1264 Investigating the mechanisms of <i>Helicobacter hepaticus</i> mediated lymphangiogenesis and tertiary lymphoid structure formation

<h3>Background</h3> Recent studies have shown that the composition of a patient’s gut microbiome¹ and the presence of tertiary lymphoid structures (TLS) are associated with improved immune checkpoint blockade response in a variety of cancers.^2–4 However, it remains unclear whether TLS are causative for better cancer outcomes or are simply a byproduct of a robust anti-cancer immune response. Our group has shown that <i>Helicobacter hepaticus</i> (<i>Hhep</i>) colonization reduces colorectal tumor burden and supports mature TLS formation. <i>Hhep</i> colonization induces a shift in local antigen presenting cell fate and function, increases genes associated with lymphangiogenesis (<i>Lyve1</i>, <i>Pdpn</i>, and <i>Vegfc</i>), and increases lymphatic vessels surrounding TLS. We hypothesize that colonization with <i>Hhep</i>, an immunogenic bacterium, alters macrophage phenotypes to support lymphangiogenesis that subsequently drives TLS maturation. <h3>Methods</h3> 4–6-week-old female C57Bl/6 SPF Jackson Labs mice were gavaged twice with <i>Hhep</i> (D-4, D0) and colons were harvested every 2 days for multispectral immunofluorescent imaging of TLS. In addition, we have assessed the temporal changes in lymphangiogenesis and TLS maturity through spatial transcriptomics (GeoMx DSP) to determine the initial driving factors of TLS. Antibody mediated depletion of macrophages (anti-CSF1R) was used to determine the importance of macrophages in <i>Hhep</i>-mediated lymphangiogenesis and TLS formation. <h3>Results</h3> <i>Hhep</i> induces TLS in the distal colon within 14 days; strikingly, morphological changes within the tissue are evident as early as day 2 post-colonization. Macrophage depletion after <i>Hhep</i> colonization significantly reduces TLS maturation. Analysis of the colon lamina propria and draining lymph nodes show that <i>Hhep</i> colonization supports inflammatory macrophage polarization (increased MHCII and CX3CR1), higher B cell infiltration, and increased T cell activation. <h3>Conclusions</h3> We have found that macrophages contribute to TLS maturity and are currently assessing whether macrophages mediate this process directly or indirectly. The <i>Hhep</i>-induced TLS model provides us the unique opportunity to elucidate the mechanisms promoting lymphangiogenesis as well as the mechanisms required for the formation and maturation of TLS in health and disease. Improving our understanding of the mechanisms governing lymphangiogenesis and TLS maturation has the potential to uncover novel immunotherapeutic targets. <h3>References</h3> Helmink BA, Reddy SM, Gao J, <i>et al</i>. B cells and tertiary lymphoid structures promote immunotherapy response. <i>Nature</i>. 2020;<b>577</b>(7791):549–555. Ruffin AT, Cillo AR, Tabib T, <i>et al</i>. B cell signatures and tertiary lymphoid structures contribute to outcome in head and neck squamous cell carcinoma. <i>Nat Commun</i>. 2021;<b>12</b>(1):3349. Wang Q, Shen X, An R, <i>et al</i>. Peritumoral tertiary lymphoid structure and tumor stroma percentage predict the prognosis of patients with non-metastatic colorectal cancer. <i>Front Immunol</i>. 2022;<b>13</b>:962056. Cabrita R, Lauss M, Sanna A, <i>et al</i>. Tertiary lymphoid structures improve immunotherapy and survival in melanoma. <i>Nature</i>. 2020;<b>577</b>(7791):561–565.