effector subsets (Figure_1B, C). This suggests continuous stimulation of T-cells. The increase in T-cell frequency was driven by the increase in TCR αβ⁺CD8⁺ and TCR Vδ1⁺ T-cell subsets (Figure_1D). No differences in T-cell frequency were observed between MC1 lesion and intra-patient controls, suggesting that MRI-normal bone marrow is also immunologically affected (Figure_1A, D). The activation marker CD69 was expressed by higher proportions of TCR Vδ1⁺ T-cells and TCR αβ⁺CD8⁺ T-cells in MC1-affected vertebrae, indicating ongoing immune response in MC1 mediated by these two T-cell subsets (Figure_1E).

DISCUSSION: TCR αβ⁺CD8⁺ and TCR Vδ1⁺ T-cells were identified as potential disease-driving immune cell subsets in MC1. Expansion of TCR αβ⁺CD8⁺ indicate antigen stimulation. Antigens could originate from the immune-privileged disc or intradiscal bacteria. Expansion of TCR Vδ1⁺ T-cells can occur without antigens though stress response, e.g. disc/endplate damaging. Yet, the antigen and mode of activation remains to be elucidated. This study provides the first evidence of specific T-cell subset accumulation in MC1. Discovery of TCR αβ⁺CD8⁺ T-cell and TCR Vδ1⁺ T-cell accumulation might represent a key step towards understanding the immune mechanisms in MC1 and ultimately to targeted treatments for MC1.