{
  "abstract": "Background Immune checkpoint inhibitor therapy (ICT), targeting CTLA-4 and/or PD-1, can induce durable remissions in previously incurable cancers. However, many patients remain refractory or develop resistance. 1–3 The tumor immune microenvironment is a critical determinant of ICT response.4–6 Recent clinical studies suggest that the timing of ICT infusions correlates with survival, but the mechanistic basis for this association remains unclear.7–9 The circadian clock—an evolutionarily conserved, cell-intrinsic transcriptional-translational feedback loop that synchronizes physiology with environmental cues—regulates innate and adaptive immunity.10–12 Yet how circadian rhythms influence the tumor immune microenvironment and ICT efficacy remains poorly understood.Methods We assessed ICT efficacy at six circadian timepoints (ZT2, ZT6, ZT10, ZT14, ZT18, ZT22) using syngeneic mouse models (e.g., MC38) treated with anti-PD-1 and/or anti-CTLA-4 antibodies. Tumor growth and survival were monitored. Tumor immune microenvironment composition and function were profiled using single-cell spatial transcriptomics (Visium HD), single-cell RNA sequencing, flow cytometry, cytokine multiplex assays, immunohistochemistry, and bulk RNA sequencing of tumor-infiltrating dendritic cells. We analyzed clinical cohorts to identify associations between the time of day of ICT initiation (first infusion), serum chemokine levels, and survival outcomes.Results ICT efficacy varied significantly by time of day. ICT was most efficacious at ZT2 (two hours after first light exposure) and least effective at ZT18 ( figure 1A, 1B). This phenotype was consistent across multiple tumor models. Circadian timing shaped tumor immune microenvironment composition, including immune cell abundance, cytokine and chemokine production, and spatial localization of myeloid and T cells in tumors (figure 1C, 1D). Deletion of the core clock gene BMAL1 in either dendritic cells (BMAL1^flox/flox × CD11c-Cre) or CD8+ T cells (BMAL1^flox/flox × CD8a-Cre) abolished time-of-day differences in ICT response and impaired anti-tumor immunity. Mechanistically, circadian regulation of CX3CL1 in dendritic cells governed recruitment of CX3CR1+ CD8+ T cells into tumors, reshaping the tumor immune microenvironment and modulating ICT response. Disruption of this axis—via CX3CL1 administration or CX3CR1 deletion—eliminated time-of-day differences in ICT efficacy. In patients, early morning ICT initiation correlated with improved survival and higher serum chemokines compared to late-afternoon treatment (figure 2).Conclusions These findings reveal a novel circadian mechanism that regulates immune cell recruitment into tumors and establish treatment timing as a critical, yet underappreciated, determinant of immunotherapy response.References Larkin J, Chiarion-Sileni V, Gonzalez R, et al. Combined nivolumab and ipilimumab or monotherapy in untreated melanoma. N Engl J Med. 2015;373:23–34.Motzer RJ, Tannir NM, McDermott DF, et al. Nivolumab plus ipilimumab versus sunitinib in first-line treatment for advanced renal cell carcinoma: extended follow-up of efficacy and safety results from a randomised, controlled, phase 3 trial. Lancet Oncol. 2019;20:1370–1385.Schmid P, Adams S, Rugo HS, et al. Atezolizumab and nab-paclitaxel in advanced triple-negative breast cancer. N Engl J Med. 2018;379:2108–2121.Giles JR, Globig AM, Kaech SM, Wherry EJ. CD8(+) T cells in the cancer-immunity cycle. Immunity. 2023;56:2231–2253.Mellman I, Chen DS, Powles T, Turley SJ. The cancer-immunity cycle: indication, genotype, and immunotype. Immunity. 2023;56:2188–2205.Zemek RM, Anagnostou V, Pires da Silva I, Long GV, Lesterhuis WJ. Exploiting temporal aspects of cancer immunotherapy. Nat Rev Cancer. 2024;24:480–497.Qian DC, Klebaner C, Yumul B, et al. Influence of time of day of immunotherapy infusion on overall survival among patients with advanced melanoma: a propensity score-matched analysis. Lancet Oncol. 2021;22:1777–1786.Deng H, Zhou R, Zhang L, et al. Chronotherapy of immune checkpoint inhibitors in metastatic non-small cell lung cancer: real-world evidence from a multicenter cohort. EBioMedicine. 