BTLA and PD-1 combined with radiotherapy for enhancing antitumor immune response in lung cancer via the regulation of memory B cells to promote T-cell infiltration

Introduction

Lung cancer is one of the most common malignant tumors and a major cause of cancer-related death globally (1). Non-small cell lung cancer (NSCLC) is the most prevalent pathological type of lung cancer, accounting for approximately 80–85% of all lung cancer cases (2). Due to the lack of specific clinical manifestations, nearly 75% of patients are diagnosed at an advanced stage, thereby missing the optimal window for surgical intervention (2). Therefore, there is an urgent need to identify and develop more effective therapeutic strategies for patients with advanced NSCLC.

Immune checkpoint inhibitors, either administered as monotherapy or in combination with chemotherapy, have become the first-line standard treatment for patients with advanced lung cancer without actionable genomic mutations (3,4). Representative immune checkpoint inhibitor targets include programmed cell death protein 1 (PD-1) and programmed cell death ligand 1 (PD-L1) (3,4). However, a portion of patients receiving first-line systemic therapy experience disease progression, which typically manifests as primary tumor enlargement, oligometastasis, and extensive metastasis (5). This suggests the need for continued exploration of alternative therapeutic strategies in patients who are refractory to PD-1/PD-L1 blockade. A growing body of evidence indicates that radiotherapy (RT) can enhance the initiation of antitumor immunity, reshape the tumor microenvironment, and exert an immune-potentiating effect, which makes it an excellent combination partner for immunotherapeutic agents (6,7). Meanwhile, dual-immune checkpoint blockade has demonstrated superior antitumor efficacy and prolonged survival as compared to single–immune checkpoint blockade (8-10). Therefore, combinations of RT with dual immune checkpoint inhibitors represents a promising strategy for patients with NSCLC who have developed resistance to PD-1/PD-L1 blockade. In addition to PD-1, a number of other immune cell surface coinhibitory molecules are capable of regulating tumor immunity. Tumor-infiltrating lymphocytes (TILs) that simultaneously express PD-1 and CTLA-4 or TIM-3 exhibit greater functional exhaustion than do other TIL subsets, and dual blockade targeting these molecules together with PD-1/PD-L1 can effectively restore effector function (11,12). Several studies have shown that combining anti-PD-1 therapy with antibodies against CTLA-4 or TIM-3 can provide stronger antitumor effects and prolong survival as compared with single-agent therapy.The meta-analysis shows the pooled objective response rate (ORR) of CTLA-4/PD-1 combinations was 35–40%, LAG-3/TIM-3/PD-1 triple blockade was 20–25% (especially in PD-1-resistant populations), and TIGIT/PD-L1 combinations achieved 37% ORR in PD-L1-high patients (13). Such immune checkpoint-based combinations thus represent promising therapeutic strategies for patients with NSCLC who develop resistance to PD-1/PD-L1 inhibitors. B- and T-lymphocyte attenuator (BTLA) belongs to the CD28 superfamily and is similar to PD-1 in structure and function (14,15). It is mainly expressed on the surface of lymphocytes, such as T cells and B cells (16,17), and its ligand, herpes virus entry mediator (HVEM), is overexpressed in patients with NSCLC with lymph node metastasis (18). The BTLA-HVEM axis recruits protein tyrosine phosphatases SHP-1 and SHP-2, which inhibit the T-cell receptor signaling pathway, impede T-cell activation, and suppress T-cell proliferation and cytokine production. In B cells, after BTLA binds to HVEM, it recruits SHP-1, reduces the activation of downstream signaling molecules of the B-cell receptor, increases the threshold for B-cell activation, and inhibits B-cell proliferation (19). However, it remains unclear whether BTLA participates in the resistance mechanism underlying the effect of PD-1/PD-L1 blockade in NSCLC treatment and whether targeting BTLA can improve the efficacy of RT combined with immunotherapy.

In this study, we first analyzed and detected the expression characteristics of BTLA in the tumor microenvironment of patients with NSCLC. Second, we examined the effects of RT combined with PD-1 and BTLA blockers on tumor growth, the microenvironment, and spleen-related lymphocytes in mice. We present this article in accordance with the ARRIVE and MDAR reporting checklists (available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2026-0449/rc).

Methods

Gene Expression Omnibus (GEO) database

Dataset GSE207422 from the GEO database was selected (18) for analysis and includes transcriptomics of approximately 92,000 single cells from 3 pretreatment and 12 posttreatment patients with NSCLC who received neoadjuvant PD-1 blockade combined with chemotherapy. The single-cell transcriptome was reanalyzed in this study. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments.

Cell line

Mouse Lewis lung carcinoma (LLC) cells were purchased from Boster Biological Technology (Pleasonton, CA, USA). For cell culture, the cells were placed in complete Dulbecco’s Modified Eagle Medium (DMEM) supplemented with fetal bovine serum and penicillin-streptomycin and then incubated in a humidified cell culture incubator at 37 °C with a gas composition of 95% air and 5% carbon dioxide for continuous proliferation. To ensure the purity of the cell culture environment, mycoplasma detection was performed regularly on cell culture flasks, the medium was replaced, and cell passage was carried out according to an established schedule.

Experimental mice

Female C57BL/6J mice (6–8 weeks old) were purchased from Vital River Laboratories (Beijing, China). All mice were housed in the animal facility of the Medical Research Center at The First Affiliated Hospital of Shandong First Medical University (Shandong Provincial Qianfoshan Hospital). The animal facility was maintained under pathogen-free conditions, with food and water provided throughout the study, and a 12-hour light-dark cycle was maintained. Animal experiments were performed under a project license (No. QFSYYPZ2022091301) granted by the Ethics Committee of The First Affiliated Hospital of Shandong First Medical University (Shandong Provincial Qianfoshan Hospital), in compliance with national guidelines for the care and use of animals.

Tumor models

LLC cells (1×10⁶) were subcutaneously injected into the right hind limb of experimental mice. When the maximum diameter of the tumor reached 6–8 mm, the mice were randomly divided into the seven following groups with 4–5 mice in each group: control; RT; RT combined with anti-PD-1 therapy (RT + P); RT combined with anti-BTLA therapy (RT + B); RT combined with anti-PD-1 and anti-BTLA therapy (RT + P + B); RT combined with anti-BTLA and LFA-1α blocker therapy (RT + B + L); and RT combined with anti-PD-1, anti-BTLA, and LFA-1α blocker therapy (RT + P + B + L). Tumor volume and mouse weight were recorded every other day.

