Radiotherapy remains a cornerstone in the treatment of advanced lung cancer, regardless of the specific pathology. For many years, the standard of care has been chemoradiotherapy (CRT), combining chemotherapy with radiotherapy (1–4). Immune checkpoint inhibitors (ICIs) have ushered in a new era in cancer treatment. ICIs represented by programmed cell death protein 1 (PD-1) inhibitors and programmed cell death protein ligand 1 (PD-L1) inhibitors have demonstrated enduring, favorable objective responses in patients diagnosed with both non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) (5, 6). ICIs have been benchmark drugs in the standard treatment of advanced lung cancer.

Besides its local tumor-killing effects, radiotherapy can stimulate immune activation, resulting in systemic changes in immune cell populations within the tumor microenvironment (7, 8), forming the foundational theory behind combining immunotherapy and radiotherapy. The PACIFIC study, a phase III randomized controlled trial of the PD-L1 inhibitor Durvalumab consolidation therapy versus placebo, showed that the Durvalumab consolidation therapy group had significantly better progression-free survival (PFS) than the placebo group after concurrent chemoradiotherapy (9, 10). In the GEMSTONE-301 study, Sugemalimab consolidation therapy significantly prolonged PFS versus placebo in patients who had not progressed on sequential chemoradiation or concurrent radiotherapy (11). Sugemalimab has been approved by the National Medical Products Administration for use in inoperable locally advanced NSCLC as a consolidation therapy after concurrent or sequential chemoradiation therapy.

While standard treatment involving immune consolidation therapy and chemoradiotherapy extends survival in patients with advanced lung cancer, approximately 40% of patients continue to experience disease progression. Reliable biomarkers for predicting efficacy and prognosis in CRT and CRIT are still lacking.

Tumor recurrence and metastasis are intricately associated with compromised immune function. Impaired immunity enables tumors to evade immune responses, thereby worsening tumor progression and dissemination, establishing a vicious cycle. Lymphocytes are critical in regulating the immune system and targeting tumor cells for destruction. The intricate interplay among lymphocyte subsets constitutes the foundation of the immune system and profoundly impacts the body’s disease response (12). Radiotherapy stimulates the generation of in situ tumor vaccines and mobilizes tumor-infiltrating lymphocytes (TILs), thereby enhancing the tumor microenvironment (13, 14).

After ionizing radiation, dendritic cells capture destroyed tumor cells by releasing tumor-associated antigens, thus serving as an in-situ tumor vaccine. This activation induces systemic immunogenicity and distant effects (15, 16). However, solely depending on the activation of tumor-resident TILs fails to fully elucidate the unified response mechanism that underlies distant effects across various metastatic tumors, which means peripheral immune cells likely play a vital role in clinical responses (17). A study discovered that patients benefiting from ICIs treatment experienced significant proliferation of PD-1-positive CD8+ T cells in peripheral blood, whereas patients with disease progression exhibited delayed or absent responses in these cells (18). Single-cell RNA sequencing and T-cell receptor repertoire sequencing experiments unveiled that the clonal replacement of exhausted CD8+ T cells in tumors does not stem from the reactivation of pre-existing TILs but chiefly arises from newly recruited cells in the peripheral blood (19). Thus, extensive T cell proliferation may originate not only from pre-existing TILs within tumors but also from recruited T cells in peripheral blood.

Although lymphocytes near tumor tissue could provide a more accurate reflection of tumor biology, tissue biopsy is hindered by the heterogeneity across various tumor regions. Moreover, the invasive nature of biopsies and the impracticality of repetitive serial examinations make them unsuitable for advanced lung cancer patients (20). Obtaining lymphocyte subsets from peripheral blood circumvents these challenges and allows repeated dynamic monitoring. Peripheral blood lymphocytes can reflect the body’s overall immune status. Several studies have shown that changes in the peripheral blood lymphocyte subset correlate with the efficacy of tumor immunotherapy (21–24). However, no studies have yet explored the influence of pre-treatment levels of peripheral blood lymphocyte subsets on standard chemoradiotherapy and subsequent immune consolidation therapy. This study collected pre-treatment distributions of peripheral blood lymphocyte subsets level (PBLSL) in unresectable lung cancer patients who received CRT or CRIT to assess their predictive potential for treatment efficacy and prognosis.

