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The immune system continuously interacts with tumors, possibly leading to systemic alterations in circulating immune cells. However, the potential of these cancer-associated changes for diagnostic purposes remains poorly explored. To investigate this, we conducted a comprehensive flow cytometric analysis of 452 peripheral blood mononuclear cell (PBMC) samples from 206 non-small-cell lung cancer (NSCLC) patients, 100 small-cell lung cancer (SCLC) patients, 94 healthy individuals, and 52 benign lung disease (BLD) patients. We focused specifically on circulating T cells, given their close interaction with tumors, and initially assessed 93 T-cell features from the flow cytometric analysis. Using a feature selection protocol, we identified five T-cell features in peripheral blood with strong diagnostic relevance. Notably, while individual alterations in these features lacked cancer specificity, simultaneous alterations were uniquely indicative of lung cancer. To comprehensively analyze these features, we developed a scoring model, IMmunoPhenotypic Analysis for Cancer deTection (IMPACT). Comprehensive analysis using the five features (IMPACT-5) demonstrated high cancer specificity and biomarker efficacy, as evidenced by the high area under the receiver operating characteristic curve values for lung cancer patients (0.9187, 0.9277, and 0.9363 for stage I NSCLC, stage IV NSCLC, and SCLC patients, respectively), in stark contrast to BLD patients (0.5212). These findings suggest that comprehensive analysis of cancer-associated changes in circulating T cells can effectively detect lung cancer from its early stages, proposing immunophenotypic analysis of circulating T cells as an innovative liquid biopsy-based diagnostic biomarker.

Defining the molecular dynamics associated with T cell differentiation enhances our understanding of T cell biology and opens up new possibilities for clinical implications. In this study, we investigated the dynamics of CD5 expression in CD8+ T cell differentiation and explored its potential clinical uses. Using PBMCs from 29 healthy donors, we observed a stepwise decrease in CD5 expression as CD8+ T cells progressed through the differentiation stages. Interestingly, we found that CD5 expression was initially upregulated in response to T cell receptor stimulation, but diminished as the cells underwent proliferation, potentially explaining the differentiation-associated CD5 downregulation. Based on the proliferation-dependent downregulation of CD5, we hypothesized that relative CD5 expression could serve as a marker to distinguish the heterogeneous CD8+ T cell population based on their proliferation history. In support of this, we demonstrated that effector memory CD8+ T cells with higher CD5 expression exhibited phenotypic and functional characteristics resembling less differentiated cells compared to those with lower CD5 expression. Furthermore, in the retrospective analysis of PBMCs from 30 non-small cell lung cancer patients, we found that patients with higher CD5 expression in effector memory T cells displayed CD8+ T cells with a phenotype closer to the less differentiated cells, leading to favorable clinical outcomes in response to immune checkpoint inhibitor (ICI) therapy. These findings highlight the dynamics of CD5 expression as an indicator of CD8+ T cell differentiation status, and have implications for the development of predictive biomarker for ICI therapy.

We recruited 50 patients with unresectable stage III NSCLC who received CCRT between March 2020 and March 2021. Durvalumab consolidation (DC) was administered to patients (n = 23) without progression after CCRT and programmed death-ligand 1 (PD-L1) ≥ 1%. Blood samples were collected before (C0) and after CCRT (C1) to calculate PBC counts and analyze CTCs. CTCs, isolated by the CD-PRIMETM system, exhibited EpCAM/CK+/CD45- phenotype in BioViewCCBSTM. At median follow-up of 27.4 months, patients with residual CTC clusters at C1 had worse median PFS than those without a detectable CTC cluster (11.0 vs. 27.8 months, p = 0.032), and this trend was noted only in the DC group (p = 0.034). Patients with high platelets at C1 (PLThi, >252 × 103/µL) had worse median PFS than those with low platelets (PLTlo) (5.9 vs. 17.1 months, p < 0.001). In multivariable analysis, PLThi and residual CTC clusters at C1 were independent risk factors for PFS, and DC group with PLThi and residual CTC clusters at C1 showed the worst median PFS (2.6 months, HR 45.16, p = 0.001), even worse than that of the CCRT alone group with PLThi (5.9 months, HR 15.39, p = 0.001). The comprehensive analysis of CTCs and PBCs before and after CCRT revealed that the clearance of CTC clusters and platelet counts at C1 might be potential biomarkers for predicting survival.

BACKGROUND: CD8+ tumor-infiltrating lymphocytes (TILs) comprise phenotypically and functionally heterogeneous subpopulations. Of these, effector memory CD45RA re-expressing CD8+ T cells (Temra) have been discovered and characterized as the most terminally differentiated subset. However, their exact ontogeny and physiological importance in association with tumor progression remain poorly understood. METHODS: We analyzed primary tumors and peripheral blood samples from 26 patients with non-small cell lung cancer and analyzed their phenotypes and functional characteristics using flow cytometry, RNA-sequencing, and bioinformatics. RESULTS: We found that tumor-infiltrating Temra (tilTemra) cells largely differ from peripheral blood Temra (pTemra), with distinct transcriptomes and functional properties. Notably, although majority of the pTemra was CD27-CD28- double-negative (DN), a large fraction of tilTemra population was CD27+CD28+ double-positive (DP), a characteristic of early-stage, less differentiated effector cells. Trajectory analysis revealed that CD8+ TILs undergo a divergent sequence of events for differentiation into either DP or DN tilTemra. Such a differentiation toward DP tilTemra relied on persistent expression of CD27 and CD28 and was associated with weak T cell receptor engagement. Thus, a higher proportion of DP Temra was correlated with lower immunogenicity of tumor antigens and consequently lower accumulation of CD8+ TILs. CONCLUSIONS: These data suggest a complex interplay between CD8+ T cells and tumors and define DP Temra as a unique subset of tumor-specific CD8+ TILs that are produced in patients with relatively low immunogenic cancer types, predicting immunogenicity of tumor antigens and CD8+ TIL counts, a reliable biomarker for successful cancer immunotherapy.