Stereotactic body radiation therapy

When the maximum diameter of the tumor reached 6–8 mm (with tumor formation assumed on day 10), RT was performed with an RS 2000 pro biological X-ray irradiator (Rad Source Technologies, Buford, GA, USA). For the RT, RT + P, RT + B, and RT + P + B groups, local RT at a dose of 8 Gy was administered on days 10, 12, and 14. For the RT + B + L and RT + P + B + L groups, RT at the same dose was administered on days 11, 13, and 15. Therefore, the total radiation dose was 24 Gy.

In vivo treatments

The in vivo antibodies used were anti-BTLA monoclonal antibody (mAb) (cat. no. BE0196; Bio X cell, Lebanon, NH, USA), anti-PD-1 mAb (cat. no. BE0146; Bio X cell), and LFA-1α (cat. no. BE0006; Bio X cell). For the RT, RT + P, RT + B, and RT + P + B groups, when the maximum diameter of the tumor reached 6–8 mm (with tumor formation being assumed to occur on day 10, anti-BTLA mAb [20 mg/kg; dissolved in 100 µL of phosphate-buffered saline (PBS)] was intraperitoneally injected on days 11, 14, and 18, and anti-PD-1 mAb (10 mg/kg; dissolved in 100 µL PBS) was intraperitoneally injected on days 12, 15, 19, and 22. For the RT + B + L and RT + P + B + L groups, anti-LFA-1α mAb (100 µg per mouse; dissolved in 100 µL PBS) was intraperitoneally injected on days 10 and 14. Anti-BTLA mAb was intraperitoneally injected on days 12, 15, and 19. Anti-PD-1 mAb was intraperitoneally injected on days 13, 16, 20, and 23. Twenty-four hours after the last intraperitoneal injection of antibodies, the mice were euthanized. Tumor tissues, spleen tissues, and eyeball serum were harvested for subsequent experiments.

Western blotting

Protein extraction from mouse tumor tissues was conducted in accordance with standard protocols. Protein concentrations were determined via BCA Protein Assay Kit (cat. no. PC0020; Solarbio Science & Technology, Beijing, China) to ensure that equal amounts of protein were loaded for immunoblotting analysis. Samples were separated by electrophoresis on a 10% sodium dodecyl sulfate (SDS)-polyacrylamide gradient gel, followed by transfer onto a polyvinylidene difluoride (PVDF) membrane (Solarbio). The membrane was then incubated sequentially with primary and secondary antibodies. The primary antibody used was anti-CD272/BTLA (cat. no. EPR20539; Abcam, Cambridge, UK) at a 1:1,000 dilution.

Flow cytometry

Spleen tissues were homogenized with a tissue grinder. For tumor samples, they were first minced manually with scissors and then subjected to digestion at 37 °C for 30 minutes in a solution containing 1 mg/mL of collagenase IV (cat. no. C8160; Solarbio) and 0.2 mg/mL of deoxyribonuclease I (cat. no. D8071; Solarbio). The digested tumor tissues were filtered through a 70-µm nylon cell sieve to prepare single-cell suspensions. The obtained single cells were centrifuged at 1,700 ×g for 5 minutes. To remove erythrocytes, the cell pellets were treated with red blood cell lysis buffer (BioGems, Westlake Village, CA, USA) and incubated at room temperature for 15 minutes in the dark. After another centrifugation at 500 ×g for 5 minutes, the supernatant was discarded, and the cells were resuspended in sample diluent. Subsequently, lymphocytes were isolated with a small volume of Percoll cell separation medium (Cytiva, Marlborough, MA, USA). After the single-cell suspension was obtained, the cells were incubated with antibodies at 4 °C for 30 minutes. They were then resuspended in PBS (with 0.05% bovine serum albumin) and fixed in 4% paraformaldehyde fixative (Solarbio). Finally, the detection was performed with a FACSAria III flow cytometer (BD, Franklin Lakes, NJ, USA), and data acquisition was conducted with FlowJo software (BD). The antibodies and accompanying labels used were as follows: CD3 mAb (cat. no. 17A2 Thermo Fisher Scientific, Waltham, MA, USA) with fluorescein isothiocyanate (1:200; Thermo Fisher Scientific), CD4 monoclonal antibody (cat. no. GK1.5 Thermo Fisher Scientific) with phycoerythrin (PE) (1:200; Thermo Fisher Scientific), CD8a mAb (cat. no. 53-6.7, Thermo Fisher Scientific) with PE-cyanine7 (1:200; Thermo Fisher Scientific), CD19 mAb (eBio1D3, Thermo Fisher Scientific) with PE-cyanine7 (1:200; Thermo Fisher Scientific), CD45.1 mAb (cat. no. A20, Thermo Fisher Scientific) with PE (1:100; Thermo Fisher Scientific), CD45.2 mAb (cat. no. 104, Thermo Fisher Scientific) with FITC (1:100; Thermo Fisher Scientific), CD11a (LFA-1alpha) mAb (cat. no. M17/4, Thermo Fisher Scientific) with eFluor 450 (1:100; Thermo Fisher Scientific), CD81 mAb (cat. no. Eat-2, BioLegend, San Diego, CA, USA) with allophycocyanin (1:100; BioLegend), and TruStain FcX PLUS (anti-mouse CD16/32; 1:200; BioLegend) with Viability Dye 780 (1:1,000; BioGems).

Enzyme-linked immunosorbent assay

Standard and sample wells were prepared on the microplate as follows: 50 µL of standard solutions at different concentrations (0, 0.5, 1, 2, 4, and 8 ng/mL) were added to standard wells. Sample wells (excluding blanks) received 10 µL of test sample plus 40 µL of diluent (fivefold dilution). Subsequently, 100 µL of horseradish peroxidase-labeled detection antibody was added to all nonblank wells, sealed, and incubated at 37 °C for 60 minutes. After five washes (procedure: fill with wash buffer, let stand for 1 minute, discard, and blot dry), 50 µL of each of substrate A and B were added, and the mixture was incubated at 37 °C in the dark for 15 minutes. The reaction was stopped with 50 µL of stop solution, and optical density values were read at 450 nm within 15 minutes. The enzyme-linked immunosorbent assay (ELISA) kits used were mouse interleukin-6 (IL-6) ELISA Kit (cat. no. MK5737A; Beyotime Biotechnology, Shanghai, China), mouse tumor necrosis factor-α ELISA Kit (cat. no. MK2868A; Beyotime Biotechnology), mouse interferon-γ (IFN-γ) ELISA Kit (cat. no. MK2918A; Beyotime Biotechnology), and mouse IL-10 ELISA Research Kit (cat. no. MK2912A; Beyotime Biotechnology).

