873 Investigating the role of NR4A1 in dendritic cell pro-tolerogenic function and its impact on anti-tumor immunity

<h3>Background</h3> Dendritic cells (DCs) are essential mediators of anti-tumor immunity and are necessary for effective checkpoint blockade responses, however within the tumor microenvironment (TME), dysfunctional DCs develop that promote tolerance.^1–3 We have recently discovered that tumor-derived lactate activates SREBP2 in DCs within the TME, promoting the development of pro-tolerogenic mature regulatory DCs (mregDCs).⁴ DC-restricted SREBP2 silencing suppressed mregDC development and inhibited tumor progression <i>in vivo</i>. Despite this work, our understanding of the transcriptional network that dictates mregDC development and function remains incomplete. Insights into these mechanisms hold great promise for the development of novel immunotherapeutic strategies. <h3>Methods</h3> To characterize DC regulatory programs, we used transgenic mouse models of melanoma and single-cell assay for transposase-accessible chromatin sequencing (scATACseq) to identify transcription factor binding motifs that are altered amongst different conventional DC sub-populations. Using CUT &amp; Tag, we characterized the transcription factor NR4A1 in DC function and then determined its contribution to regulating anti-tumor immunity using both <i>in vitro</i> and <i>in vivo</i> models. <h3>Results</h3> scATACseq and transcription factor binding motif analysis of DCs isolated from transgenic BRAF^V600EPTEN^-/- melanoma-bearing mice revealed an enrichment in the accessibility of NR4A1 motifs in mregDCs. We demonstrated that lactate released by tumor cells via monocarboxylate transporter 1 (MCT1) induces DC <i>Nr4a1</i> gene expression in a SREBP2-dependent manner utilizing a <i>Nr4a1</i>-GFP reporter model. ChIP-qPCR verified that SREBP2 binds to the <i>Nr4a1</i> promoter and CUT &amp; Tag sequencing identified significant differences in NR4A1 genomic localization in mregDCs relative to other cDCs. Given this SREBP2-NR4A1 signaling axis, we conducted additional T cell cross-priming studies demonstrating that inhibition of NR4A1 reversed lactate-mediated suppression of DC-dependent CD8⁺ T cell activation. Additional studies revealed NR4A1 inhibition to suppress mregDC expression of <i>Ido1</i> and <i>Il4i1</i> while also inhibiting mregDC-dependent FoxP3⁺-regulatory T cell development and activation. Ongoing experiments are identifying the extent to which DC-specific Nr4a1 drives tumor-mediated immune tolerance and tumor progression in vivo. <h3>Conclusions</h3> Tolerogenic DCs are well-established contributors to immunosuppression during tumor progression, but how they develop and function to prevent adaptive immunity is poorly understood. Here, we have expanded our understanding of the development and function of the pro-tolerogenic mregDC population by determining that NR4A1 functions downstream of a lactate-SREBP2 axis to regulate antigen cross-presentation and Treg differentiation. Given the ability of mregDCs to block antigen cross-presentation while driving Treg development in the TME, targeting the SREBP2-NR4A1 signaling axis represents a promising strategy for overcoming anti-PD-1 resistance in traditionally refractory tumors. <h3>References</h3> Gardner A, Ruffell B. Dendritic cells and cancer immunity. <i>Trends Immunol</i> 2016;<b>37</b>:855–865. doi:10.1016/j.it.2016.09.006. DeVito NC, Plebanek MP, Theivanthiran B, Hanks BA. Role of tumor-mediated dendritic cell tolerization in immune evasion. <i>Front Immunol</i> 2019;<b>10</b>:2876. doi:10.3389/fimmu.2019.02876. Gerhard GM, Bill R, Messemaker M, Klein AM, Pittet MJ. Tumor-infiltrating dendritic cell states are conserved across solid human cancers. <i>J Exp Med</i> 2021;<b>218</b>:doi:10.1084/jem.20200264. Plebanek MP, <i>et al</i>. A lactate-SREBP2 signaling axis drives tolerogenic dendritic cell maturation and promotes cancer progression. <i>Sci Immunol</i> 2024;<b>9</b>:eadi4191.doi:10.1126/sciimmunol.adi4191.