Table 1 has summarized the baseline characteristics of the 77 patients, including gender, age, histological type, and radiotherapy received, presented as mean ± standard deviation or median (range). Table 2 showed the relationship between PBLSLs and pre-treatment baseline characteristics. No significant differences in PBLSLs have indicated well-matched samples, minimizing group differences’ impact on analysis. Ultimately, 48 patients have been in the responder group, and 29 have been in the non-responder group.

In 2022, the International Agency for Research on Cancer, operating under the World Health Organization, published the most recent global cancer estimates. The findings showed that lung cancer retained its position as the most common and deadliest cancer globally. The phase III PACIFIC trial, compared durvalumab with placebo in patients with unresectable, stage III NSCLC and no disease progression after concurrent chemoradiotherapy, has reported 5-year overall survival outcomes, demonstrating a 43% overall survival rate in the durvalumab arm (25). This reality emphasizes the critical need to improve objective clinical response rates to standard treatments and the precision in selecting favorable patient populations.

Now, research on predictive biomarkers mainly focuses on imaging features, genetic detection, and liquid biopsy (26–30). Imaging technology is improving but needs to provide a complete view of immune changes. Genetic testing is invasive and may not help late-stage patients. Liquid biopsy, like circulating tumor DNA and molecular residual disease, is gaining popularity for recurrence detection and therapy response prediction. Negative molecular residual disease after chemotherapy suggests a potential cure (31). However, liquid biopsy needs improvements in sensitivity and struggles to predict outcomes for specific patients; its use is also limited by cost.

Much of the research mainly focused on the changes in peripheral blood lymphocyte levels in immunotherapies (21–24). Nonetheless, the correlation between initial peripheral blood lymphocyte levels and radiotherapy remains inadequately understood. This study aims to address this gap. Our specific analysis across different pathological types, treatments, and age Groups in advanced lung cancer patients undergoing CRT or CRIT revealed varied lymphocyte correlations. These findings collectively emphasize the significance of the research on peripheral blood lymphocyte subsets.

Classic lymphocyte subsets, including CD3+, CD4+, CD8+, B cells, and natural killer cells, play crucial roles in regulating immunity and specifically targeting tumor cells for destruction. As proficient antigen-presenting cells, B cells can initiate and amplify antigen-specific T cells, triggering anti-tumor responses. Animal models of B-cell depletion have shown a notable decline in the generation of effector-memory and IFNγ or TNFα- secreting CD4+ and CD8+ T cells, along with diminished activity of both CD4+ and CD8+ T cells in tumors and lymph nodes (32). B cells also promote the development of CD4 T follicular helper cells, enhancing the toxic effects of CD8+ T cells by producing IL-21 (33). On the other hand, peripheral B lymphocytes efficiently encoded antigen mRNA and expanded tumor antigen specific CD8 T cells in vitro. Additionally, B lymphocytes also mediated the presentation of plasmid antigen-induced robust Th1-biased immunity, effectively eliciting an anti-tumor response in vivo (34). Immune activation has been shown to depend on the collaborative relationship between B and T cells. An increase in infiltrating B cells was observed after tumors were treated with radiotherapy (35). Host plasma cells and class-switched memory B cells were highly resistant to radiotherapy (36); this suggests that the remaining B cells, after radiation, are likely to carry more potent B-cell antigen receptors responsible for the development and differentiation ensuring effective subsequent immune interactions. Our findings indicated that elevated B-cell levels correlated with better radiotherapy efficacy. Higher B-cell levels may possess a more active modulation of tumor immune changes in response to radiotherapy and immunotherapy.