Statistical analysis

All results were obtained from three experimental biological replicates. Preliminary analysis of flow cytometry data was conducted using FlowJo software (BD), and GraphPad Prism 9.0 (Dotmatics, Boston, MA, USA) was employed for the final statistical analysis. Statistical significance (P values) was assessed via the Student t-test and one-way analysis of variance (ANOVA). All data are presented as the mean ± standard deviation (SD), with statistical significance defined as P<0.05.

Results

Increased expression level of BTLA was associated with immunotherapy resistance in patients with NSCLC

In this study, GEO dataset GSE207422, which includes transcriptomics of approximately 92,000 single cells from 3 pretreatment and 12 posttreatment patients with NSCLC who received neoadjuvant PD-1 immunotherapy combined with chemotherapy, was analyzed (20). The 12 posttreatment samples were divided into a major pathological response group (MPR; n=4) and a nonmajor pathological response group (NMPR; n=8) based on the pathological response after treatment. The analysis revealed that compared with the MPR group, the NMPR group had a higher expression of BTLA in the tumor microenvironment (Figure 1A). This indicated that the increased expression of BTLA was associated with the development of resistance to immunotherapy in some patients and provides an important basis for further examination of the related mechanisms and optimization of treatment strategies.

Figure 1 Increased expression level of BTLA mediated resistance to immunotherapy and radiotherapy. Results of analysis from the GEO database: (A) scatter plot of gene expression differences; (B) the expression level of BTLA in the tumor microenvironment of mice: when the tumors grew to 6–8 mm, the mice were randomly divided into a control group and an RT group. The RT group received RT every other day. After the completion of RT treatment, tumor tissues were extracted from mice, and the expression level of BTLA was determined via Western blotting. Statistical significance (P values) was assessed via the Student t-test and one-way analysis of variance. *, P<0.05. BTLA, B- and T-lymphocyte attenuator; FDR, false discovery rate; GEO, Gene Expression Omnibus; RT, radiotherapy.

RT induced increased BTLA expression in the tumor microenvironment of mice

RT or dual immunotherapy was to found to effectively overcome the resistance to immunotherapy in patients with NSCLC. Moreover, the combination of immunotherapy and radiation therapy exhibited a synergistic effect for local and distant tumor control, thereby improving patients’ survival. Hypofractionated RT, particularly stereotactic body radiation therapy, can induce a more significant abscopal effect. In this study, LLC cells (1×10⁶) were subcutaneously implanted into the left inguinal region of C57BL/6 mice. When the tumors grew to 6–8 mm, the mice were randomly divided into a control group and an RT group. The RT group received hypofractionated radiation (3×8 Gy) every other day. After the completion of RT treatment, tumor tissues were extracted from mice, and the expression level of BTLA was determined via Western blotting. We found that the expression level of BTLA in the RT group was significantly higher than that in the control group (Figure 1B).

Anti-BTLA blockade enhanced the antitumor efficacy of RT combined with immunotherapy

In clinical practice, RT combined with immunotherapy has shown significant efficacy in patients with NSCLC. Given that BTLA was associated with resistance to immunotherapy in patients with NSCLC and RT could upregulate the expression of BTLA in the mouse lung cancer models in this study, we speculated that the application of BTLA blockers can further enhance the antitumor efficacy of immunotherapy and RT. To verify this, we constructed a mouse lung cancer model which was treated with RT combined with drug immunotherapy, aiming to observe the inhibitory effect of the combined therapy on tumor growth.

LLC cells (1×10⁶) were subcutaneously implanted into the left inguinal region of C57BL/6 mice, and the tumor size of the tumor-bearing mice was measured. When the tumors grew to 6–8 mm, the mice were randomly divided into five groups for experimental intervention (Figure 2A,2B). The tumor volume of the mice was measured every 2 days during the intervention period (Figure 2B). After completion of the intervention, the tumor volumes of each group were as follows: control group, 1,801.534±370.007 mm³; RT group, 557.903±78.800 mm³; RT + P group, 279.461±66.212 mm³; RT + B group, 379.542±41.269 mm³; and RT + P + B group, 155.435±29.400 mm³ (Figure 2C).

Figure 2 Anti-BTLA treatment enhanced the antitumor efficacy of radiotherapy combined with immunotherapy and further promoted T-cell infiltration. (A) Experimental intervention process and grouping: when the maximum diameter of the tumor reached 6–8 mm (with tumor formation being assumed to occur on day 10), local RT at a dose of 8 Gy was administered on days 10, 12, and 14. Anti-BTLA mAb (20 mg/kg; dissolved in 100 µL PBS) was intraperitoneally injected on days 11, 14 and 18, and anti-PD-1 mAb (10 mg/kg; dissolved in 100 µL PBS) was intraperitoneally injected on days 12, 15, 19, and 22. Grouping: control, RT, RT + P, RT + B, and RT + P + B. (B-D) Tumor growth kinetics and mouse body weight in LLC tumor-bearing mice. (E-G) Flow cytometry analysis of tumor-infiltrating CD8⁺ T cells and CD4⁺ T cells. Statistical significance (P values) was assessed via the Student t-test and one-way analysis of variance. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001; ns, not significant. RT + P, RT combined with anti-PD-1 therapy; RT + B, RT combined with anti-BTLA therapy; RT + P + B, RT combined with anti-PD-1 and anti-BTLA therapy. BTLA, B- and T-lymphocyte attenuator; PBS, phosphate-buffered saline; PD-1, programmed cell death protein 1; LLC, Lewis lung carcinoma; RT, radiotherapy.

The results showed that compared with the RT group, the RT + P and RT + B groups have significantly improved antitumor efficacy (RT vs. RT + P: P<0.0001; RT vs. RT + B: P=0.004). The RT + P + B triple-therapy group had an even greater antitumor efficacy as compared to the RT + P and RT + B groups (RT + P vs. RT + P + B: P=0.04; RT + B vs. RT + P + B: P<0.001). However, there were no significant differences in the changes of mouse body weight (Figure 2D).

Triple therapy further enhanced T-cell infiltration in the tumor microenvironment of mice

A previous study (21) has shown that T cells play a crucial role in the antitumor immune response induced by RT combined with immunotherapy. In this study, tumor tissues were processed into single-cell suspensions after the completion of treatment, and the infiltration level of CD4⁺ T cells and CD8⁺ T cells in tumor tissues was analyzed through flow cytometry. The experimental results showed that the RT + P group had a significant effect in increasing the infiltration level of CD8⁺ T cells as compared with the control and the RT groups (control vs. RT + P: P<0.0001; RT vs. RT + P: P=0.02). More importantly, the RT + P + B triple-therapy group had a significantly greater infiltration level of CD8⁺ T cells as compared with the RT + P group (P=0.01) (Figure 2E,2F), confirming the synergistic effect of triple therapy. In addition, the analysis of CD4⁺ T-cell infiltration revealed a similar pattern for CD8⁺ T cells (Figure 2G). These findings indicate that both CD4⁺ T cells and CD8⁺ T cells are involved in the antitumor immune response induced by the triple therapy and that they jointly influence the tumor immune microenvironment and treatment outcomes.

