CXCL9-Dependent Immune Networks of Spontaneous Tertiary Lymphoid Structures in a Murine Subcutaneous Tumor Model

Abstract

Recent discoveries have identified human tumor tertiary lymphoid structures (TLS) as organized clusters of innate and adaptive immune cells and stromal cells. Cancer-associated TLS can promote tumor clearance and enhance responses to immune checkpoint blockade (ICB) immunotherapy. However, the absence of TLS in conventional mouse tumor models, particularly subcutaneous transplant models, limits experimental manipulation and study of TLS development. By tuning tumor antigen expression, we have generated a subcutaneous tumor model that naturally forms TLS within the immune-hot, vasculature-rich peritumoral adipose tissue. These TLS were identified as clusters of B and T cells in close proximity to high endothelial venules. TLS organization was underpinned by spatially compartmentalized chemokine expression, with CCL21 produced in the T cell zone of perivascular TLS and CXCL13 present in the B cell region. Single cell transcriptomic sequencing showed that the immune composition in the TLS forming peritumoral tissue resembled human cancer TLS signatures. Upon anti-PD-L1 ICB treatment, TLS underwent expansion, which was associated with increased interferon responses in macrophages and coincided with tumor clearance. We further identified the interferon-inducible chemokine CXCL9 as a key regulator of the TLS microenvironment. CXCL9-expressing macrophages populated TLS, interacting with B cells following ICB treatment. CXCL9 deficiency reduced B cell recruitment to peritumoral tissue and TLS formation. Further T and B cell receptor sequencing indicated a CXCL9-dependent promotion of antigen-specific responses within the tumor and TLS niche. Combined, these findings establish a tractable subcutaneous model of naturally arising tumor TLS and define the cellular and molecular networks that regulate their formation and function. This provides a platform to dissect how TLS develop and test strategies that promote TLS formation to enhance cancer therapy.