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The utility of circulating CX3CR1+ CD8+ T cells as a predictive correlate of response to chemo-immunotherapy in patients with non-small cell lung cancer

ABSTRACT: Lack of reliable predictive biomarkers is a major limitation of combination therapy with chemotherapy and anti-PD-1 therapy (chemo-immunotherapy). The increase of peripheral blood (PB) CD8+ T cells expressing CX3CR1, a marker of differentiation, correlates with response to anti-PD-1 therapy; however, the predictive and prognostic value of T-cell CX3CR1 expression during chemo-immunotherapy remains elusive. Here, we evaluated the utility of PB CX3CR1+CD8+ T cells as a predictive correlate of response to chemo-immunotherapy in non-small cell lung cancer (NSCLC) patients. At least 10% increase of the CX3CR1+ subset in circulating CD8+ T cells from baseline (CX3CR1 score) was associated with response to chemo-immunotherapy at 6 weeks (p = 0.0382) and 9 weeks (p = 0.0045). Sensitivity, specificity, positive and negative predictive value of at least 10% increase of the CX3CR1 score was 83.3%, 88.2%, 83.3% and 88.2%, respectively with 86.2% overall accuracy of predicting response at 9 weeks. Furthermore, at least 10% increase of the CX3CR1 score correlated with improved overall (p = 0.0105) and progression-free survival (p = 0.0265). Combined T-cell receptor (TCR) sequencing of tumor tissue and single-cell RNA/TCR sequencing of longitudinal PB samples from a patient who received a long-term benefit from chemo-immunotherapy demonstrated that circulating clonally expanded CD8+ T cells were terminal effector T cells expressing high levels of CX3CR1, and contained frequent tumor-infiltrating T-cell colonotypes early-on post treatment. Collectively, these findings suggest the potential utility of T-cell CX3CR1 expression as a dynamic blood-based biomarker during the course of chemo-immunotherapy and a marker to identify frequent tumor-infiltrating lymphocyte repertoires.

ORGANISM(S): Homo sapiens

PROVIDER: GSE213902 | GEO | 2023/04/05

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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The utility of circulating CX3CR1+ CD8+ T cells as a predictive correlate of response to chemo-immunotherapy in patients with non-small cell lung cancer

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