The expression level of BTLA on memory B cells was elevated in the NMPR group

The results of in vivo experiments on mice showed that anti-BTLA blockade could further enhance the antitumor efficacy of RT combined with immunotherapy.

BTLA expressed in B cells is associated with inhibition of B-cell activation and antitumor immunity. The expression level of BTLA on B-cell subsets was analyzed via the GEO dataset. The results showed that compared with the pretreatment and MPR groups, the NMPR group had a higher expression level of BTLA in memory B cells (Figure 3).

Figure 3 The expression level of BTLA on memory B cells was increased in the nonmajor pathological response group. (A) Single-cell dimensionality reduction and clustering plot. (B) Dimensionality reduction and clustering plot of BTLA+ memory B cells. BTLA, B- and T-lymphocyte attenuator; MPR, major pathological response; NMPR, nonmajor pathological response; UMAP, uniform manifold approximation and projection.

Anti-BTLA blockade increased the proportion of tumor memory B cells in the mouse spleen

A previous study has shown that when tumor antigens are released into the bloodstream, B cells in peripheral lymphoid organs, among which the spleen is the largest, are selectively activated and produce antibodies, which promote the proliferation of lymphocytes and their differentiation into effector T cells and memory B cells. These cells then migrate to the tumor microenvironment and exert immune effects. To investigate the role of anti-BTLA therapy in B cells, we first detected the proportion of specific immune cell subsets in mouse spleens using flow cytometry.

The results showed that there was no significant difference in the proportion of total B cells among lymphocytes in the four groups (RT group, RT + P group, RT + B group, and RT + P + B group) (Figure 4A,4B), while the proportions of memory B cells in the RT + B group and RT + P + B group were significantly higher as compared with that of the RT + P group (RT + P vs. RT + B: P=0.0322; RT + P vs. RT + P + B: P=0.0008) (Figure 4A,4B). These results indicated that the mice treated with anti-BTLA therapy had an increased proportion of memory B cells among B cells in the spleen.

Figure 4 Anti-BTLA blockade increased the proportion of tumor memory B cells in the mouse spleen. (A,B) Flow cytometry analysis of splenic B cells and memory B cells. (C,D) Flow cytometry analysis of splenic CD8⁺ T cells and CD4⁺ T cells. Statistical significance (P values) was assessed with the Student t-test and one-way analysis of variance. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001; ns, not significant. RT + P, RT combined with anti-PD-1 therapy; RT + B, RT combined with anti-BTLA therapy; RT + P + B, RT combined with anti-PD-1 and anti-BTLA therapy. BTLA, B- and T-lymphocyte attenuator; PD-1, programmed cell death protein 1; RT, radiotherapy.

Subsequently, we further analyzed the proportions of CD8⁺ T cells and CD4⁺ T cells in spleen tissues. The proportions of CD8⁺ T and CD4⁺ T cells were significantly higher in the RT + P group than in the control group (CD8: P<0.001; CD4: P=0.008), RT group (CD8: P<0.001; CD4: P=0.002) (Figure 4C,4D), RT + B group (CD8: P=0.02; CD4: P=0.002), and RT + P + B group (CD8: P=0.01; CD4: P=0.006; Figure 4C,4D). The analysis indicated that, compared with anti-PD-1 therapy, anti-BTLA therapy had no significant effect on regulating the proportions of CD8⁺ T cells and CD4⁺ T cells in the spleen.

Depletion of memory B cells abrogates the antitumor effects induced by triple combination therapy

Mouse tumor models were used to further verify the antitumor role of memory B cells in the groups treated with anti-BTLA therapy. When the tumors grew to 6–8 mm, the mice were randomly divided into the following three groups for experimental intervention (Figure 5A,5B): control, RT + anti-BTLA + LFA blocker (RT + B + L), and RT + anti-PD-1 + anti-BTLA + LFA blocker treatment (RT + P + B + L). The tumor volume of the mice was measured every 2 days during the intervention period (Figure 5B). After the intervention, the tumor volumes of each group were as follows: control group, 1,857.394±481.966 mm³; RT + B + L group, 388.778±41.531 mm³; and RT + P + B + L group, 267.801±56.415 mm³. Compared with the RT + P + B group, whose mean tumor volume after intervention was 155.435±29.400 mm³, the RT + P + B + L group had a higher tumor volume at the end of the treatment period (Figures 2B,5B,5C). These results indicate that, in terms of mouse tumor volume, blocking memory B cells can reverse the antitumor effect of triple therapy.

Figure 5 Blocking memory B cells reverse the antitumor effect of triple therapy. (A) Experimental intervention process and grouping: when the maximum diameter of the tumor reached 6–8 mm (with tumor formation being assumed to occur on day 10), local RT at a dose of 8 Gy was administered on days 11, 13, and 15. Anti-LFA-1α mAb (100 μg per mouse; dissolved in 100 μL PBS) was intraperitoneally injected on days 10 and 14. Anti-BTLA mAb (20 mg/kg; dissolved in 100 μL PBS) was intraperitoneally injected on days 12, 15, and 19, and anti-PD-1 mAb (10 mg/kg; dissolved in 100 μL PBS) was intraperitoneally injected on days 13, 16, 20, and 23. Grouping: control; RT + B + L; and RT + P + B + L. (B,C) Tumor growth kinetics of LLC tumor-bearing mice. (D,E) Flow cytometry analysis of splenic B cells and memory B cells. (F,G) Flow cytometry analysis of splenic CD8⁺ T cells and CD4⁺ T cells. Statistical significance (P values) was assessed with the Student t-test and one-way analysis of variance. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001; ns, not significant. RT + B + L, RT combined with anti-BTLA and anti-LFA-1α blocker therapy; RT + P + B + L, RT combined with anti-PD-1, anti-BTLA and anti-LFA-1α blocker therapy. BTLA, B- and T-lymphocyte attenuator; LFA-1α, lymphocyte function-associated antigen 1 alpha; LLC, Lewis lung carcinoma; PBS, phosphate-buffered saline; PD-1, programmed cell death protein 1; RT, radiotherapy.