CD8+ T cells are typically considered to be directly involved in anti-tumor immunity due to their ability to efficiently destroy tumors through potent cytotoxicity (37). Nevertheless, the unique role of CD4+ T cells in optimizing anti-tumor immunity is becoming increasingly well-established. CD4+ T cells are indispensable support for T-cell initiation. They facilitate the initiation of the CD8+ gene expression program through various mechanisms and enhance the clonal expansion, differentiation, and memory formation of cytotoxic T-lymphocytes (CTL) (38, 39). Second, CD4+ effector T cells can indirectly kill major histocompatibility complex (MHC)-deficient tumor cells that evade CD8+ T cell targeting, thereby independently eradicating established tumors (40). To boost CTL production, delivery, and killing activity, systemic involvement of CD4+ T cells may be necessary. The study by Li et al. demonstrated that CD4+ T cells were significantly linked to response across all lung cancer patients received radiotherapy. They also revealed that the higher CD4+/CD8+ T cell ratios before treatment was correlated with more prolonged PFS (22). Liu et al. reported that recurrence of NSCLC was associated with decreased levels of CD4+ T cells and the CD4/CD8 ratio (41). Additionally, Miao et al. identified CD4+ CD45RA-T cells in the CD4+ T cell subsets as protective and risk factors influencing the prognosis of NSCLC patients undergoing ICI (24). Patients exhibiting a notably high pre-treatment proportion of CD62Llow CD4+ T cells experienced improved treatment response and long-term remission (42). Combining radiotherapy with ICIs enhanced non-redundant immune mechanisms in anti-tumor responses, a trend observed in patients undergoing CRT and CRIT in our study. This highlights the importance of maintaining stable CD4+ T cell and the CD4+/CD8+ T cell ratios for optimal immune function. Regular monitoring of CD4+ T cell levels and the CD4+/CD8+ T cell ratios in peripheral blood could become a critical strategy for guiding treatment decisions in lung cancer patients, whether they receive radiotherapy or immunotherapy.

Conversely, our study did not find the correlation between CD8+ T cells in peripheral blood and treatment efficacy. Similarly, other studies did not observe a significant role for CD8+ T cells (22–24). Instead, the proliferation of CD8+ T cells in peripheral blood during treatment correlated with sustained clinical benefit (18). In the Impassion130 study, CD8+ TILs were identified as a stratified feature associated with improved clinical outcomes in immunotherapy (43). A meta-analysis further illustrated that increased CD8+ TIL density was a favorable prognostic indicator for ICIs therapy and combined therapy, rather than peripheral blood (44). This suggests that CD8+ T cell activity may be more significant than peripheral blood levels measured at a single time point. CD8+ T cells expand and differentiate into CTLs, migrating from peripheral blood to the tumor site to exert their effect. Elevated levels of CD8+ T cells in peripheral blood may imply a lack of lymphocyte infiltration within the tumor, indicating a weakened antagonistic effect against the tumor. We did not observe a predictive benefit of baseline NK cell levels in peripheral blood for CRT and CRIT outcomes. There is growing evidence that NK cells can display adaptive immune characteristics (45). NK cells are divided into two subsets based on CD56 expression: CD56dim and CD56bright. In peripheral blood, CD56dim NK cells comprise 90% of the population. While CD56dim NK cells have antitumor cytotoxicity, some studies have found that the cytotoxic CD56dim CD16+ NK cell subset frequency is lower in cancer patients compared to healthy individuals (46, 47). In addition, regulatory CD56bright NK cells isolated from cancer patients exhibited significantly lower NKp46 expression and showed varying degrees of positive or negative correlation with MCP-1, IP-10, VEGF, and Eotaxin (47). These results indicate that cancer patients exhibit a shift in NK characteristics with lower cytotoxicity and activation, which may be the underlying reason why NK cells have not been excellent predictors in some peripheral blood studies (23, 24, 48), including this study. A clearer understanding of the detailed phenotype and temporal changes of inhibitory lymphocyte markers could provide further insight. Considering the potential effects of cell dynamics, understanding of the detailed phenotype and continuous monitoring of lymphocyte changes during treatment could provide valuable insights.