2024;101:105630.Patel JS, Woo Y, Draper A, et al. Impact of immunotherapy time of day infusion on survival and immunologic correlates in metastatic renal cell carcinoma: a multicenter cohort analysis. J Immunother Cancer. 2024;12:e008011.Takahashi JS. Transcriptional architecture of the mammalian circadian clock. Nat Rev Genet. 2017;18:164–179.Brooks JF 2nd, Hooper LV. Interactions among microbes, the immune system, and the circadian clock. Semin Immunopathol. 2020;42:697–708.Wang C, Lutes LK, Barnoud C, Scheiermann C. The circadian immune system. Sci Immunol. 2022;7:eabm2465.Ethics Approval Experiments using animals were performed using protocols (APN 2017-102122) approved by the Institutional Animal Care and Use Committees (IACUC) of the UT Southwestern Medical Center. Clinical samples and data were collected as part of a prospective biospecimen registry study approved by the University of Texas Southwestern Institutional Review Board (IRB #STU 082015–053). Patients provided written informed consent prior to undergoing any study-specific procedures.Abstract 461 Figure 1ICT efficacy is time-of-day dependentAbstract 461 Figure 2Initial ICT infusions in the early morning improve survival in cancer patients",
  "authors": [
    {
      "affiliations": [
        "University of Texas Southwestern Medical Center, Dallas, TX, USA"
      ],
      "name": "Jake N Lichterman"
    },
    {
      "affiliations": [
        "University of Texas Southwestern Medical Center, Dallas, TX, USA"
      ],
      "name": "Tarun Srinivasan"
    },
    {
      "affiliations": [
        "University of Texas Southwestern Medical Center, Dallas, TX, USA"
      ],
      "name": "Ruheng Wang"
    },
    {
      "affiliations": [
        "University of Texas Southwestern Medical Center, Dallas, TX, USA"
      ],
      "name": "Chaitanya Dende"
    },
    {
      "affiliations": [
        "University of Texas Southwestern Medical Center, Dallas, TX, USA"
      ],
      "name": "Mitchell S von Itzstein"
    },
    {
      "affiliations": [
        "University of Texas Southwestern Medical Center, Dallas, TX, USA"
      ],
      "name": "Jialiang Liu"
    },
    {
      "affiliations": [
        "University of Texas Southwestern Medical Center, Dallas, TX, USA"
      ],
      "name": "Josue Ramirez"
    },
    {
      "affiliations": [
        "University of Texas Southwestern Medical Center, Dallas, TX, USA"
      ],
      "name": "Wenling Li"
    },
    {
      "affiliations": [
        "University of Texas Southwestern Medical Center, Dallas, TX, USA"
      ],
      "name": "Laura Coughlin"
    },
    {
      "affiliations": [
        "University of Texas Southwestern Medical Center, Dallas, TX, USA"
      ],
      "name": "Nicole Poulides"
    },
    {
      "affiliations": [
        "University of Texas Southwestern Medical Center, Dallas, TX, USA"
      ],
      "name": "Parastoo Sabaeifard"
    },
    {
      "affiliations": [
        "University of Texas Southwestern Medical Center, Dallas, TX, USA"
      ],
      "name": "Tian Zhang"
    },
    {
      "affiliations": [
        "University of Texas Southwestern Medical Center, Dallas, TX, USA"
      ],
      "name": "Hans Hammers"
    },
    {
      "affiliations": [
        "University of Texas Southwestern Medical Center, Dallas, TX, USA"
      ],
      "name": "Xin Li"
    },
    {
      "affiliations": [
        "University of Texas Southwestern Medical Center, Dallas, TX, USA"
      ],
      "name": "Bret Evers"
    },
    {
      "affiliations": [
        "University of Texas Southwestern Medical Center, Dallas, TX, USA"
      ],
      "name": "Xiaowei Zhan"
    },
    {
      "affiliations": [
        "University of Texas Southwestern Medical Center, Dallas, TX, USA"
      ],
      "name": "David E Gerber"
    },
    {
      "affiliations": [
        "University of Texas Southwestern Medical Center, Dallas, TX, USA",
        "Howard Hughes Medical Institute, Dallas, TX, USA"
      ],
      "name": "Lora V Hooper"
    },
    {
      "affiliations": [
        "University of Texas Southwestern Medical Center, Dallas, TX, USA"
      ],
      "name": "Andrew Y Koh"
    }
  ],
  "title": "461 Circadian control of the tumor immune microenvironment determines immune checkpoint inhibitor efficacy",
  "uid": "6869449e-7b90-5669-90d6-da50520ee204"
}