Subsequently, we extracted the spleen tissues of mice, prepared them into single-cell suspensions, and detected the blocking effect on memory B cells by means of flow cytometry (Figure 5D). Compared with the RT + B and RT + P + B groups, the RT + P + B + L had a lower proportion of memory B cells (Figures 4C,5E). Similarly, compared with the RT + B group, the RT + B + L group had a lower proportion of memory B cells (Figures 4C,5E). The proportions of memory B cells in each group were as follows: RT + B, 4.407±0.283; RT + P + B; 5.543±1.054; RT + B + L, 3.167±0.676; and RT + P + B + L, 3.210±0.392. These results indicate that we successfully achieved the blocking of memory B cell expression in mice.

Next, we assessed the impact of blocking memory B cells on reversing the antitumor efficacy of triple therapy at the level of T-cell tumor infiltration as determined by flow cytometry (Figure 5F). Compared with the RT + B and RT + P + B groups, the RT + P + B + L group had significantly lower proportions of CD4⁺ T and CD8⁺ T cells. Similarly, the RT + B + L group had significantly lower proportions of CD4⁺ T and CD8⁺ T cells than did the RT+B group (Figure 5G). The proportions of CD4⁺ T cells in the tumor microenvironment of each group were as follows: RT + B, 14.700±2.100; RT + P + B, 20.200±2.606; RT + B + L, 8.420±1.538; and RT + P + B + L, 6.003±1.103. The proportions of CD8⁺ T cells in the tumor microenvironment of each group were as follows: RT + B, 21.167±3.361; RT + P + B, 34.167±3.556; RT + B + L, 11.700±0.400; and RT + P + B + L, 14.433±2.854.

These findings indicated that blocking memory B cells reverses the original antitumor effect of triple therapy. At the level of mouse tumor volume, after memory B cells were blocked (RT + B + L and RT + P + B + L groups), the tumor volume larger in the nonblocking groups (RT + B and RT + P + B). At the cellular level, blocking also reversed the infiltration levels of CD4⁺ T and CD8⁺ T cells in tumor tissues. This finding reveals the key role of memory B cells in regulating T-cell infiltration in the tumor microenvironment.

Triple therapy promoted the expression levels of antitumor cytokines

To further investigate the antitumor mechanism of triple therapy, we analyzed the expression levels of the relevant cytokines in serum. First, the levels of cytokines closely related to the activity and function of immune cells were analyzed. The results showed that compared with the RT + P group and RT + B group, the RT + P + B triple-therapy group had significantly increased expression levels of IFN-γ and IL-10. It is worth noting that after administration of the LFA blocker, the high expression levels of IFN-γ and IL-10 decreased (Figure 6A,6B).

Figure 6 Triple therapy promoted the expression levels of antitumor cytokines. (A) The expression level of IFN-γ cytokines in serum. (B) The expression level of IL-10 cytokines in serum. Statistical significance (P values) was assessed with the Student t-test and one-way analysis of variance. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001; ns, not significant. RT + P, RT combined with anti-PD-1 therapy; RT + B, RT combined with anti-BTLA therapy; RT + P + B, RT combined with anti-PD-1 and anti-BTLA therapy; RT + B + L, RT combined with anti-BTLA and anti-LFA-1α blocker therapy; RT + P + B + L, RT combined with anti-PD-1, anti-BTLA and anti-LFA-1α blocker therapy. BTLA, B- and T-lymphocyte attenuator; IFN-γ, interferon-γ; LFA-1α, lymphocyte function-associated antigen 1 alpha; IL-10, interleukin 10; PD-1, programmed cell death protein 1; RT, radiotherapy.

Discussion

In this study, the expression level of BTLA in patients with NSCLC was associated with resistance to immunotherapy, and RT was found to increase the expression level of BTLA in the tumor microenvironment of a mouse lung cancer model. Based on the above findings, we, for the first, time incorporated anti-BTLA blockade into the classic antitumor therapy of RT combined with anti-PD-1 blockade. Anti-BTLA blockade further enhanced the therapeutic effect of RT combined with anti-PD-1 therapy on tumors in the mouse lung cancer model. This triple therapy exerted an improved antitumor effect by enhancing the infiltration level of T cells into the tumor microenvironment. Moreover, the increase in T-cell infiltration level appears to be related to the fact that anti-BTLA blockade increased the proportion of memory B cells in the spleen of mice. In conclusion, blockade of BTLA enhances the antitumor efficacy of RT in combination with anti–PD-1 therapy, and the RT + P + B regimen may represent a novel approach for the treatment of patients with advanced NSCLC with resistance to PD-1 inhibitors.

Although immunotherapy has become a standard of care in the treatment of lung cancer, resistance remains a major challenge. Dual immunotherapy alone or combined with chemotherapy has demonstrated superior efficacy in terms of overall survival (OS) and progression-free survival (PFS) as compared to chemotherapy in patients with advanced or metastatic NSCLC. The combination of anti-CTLA-4 and anti-PD-1 therapy was approved by the US Food and Drug Administration (FDA) in 2020 as a first-line dual-immunotherapy regimen for patients with metastatic NSCLC without gene mutations (9). In the CheckMate 227 (9) and CheckMate 9LA (22) trials, dual immunotherapy alone or in combination with chemotherapy yielded better survival outcomes than did chemotherapy alone. A meta-analysis (23) on dual-immunotherapy combinations targeting different molecules in patients with advanced NSCLC showed that, in addition to therapies targeting PD-1/PD-L1 and CTLA-4, the combination of anti-PD-1/PD-L1 and anti-TIGIT therapy can prolong OS and PFS. Furthermore, studies have shown that RT, administered after the development of immunotherapy resistance, can reduce resistance by reshaping the tumor microenvironment (24-26). Other research indicates that the expression of BTLA is increased in NSCLC and that its overexpression is associated with poor prognosis, suggesting that BTLA may be a potential target for immunotherapy in patients with NSCLC (27). Zhang et al. (28) reported that, in their study, the percentage of BTLA⁺CD8⁺ T cells was increased in the malignant pleural effusion and tumors of patients with NSCLC refractory to anti–PD-1 therapy. In other studies involving animal experiments, dual blockade of BTLA and PD-1 pathways reversed the exhaustion of CD8⁺ T cells and increased the infiltration levels of CD8⁺ and CD4⁺ T cells in the tumor microenvironment (29,30). In phase I or phase II clinical trials, the world’s first humanized IgG4 monoclonal ani-BTLA antibody, tifcemalimab, exhibited a manageable safety profile and exerted preliminary antitumor activity in the treatment of advanced solid tumors and lymphomas in, either in monotherapy or in combination with anti-PD-1 monoclonal antibody and chemotherapy (31-34). In the recent NCT05000684 study, anti-BTLA combined with anti-PD-1 antibodies exhibited encouraging efficacy and favorable safety in previously treated patients with advanced lung cancer, particularly in patients with extensive-stage small-cell lung cancer, with an ORR as high as 35.0% (35). In our study, data from the GEO database indicated that the expression level of BTLA was correlated with the pathological response of patients with NSCLC receiving immunotherapy. In mouse animal models, RT increased BTLA expression in the tumor microenvironment of mice. In addition, we discovered that the addition of anti-BTLA blockade further inhibited tumor growth in mice compared with RT combined with anti-PD-1 therapy. Other mechanistic investigations have reported increased infiltration of CD8⁺ and CD4⁺ T cells in the tumor microenvironment and an elevated level of cytokine release in serum (36-38).