The peak demographic for advanced lung cancer comprises the elderly, yet the survival benefits of radiotherapy remain contentious (49). Older patients (>70 years old) undergoing chemoradiotherapy were considered to exhibit poorer survival and heightened mortality during treatment (50). In contrast, recent retrospective studies suggest no disparity in survival between older and younger patients receiving chemoradiotherapy (51, 52). Age impacts the immune system, a gradual process known as immunosenescence. A peripheral blood study of 1,068 healthy individuals, including young (19-44 years), middle-aged (45-64 years), and elderly (65-80 years) participants, showed that lymphocytes decline with age, both in number and function (53). Patients with tumors experience immune compromise. A comparison of lymphocyte subpopulations in NSCLC patients and healthy individuals revealed a significant decrease in lymphocyte counts across different stages of NSCLC compared to healthy controls (54). Advanced age is accompanied by decreased T and B lymphocytes and reduced telomerase activity, ultimately weakening immune effector function (55, 56). Yang et al. observed opposing apoptotic gene expression for CD4+ and CD8+ T cells with age (57). T lymphocyte distribution may exacerbate with aging, potentially disrupting immune equilibrium. Moreover, a study found that heightened CD8+ T cell subsets expression has been linked to severe immune-related adverse reactions (24). CD8+ cells, in particular, fluctuate more strongly under age changes than CD4+ (58). Our study identified that older patients with higher CD4+ T cell levels and higher CD4+/CD8+ T cell ratios exhibited improved efficacy, while in younger patients, B-cell levels predicted efficacy. This highlights the importance of lymphocyte subset levels for treatment evaluation and their potential as key indicators for treatment screening.

Lymphocytes have been found to differentially contribute to the shaping of the tumor microenvironment and to the promotion of immune interactions between SCC and AD. Single-cell RNA sequencing revealed that SCC tissues exhibited higher infiltration of mast cells, B cells, CD8 cells, and Treg cells. In contrast, AD showed a prevalence of effector and activated cells and a lower number of Tregs (59). Our findings indicate that efficacy in SCCs correlates more strongly with B-cell levels, while in ADs, the CD4+/CD8+ T cell ratios play a more critical role in predicting efficacy; however, neither is assessed as a prognostic marker. The heterogeneity of the microenvironment may influence the variations in intratumoral recruitment of lymphocytes to the peripheral blood. Additionally, we observed no relevance of lymphocyte subsets in small-cell lung cancer. Generally, SCLC is considered to be an immune desert tumor with low MHC expression (60). Our negative results possibly suggest that immune cell infiltration is hindered in SCLC, indicating the sluggish mobilization of intratumoral or circulating lymphocyte subpopulations to exert immune effects in this cancer type.

However, there is still room for further research. First, increasing the number of patients, possibly needing more research centers, is essential to validate these findings in a larger sample population. Additionally, our continued focus on detailed immunophenotyping of cell subsets and monitoring their dynamic changes over time is necessary to better understand the potential of lymphocyte subsets as reliable biomarkers.

The relationship between CRT or CRIT treatment and peripheral blood immune cells is becoming better understood. We investigated the efficacy and prognostic value of CRT and CRIT in advanced lung cancer patients by analyzing peripheral lymphocyte subsets. We found varying lymphocyte subsets reflecting efficacy in distinct patient subgroups, an aspect that has received limited attention previously. Monitoring lymphocyte subsets in peripheral blood may aid in determining optimal treatment strategies for advanced lung cancer, offering tailored and personalized approaches for patients.