Overview of study design and representative images.

(A) Flow diagram of the study. (B) Trial schema of the study. Patients with NSCLC received modified neoadjuvant chemo-immunotherapy, followed by surgical resection and modified adjuvant chemo-immunotherapy. Tumor tissues were collected at baseline and the surgery phase. Serial blood samples were collected at baseline and each time-point for TCR and ctDNA sequencing. (C) Representative hematoxylin and eosin staining of tumor specimens obtained from patient 2 (P2), who obtained pCR. (D) Representative computed tomographic imaging of lung lesions of P2. NSCLC, non-small cell lung cancer; TCR, T-cell receptor; ctDNA, circulating tumor DNA; pCR, pathologic complete response.

Baseline clinical characteristics of patients.

Clinical efficacy of modified neoadjuvant chemo-immunotherapy.

(A) Tumor size changes from baseline based on radiological imaging. (B) Ring diagram showed the rate of overall response according to RECIST criteria (CR, PR, and SD) in patients. (C) Swimmer plot of 31 patients who underwent R0 resection involved in this trial. (D) Barplot showed the number of patients distributed in pathological response (pCR in blue, MPR in green, and non-MPR in red, respectively). (E) DFS curve for patients with modified treatment. CR, complete response; PR, partial response; SD, stable disease; pCR, completed pathological response; MPR, major pathological response; DFS, disease free survival.

Response to modified neoadjuvant chemo-immunotherapy

Summary of treatment-related adverse events

Dynamic changes in T cell repertoire during therapy.

(A) The schematic of samples collection and analyses performed for each patient. Top boxes represent therapeutic phases, blue for chemotherapy, yellow for immunotherapy, and purple for operation. TCR sequencing is indicated as circle. Square and triangle represents ctDNA and RNA sequencing, respectively. Solid shapes are defined as available data, while open shapes are defined as unavailable data. (B) Comparison of top 20 TCR clones at different therapeutic phases. (C) Comparison of top 20 TCR clones at different therapeutic cycles. For boxplots, each box indicates the first quartile (Q1) and third quartile (Q3), and the black horizontal line represents the median; the upper whisker is the min[max(x), Q3 + 1.5 × IQR], and the lower whisker is the max[min(x), Q1 - 1.5 × IQR], where x represents the data, Q3 is the 75th percentile, Q1 is the 25th percentile, and IQR = Q3 - Q1. (D) Comparison of large TCR clones at different therapeutic phases. (E) Comparison of large TCR clones at different therapeutic cycles. Average and SD are shown. Wilcoxon’s Signed-Rank test was used for comparison in B-E (*p < 0.05, **p < 0.01, ***p < 0.001, **** p < 0.0001). (F) Changes in diversity (Shannon index) of TCR clones for each patient. TCR, T-cell receptor; ctDNA, circulating tumor DNA; IQR, interquartile range.

Characterization of ctDNA and the alteration of tumor burden during treatment.

(A) Genomic landscape of mutations detected in plasma via ctDNA sequencing. Frequency of mutated genes were shown on the bar of middle and the proportion was shown in numerical form. Counts of mutations for each sample were displayed on top bar. Frequency for each of the six substitutions of the bases spectrum was shown on right. The scale lines below show the proportion of various mutation substitutions. (B) Changes in maxVAF across the entire course of therapy. Wilcoxon’s Signed-Rank test was used for comparison. (*p < 0.05). ctDNA, circulating tumor DNA; maxVAF, maximal somatic variant allelic frequency.

Dynamic change of top 20 TCR clones in the distinct phases.

(A) Changes of top 20 TCR clones of P1, P2, P3, P10, P11, and P12 in T0 to T1 (PH 1). (B) Changes of top 20 TCR clones of P1, P2, P5, P10, P11, and P12 in T1 to T2 (PH 2). (C) Changes of top 20 TCR clones of P2, P4, P10, P11, and P12 in T2 to T3 (PH 3). For boxplots, each box indicates the first quartile (Q1) and third quartile (Q3), and the black horizontal line represents the median; the upper whisker is the min[max(x), Q3 + 1.5 × IQR], and the lower whisker is the max[min(x), Q1 - 1.5 × IQR], where x represents the data, Q3 is the 75th percentile, Q1 is the 25th percentile, and IQR = Q3 - Q1. Wilcoxon’s Signed-Rank test was used for comparison (*p < 0.05, **p < 0.01, ***p < 0.001, **** p < 0.0001). TCR, T-cell receptor; IQR, interquartile range.

Characterization of temporal dynamics of the T cell repertoire in the longitudinal blood.

(A) Changes of the D50 index among patients. (B) Changes of Pielou index (evenness) among patients. (C) Changes of clonality index among patients.

Infiltration of CD4+ and CD8+ T lymphocytes after modified neoadjuvant therapy.

(A) Relative proportion of lymphocytes was assessed by ImmunCellAI. (B) Relative proportion of lymphocytes was assessed by xCell. For boxplots, each box indicates the first quartile (Q1) and third quartile (Q3), and the black horizontal line represents the median; the upper whisker is the min[max(x), Q3 + 1.5 × IQR], and the lower whisker is the max[min(x), Q1 - 1.5 × IQR], where x represents the data, Q3 is the 75th percentile, Q1 is the 25th percentile, and IQR = Q3 - Q1. Wilcoxon’s Signed-Rank test was used for comparison. Tcm, central memory T cells; Tem, Effector memory T cells; IQR, interquartile range.

Change in maxVAF across different phases.

For boxplots, each box indicates the first quartile (Q1) and third quartile (Q3), and the black horizontal line represents the median; the upper whisker is the min[max(x), Q3 + 1.5 × IQR], and the lower whisker is the max[min(x), Q1 - 1.5 × IQR], where x represents the data, Q3 is the 75th percentile, Q1 is the 25th percentile, and IQR = Q3 - Q1. Wilcoxon’s Signed-Rank test was used for comparison. IQR, interquartile range.