Although T cells play a critical role in the antitumor immune responses induced by immunotherapy and RT (39,40), studies have shown that functionally exhausted or dysfunctional CD8⁺ and CD4⁺ TILs express C-X-C motif chemokine ligand 13 (CXCL13) that recruits B cells, indicating that they are programmed to seek help from B cells in the face of tumor persistence (41-43). At present, the role of B cells in antitumor immunotherapy is not clear, but studies have widely recognized the value of B cells [especially tumor-infiltrating B cells (TIL-Bs)] in immunotherapy, which is reflected in several aspects: first, TIL-Bs, together with T cells and myeloid cells, construct an immune “hot” tumor microenvironment, enhance T cells’ antitumor responses through antigen presentation, and the tertiary lymphoid structures (TLSs) they form are associated with better treatment prognosis, especially in immune checkpoint blockade therapy where the presence of TLSs can predict treatment response. Second, TIL-Bs exert various effects by producing antibodies, including antibody-dependent cellular cytotoxicity, antibody-dependent cellular phagocytosis, complement-dependent cytotoxicity, and can directly affect tumor cell functions through mechanisms such as antibody-mediated signal interference (44). In addition, when tumor antigens are released into the blood, B cells in peripheral lymphoid organs are selectively activated to produce antibodies, promoting lymphocyte proliferation and differentiation into effector T cells and memory B cells, which then migrate to the tumor microenvironment to exert immune effects (45,46).

Studies have also demonstrated that the expression level of BTLA on the surface of B cells is higher than that on T cells (47,48). Therefore, we further analyzed B cell-related subsets in the GEO database and found that the expression level of BTLA in memory B cells was higher in the NMPR group than in the MPR group. Based on this, we further investigated the reasons why triple therapy increased the infiltration level of T cells in tumors. Since the spleen is the largest peripheral lymphoid organ and harbors a high density of antigen-presenting cells and lymphocytes (49), we first examined the proportion of B cell-related subsets in mouse spleens. We found that the addition of anti-BTLA blockade significantly increased the proportion of memory B cells among B cells in the spleen, without increasing the proportion of related of T cells. Regarding the decrease in the proportion of T cells in the spleen, when the body responds to tumors, the distribution of immune system cells undergoes dynamic changes. The chemotactic effect of the tumor microenvironment on T cells may prompt a greater number of CD8⁺ T and CD4⁺ T cells to leave lymphoid organs such as the spleen and migrate to tumor tissues (50). Further experiments in our study showed that blocking memory B cells reversed the antitumor efficacy in mouse tumors, and the infiltration levels of CD8⁺ T and CD4⁺ T cells in the tumor microenvironment decreased. This observation suggests that anti-BTLA blockade exerts anti-tumor immune effects not mainly by regulating the proportion of T cells in the spleen but also by regulating the proportion of memory B cells in the spleen. In addition to increasing the expression level of IFN-γ (a T-cell activation marker), the triple therapy also elevated the expression of IL-10 and reduced the levels of pro-inflammatory cytokines TNF-α and IL-6. Notably, IL-10 can be secreted and released by B cell-related subsets (51). In the present study, the triple therapy-induced elevation of IL-10 expression was reversed after blocking memory B cells. Previous studies have demonstrated that IL-10, as an inhibitory regulator of inflammatory responses, can suppress the expression levels of pro-inflammatory cytokines TNF-α and IL-6 (51). This effect may be related to the fact that increased IL-10 release after BTLA blockade activates STAT3, thereby promoting the development, survival of tumor-specific terminal CD8⁺ T cells, and enhancing their effector functions (48,52); however, the related mechanisms remain to be further clarified.

This study involved certain limitations that should be addressed. To begin, we did not investigate the infiltration level of B cells in the mouse tumor microenvironment, the release level of related cytokines, or the underlying mechanisms. Additionally, in the experiment in which blocking memory B cells reversed the effect of triple therapy, it seems that comparing RT + P + B with RT + P + B + L would have provided more valuable insights. However, the results indicate that anti-BTLA blockade further enhances the antitumor efficacy of RT combined with anti-PD-1 therapy in the mouse lung cancer model. We endeavor to conduct subsequent in-depth studies. Overall, this study provides a novel perspective for the optimization of RT combined with immunotherapy.

Conclusions

In this study, RT increased the expression level of BTLA in the tumor microenvironment of mice. Moreover, anti-BTLA blockade promoted the infiltration of T cells into the tumor microenvironment by regulating the proportion of memory B cells in the spleen of mice, thereby further enhancing the antitumor efficacy of RT combined with anti-PD-1 therapy. Future studies should further investigate the mechanisms underlying this effect, as well as its clinical role in treating patients and its impact on the tumor microenvironment.

Acknowledgments

The authors are grateful to the GEO database for providing access to its shared data and the Ethics Committee of The First Affiliated Hospital of Shandong First Medical University.

