Neoadjuvant immunochemotherapy (NIC) has emerged as a promising strategy for esophageal squamous cell carcinoma (ESCC). However, its impact on tertiary lymphoid structures (TLSs) and the immune microenvironment remains to be fully elucidated. We retrospectively analyzed 50 ESCC patients treated with NIC ( n = 25) or chemotherapy alone ( n = 25) between 2021 and 2023. TLSs and lymphocyte subpopulations in paired tumor tissues were assessed via multiplex immunofluorescence. Disease‐free survival (DFS) was evaluated using Cox regression, and correlations with peripheral blood lymphocytes were analyzed. NIC significantly increased the abundance and maturity of TLSs compared to chemotherapy alone. Specifically, NIC‐induced TLSs were enriched with CD3 + CD8 + PD‐1+ T cells and CD103 + CXCL13+ dendritic cells (DCs). Clinically, the NIC group demonstrated a significantly prolonged median DFS compared to the chemotherapy group (17.4 vs. 13.5 months, p = 0.009). High abundance of mature TLSs was an independent predictor of improved DFS (HR = 0.45). Furthermore, elevated proportions of CD3 + CD8 + PD‐1+ T cells and CD103 + CXCL13+ DCs within TLSs were strongly associated with longer survival. These local TLS alterations also correlated with specific changes in peripheral blood lymphocyte subsets. Our preliminary data suggest that NIC promotes TLS maturation and is associated with enrichment of specific immune subsets within TLSs. CD3 + CD8 + PD‐1+ T cells and CD103 + CXCL13+ DCs may serve as exploratory biomarkers associated with prognosis and immunotherapy response.

Background: Despite limited research on refractory and/or endocrine therapy failure in elderly metastatic breast cancer (MBC) patients, a prior study did show that low-dose oral cyclophosphamide (CY) can improve the overall survival rate of MBC, possibly through immunoregulation of regulatory T cells (Tregs). We performed a preliminary investigation of the combination of endocrine therapy (ET) with oral low-dose CY as salvage therapy in elderly patients with peripheral blood regulatory T cell analyses. In addition, we evaluated the association of tumor tertiary lymphoid structures (TLSs) with therapeutic outcomes. Patients and Methods: HR+/HER2˗ advanced breast cancer patients who received low-dose CY combined with ET or ET only from April 2015 to August 2021 were enrolled in this retrospective study. The primary outcome was the clinical control rate (CCR), and the secondary outcome was progression-free survival (PFS). Circulating T lymphocyte subpopulations represented by Tregs were monitored during treatment by flow cytometry methods. TLS diagnosis was confirmed by hematoxylin–eosin staining of pretreatment specimens, and CD3, CD4, and Foxp3 were stained using Opal multicolor immunofluorescence. Results:85 patients received CY + ET and 50 patients received ET only were enrolled, CCR was 73% (62/85) vs. 70% (45/50), and objective response rate (ORR) was 28% (24/85) vs. 24% (12/50). No deaths occurred during the study period. The median PFS time was 13 vs. 11 months (P = 0.03). In the CY + ET group, the decreases in CD4+/CD25+/Foxp3+ T cells (P<0.001) were favorable for both clinical control and prolonged PFS (P < 0.001), compared with patients without TLSs, those with TLSs were more likely to have better clinical control and PFS (mean time=6 months), and a higher level of Treg cells in TLSs pretreatment correlated with longer PFS (P=0.043). Conclusions: Oral low-dose CY combined with standard ET exerts immunological regulation by decreasing Treg to achieve improved clinical responses. Moreover, patients with TLSs might benefit more from such therapy than those without TLSs, and a high Treg count in TLSs before treatment predicts better therapy efficacy.