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Circadian tumor infiltration and function of CD8+ T cells dictate a temporal response to immunotherapy

ABSTRACT: The quantity and quality of tumor-infiltrating lymphocytes (TILs), particularly CD8+ T cells, are parameters linked to tumor control and response to immunotherapy. Yet, it is unknown whether these parameters are controlled in a circadian manner. Here, we show in murine and human cancers that the phenotype and number of TILs show time-of-day differences, which are driven by rhythmic leukocyte infiltration and depend on the circadian clock machinery. These rhythms can be harnessed therapeutically: chimeric antigen receptor (CAR) T cells or immune checkpoint blockers produce significant antitumor benefits when administered at the optimal time of the day, but have little effect when administered at other times. Furthermore, time-of-day-dependent T cell signatures in murine tumor models predict overall survival in melanoma patients and correlate with response to anti-PD-1 therapy. Our data provide unexpected evidence of circadian dynamics in the tumor microenvironment (TME), and suggest the importance of exploiting them in prospective clinical trials.

ORGANISM(S): Mus musculus

PROVIDER: GSE260641 | GEO | 2024/05/17

REPOSITORIES: GEO

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Project description:We investigated the frequency of CX3CR1+CD8+ T cells in peripheral blood (PB) before and during immune checkpoint inhibitor (ICI) therapy, and delineated the TCR repertoire in peripheral CX3CR1+CD8+ T-cell subsets and CD8+ tumor-infiltrating lymphocytes (TILs) using preclinical models. To understand the clinical utility of CX3CR1 as a circulating T-cell biomarker, we analyzed longitudinal PB samples from non-small cell lung cancer (NSCLC) patients undergoing anti-PD-1 therapy, and evaluated predictive and prognostic value of changes in the frequency of PB CX3CR1+ CD8+ T cells Methods: Female C57BL/6 mice were inoculated subcutaneously with 5 x 10^5 MC38 tumor cells per mouse on the right flank on day 0. When tumor volume reached approximately 50 mm^3, 200 µg of anti-PD-L1 Ab (clone 10F.9G2, BioXcell) and 100 µg of anti-CTLA-4 Ab (clone 9H10, BioXcell) were administered intraperitoneally every 3 days and every other day, respectively. MC38 tumor-bearing mice were treated for 14 days, and spleens and tumors were harvested. [Tumor cells] Tumors were cut into small pieces of 2-4 mm. Single-cell suspensions were obtained by mechanical dispersion consisting of two 30-min incubations at 37°C, 5% CO2 in 5 ml RPMI 1640 (Gibco) and tumor dissociation kit (Miltenyi Biotec) in C Tubes (Miltenyi Biotec) interspersed with three mechanical dispersions on a GentleMACS dissociator (Miltenyi Biotec). The tumor cell suspensions were then filtered through a cell strainer (70 μm; BD Biosciences). An EasySep Mouse CD8a Positive Selection Kit II (STEMCELL Technologies) was used to isolate murine CD8+ tumor-infiltrating lymphocytes (TILs) for TCR sequencing. [Splenocytes] Spleens were homogenized by forcing the tissue through a cell strainer (70 μm; BD Biosciences). Red blood cells in blood and spleen were lysed using ACK Lysis Buffer (Gibco). For TCR sequencing of murine splenic CD8+ T cells, single cell suspensions from freshly isolated splenocytes were stained. CD45+ CD3+ CD8+ T cells were gated, and CD27lo CX3CR1-, CD27hi CX3CR1-, and CX3CR1+ CD8+ T cells were sorted using BD FACSAria II Cell Sorter. DNA from flow-isolated murine splenic CD8+ T cells and CD8+ TILs, and PB CD8+ T cells was extracted using QIAamp DNA Micro Kit (QIAGEN). DNA was quantified using Qubit dsDNA BR Assay (Invitrogen). Amplification and sequencing of TCRβ CDR3 regions was performed using ImmunoSEQ immune profiling system at the survey level (Adaptive Biotechnologies). Sequencing was performed on an Illumina NextSeq system using 150 cycle mid-output kit (Illumina Inc.). Processed data were uploaded to the ImmunoSEQ Platform (Adaptive Biotechnologies) for bioinformatics analysis. Processed data were downloaded and frequencies/counts for TCR clonotypes and diversity were examined by nucleotide sequences after non-productive reads were filtered out. Results: Clonally expanded TCR repertoires of CD8+ TILs are enriched in the peripheral CX3CR1+ subset during ICI therapy. ICI therapy induces high degree of TCR sequence similarity and clonality between CD8+ TILs and the peripheral CX3CR1+ CD8+ T cells.

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Circadian tumor infiltration and function of CD8+ T cells dictate a temporal response to immunotherapy

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