Footnote

Reporting Checklist: The authors have completed the ARRIVE and MDAR reporting checklists. Available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2026-0449/rc

Data Sharing Statement: Available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2026-0449/dss

Peer Review File: Available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2026-0449/prf

Funding: This study was funded by the National Natural Science Foundation of China (No. 81803043), the Shandong Natural Science Foundation (Nos. ZR2021LSW023, ZR2021QH356, and ZR2022QH351), China Health & Medical Development Foundation (No. HX2024030), and the Beijing Huikang Ren’ai Public Welfare Foundation (No. HKRA2025050093).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2026-0449/coif). All authors report funding support from the National Natural Science Foundation of China (No. 81803043), the Shandong Natural Science Foundation (Nos. ZR2021LSW023, ZR2021QH356, and ZR2022QH351), China Health & Medical Development Foundation (No. HX2024030), and the Beijing Huikang Ren’ai Public Welfare Foundation (No. HKRA2025050093). The authors have no other conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. Animal experiments were performed under a project license (No. QFSYYPZ2022091301) granted by the Ethics Committee of The First Affiliated Hospital of Shandong First Medical University (Shandong Provincial Qianfoshan Hospital), in compliance with national guidelines for the care and use of animals.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

References

1. Siegel RL, Giaquinto AN, Jemal A. Cancer statistics, 2024. CA Cancer J Clin 2024;74:12-49. [Crossref] [PubMed]
2. Herbst RS, Morgensztern D, Boshoff C. The biology and management of non-small cell lung cancer. Nature 2018;553:446-54. [Crossref] [PubMed]
3. Herbst RS, Giaccone G, de Marinis F, et al. Atezolizumab for First-Line Treatment of PD-L1-Selected Patients with NSCLC. N Engl J Med 2020;383:1328-39. [Crossref] [PubMed]
4. Garassino MC, Gadgeel S, Speranza G, et al. Pembrolizumab Plus Pemetrexed and Platinum in Nonsquamous Non-Small-Cell Lung Cancer: 5-Year Outcomes From the Phase 3 KEYNOTE-189 Study. J Clin Oncol 2023;41:1992-8. [Crossref] [PubMed]
5. Gomez DR, Blumenschein GR Jr, Lee JJ, et al. Local consolidative therapy versus maintenance therapy or observation for patients with oligometastatic non-small-cell lung cancer without progression after first-line systemic therapy: a multicentre, randomised, controlled, phase 2 study. Lancet Oncol 2016;17:1672-82. [Crossref] [PubMed]
6. Kalbasi A, Ribas A. Tumour-intrinsic resistance to immune checkpoint blockade. Nat Rev Immunol 2020;20:25-39. [Crossref] [PubMed]
7. Lhuillier C, Rudqvist NP, Elemento O, et al. Radiation therapy and anti-tumor immunity: exposing immunogenic mutations to the immune system. Genome Med 2019;11:40. [Crossref] [PubMed]
8. Limagne E, Richard C, Thibaudin M, et al. Tim-3/galectin-9 pathway and mMDSC control primary and secondary resistances to PD-1 blockade in lung cancer patients. Oncoimmunology 2019;8:e1564505. [Crossref] [PubMed]
9. Hellmann MD, Paz-Ares L, Bernabe Caro R, et al. Nivolumab plus Ipilimumab in Advanced Non-Small-Cell Lung Cancer. N Engl J Med 2019;381:2020-31. [Crossref] [PubMed]
10. Boutros C, Tarhini A, Routier E, et al. Safety profiles of anti-CTLA-4 and anti-PD-1 antibodies alone and in combination. Nat Rev Clin Oncol 2016;13:473-86. [Crossref] [PubMed]
11. Kim JE, Patel MA, Mangraviti A, et al. Combination Therapy with Anti-PD-1, Anti-TIM-3, and Focal Radiation Results in Regression of Murine Gliomas. Clin Cancer Res 2017;23:124-36. [Crossref] [PubMed]
12. Hellmann MD, Ciuleanu TE, Pluzanski A, et al. Nivolumab plus Ipilimumab in Lung Cancer with a High Tumor Mutational Burden. N Engl J Med 2018;378:2093-104. [Crossref] [PubMed]
13. Li Y, Liang X, Li H, et al. Efficacy and safety of immune checkpoint inhibitors for advanced non-small cell lung cancer with or without PD-L1 selection: A systematic review and network meta-analysis. Chin Med J (Engl) 2023;136:2156-65.
14. Watanabe N, Gavrieli M, Sedy JR, et al. BTLA is a lymphocyte inhibitory receptor with similarities to CTLA-4 and PD-1. Nat Immunol 2003;4:670-9. [Crossref] [PubMed]
15. Chang MC, Lee WC, Tai YJ, et al. BTLA: An Emerging Immune Checkpoint Target in Cancer Immunotherapy. Pharmaceuticals (Basel) 2025;18:1784. [Crossref] [PubMed]
16. Ning Z, Liu K, Xiong H. Roles of BTLA in Immunity and Immune Disorders. Front Immunol 2021;12:654960. [Crossref] [PubMed]
17. Murphy TL, Murphy KM. Slow down and survive: Enigmatic immunoregulation by BTLA and HVEM. Annu Rev Immunol 2010;28:389-411. [Crossref] [PubMed]
18. Ren S, Tian Q, Amar N, et al. The immune checkpoint, HVEM may contribute to immune escape in non-small cell lung cancer lacking PD-L1 expression. Lung Cancer 2018;125:115-20. [Crossref] [PubMed]
19. Wojciechowicz K, Spodzieja M, Wardowska A. The BTLA-HVEM complex - The future of cancer immunotherapy. Eur J Med Chem 2024;268:116231. [Crossref] [PubMed]
20. Hu J, Zhang L, Xia H, et al. Tumor microenvironment remodeling after neoadjuvant immunotherapy in non-small cell lung cancer revealed by single-cell RNA sequencing. Genome Med 2023;15:14. [Crossref] [PubMed]
21. Kalbasi A, June CH, Haas N, et al. Radiation and immunotherapy: a synergistic combination. J Clin Invest 2013;123:2756-63. [Crossref] [PubMed]
22. Paz-Ares L, Ciuleanu TE, Cobo M, et al. First-line nivolumab plus ipilimumab combined with two cycles of chemotherapy in patients with non-small-cell lung cancer (CheckMate 9LA): an international, randomised, open-label, phase 3 trial. Lancet Oncol 2021;22:198-211. [Crossref] [PubMed]
23. Yang Y, Xin D, Guan L, et al. Dual immunotherapy in advanced or metastatic non-small cell lung cancer: A network meta-analysis. Heliyon 2024;10:e27576. [Crossref] [PubMed]
24. Tachihara M, Tsujino K, Ishihara T, et al. Durvalumab Plus Concurrent Radiotherapy for Treatment of Locally Advanced Non-Small Cell Lung Cancer: The DOLPHIN Phase 2 Nonrandomized Controlled Trial. JAMA Oncol 2023;9:1505-13. [Crossref] [PubMed]
25. Shang S, Liu J, Verma V, et al. Combined treatment of non-small cell lung cancer using radiotherapy and immunotherapy: challenges and updates. Cancer Commun (Lond) 2021;41:1086-99. [Crossref] [PubMed]
26. Demaria S, Golden EB, Formenti SC. Role of Local Radiation Therapy in Cancer Immunotherapy. JAMA Oncol 2015;1:1325-32. [Crossref] [PubMed]
27. Li X, Xu Z, Cui G, et al. BTLA Expression in Stage I-III Non-Small-Cell Lung Cancer and Its Correlation with PD-1/PD-L1 and Clinical Outcomes. Onco Targets Ther 2020;13:215-24. [Crossref] [PubMed]
28. Zhang Y, Yang Y, Zeng Y, et al. B and T lymphocyte attenuator (BTLA) and PD-1 pathway dual blockade promotes antitumor immune responses by reversing CD8(+) T-cell exhaustion in non-small cell lung cancer. Front Immunol 2025;16:1553042. [Crossref] [PubMed]
29. Mortezaee K. Targeting B and T lymphocyte attenuator in cancer immunotherapy. Int J Biol Macromol 2025;318:144953. [Crossref] [PubMed]
30. Nishizaki D, Choi S, Pandya C, et al. Pan-Cancer Landscape of B- and T-Lymphocyte Attenuator: Implications for Potential Immunotherapy Combinations. JCO Precis Oncol 2025;9:e2500304. [Crossref] [PubMed]
31. Ma J, Xie Y, Zhang H, et al. Phase I study of the anti-BTLA antibody icatolimab as a single agent or in combination with toripalimab in relapsed/refractory lymphomas. J Clin Oncol 2022;40:7578.
32. Yu Y, Yu Y, Zhang Y, et al. Tifcemalimab combined with toripalimab and chemotherapy as 1st line treatment for extensive-stage small cell lung cancer (ES-SCLC): A phase Ib/II, open-label study. J Clin Oncol 2024;42:8089.
33. Cheng Y, Wang J, Yu Y, et al. Phase I/II combination study of tifcemalimab with toripalimab in patients with refractory extensive stage small cell lung cancer (ES-SCLC). J Clin Oncol 2023;41:8579.
34. Ma VT, Milhem MM, Hidalgo M, Shields AF, Segar JM, Khalil M, et al. Phase I dose-escalation and cohort expansion study of the anti-BTLA antibody, tifcemalimab, in combination with toripalimab (anti-PD-1) in heavily pretreated patients (pts) with advanced malignancies. J Clin Oncol 2024;42:2596.
35. Cheng Y, Wang J, Yu Y, et al. Phase I/II Study of Tifcemalimab, an Anti-B- and T-lymphocyte Attenuator Antibody, in Combination with Toripalimab in Previously Treated Advanced Lung Cancer. Clin Cancer Res 2025;31:2926-34. [Crossref] [PubMed]
36. Zagorulya M, Yim L, Morgan DM, et al. Tissue-specific abundance of interferon-gamma drives regulatory T cells to restrain DC1-mediated priming of cytotoxic T cells against lung cancer. Immunity 2023;56:386-405.e10. [Crossref] [PubMed]
37. St Paul M, Ohashi PS. The Roles of CD8(+) T Cell Subsets in Antitumor Immunity. Trends Cell Biol 2020;30:695-704. [Crossref] [PubMed]
38. Garris CS, Arlauckas SP, Kohler RH, et al. Successful Anti-PD-1 Cancer Immunotherapy Requires T Cell-Dendritic Cell Crosstalk Involving the Cytokines IFN-γ and IL-12. Immunity 2018;49:1148-61.e7.
39. Oliveira G, Wu CJ. Dynamics and specificities of T cells in cancer immunotherapy. Nat Rev Cancer 2023;23:295-316. [Crossref] [PubMed]
40. Chow A, Perica K, Klebanoff CA, et al. Clinical implications of T cell exhaustion for cancer immunotherapy. Nat Rev Clin Oncol 2022;19:775-90. [Crossref] [PubMed]
41. Li H, van der Leun AM, Yofe I, et al. Dysfunctional CD8 T Cells Form a Proliferative, Dynamically Regulated Compartment within Human Melanoma. Cell 2019;176:775-789.e18. [Crossref] [PubMed]
42. Workel HH, Lubbers JM, Arnold R, et al. A Transcriptionally Distinct CXCL13(+)CD103(+)CD8(+) T-cell Population Is Associated with B-cell Recruitment and Neoantigen Load in Human Cancer. Cancer Immunol Res 2019;7:784-96. [Crossref] [PubMed]
43. Thommen DS, Koelzer VH, Herzig P, et al. A transcriptionally and functionally distinct PD-1(+) CD8(+) T cell pool with predictive potential in non-small-cell lung cancer treated with PD-1 blockade. Nat Med 2018;24:994-1004. [Crossref] [PubMed]
44. Laumont CM, Banville AC, Gilardi M, et al. Tumour-infiltrating B cells: immunological mechanisms, clinical impact and therapeutic opportunities. Nat Rev Cancer 2022;22:414-30. [Crossref] [PubMed]
45. Sautès-Fridman C, Petitprez F, Calderaro J, et al. Tertiary lymphoid structures in the era of cancer immunotherapy. Nat Rev Cancer 2019;19:307-25. [Crossref] [PubMed]
46. Sharonov GV, Serebrovskaya EO, Yuzhakova DV, et al. B cells, plasma cells and antibody repertoires in the tumour microenvironment. Nat Rev Immunol 2020;20:294-307. [Crossref] [PubMed]
47. Mintz MA, Felce JH, Chou MY, et al. The HVEM-BTLA Axis Restrains T Cell Help to Germinal Center B Cells and Functions as a Cell-Extrinsic Suppressor in Lymphomagenesis. Immunity 2019;51:310-323.e7. [Crossref] [PubMed]
48. Chen YL, Lin HW, Chien CL, et al. BTLA blockade enhances Cancer therapy by inhibiting IL-6/IL-10-induced CD19(high) B lymphocytes. J Immunother Cancer 2019;7:313. [Crossref] [PubMed]
49. He X, Wang J, Tang Y, et al. Recent Advances of Emerging Spleen-Targeting Nanovaccines for Immunotherapy. Adv Healthc Mater 2023;12:e2300351. [Crossref] [PubMed]
50. Morgan DM, Horton BL, Bhandarkar V, et al. Expansion of tumor-reactive CD8(+) T cell clonotypes occurs in the spleen in response to immune checkpoint blockade. Sci Immunol 2024;9:eadi3487. [Crossref] [PubMed]
51. Salkeni MA, Naing A. Interleukin-10 in cancer immunotherapy: from bench to bedside. Trends Cancer 2023;9:716-25. [Crossref] [PubMed]
52. Sun Q, Zhao X, Li R, et al. STAT3 regulates CD8+ T cell differentiation and functions in cancer and acute infection. J Exp Med 2023;220:e20220686. [Crossref] [PubMed]

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