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Clinical/translational cancer immunotherapy
Original research
Dual inhibition of BTLA and PD-1 can enhance therapeutic efficacy of paclitaxel on intraperitoneally disseminated tumors
  1. Wei-Zen Sun1,
  2. Han-Wei Lin1,
  3. Wan-Yu Chen2,
  4. Chung-Liang Chien3,
  5. Yen-Ling Lai4,5,
  6. Jung Chen4,
  7. Yu-Li Chen4,6 and
  8. Wen-Fang Cheng2,4,7
  1. 1Department of Anesthesiology, College of Medicine, National Taiwan University, Taipei, Taiwan
  2. 2Graduate Institute of Oncology,College of Medicine, National Taiwan University, Taipei, Taiwan
  3. 3Graduate Institute of Anatomy and Cell Biology,College of Medicine, National Taiwan University, Taipei, Taiwan
  4. 4Department of Obstetrics and Gynecology,College of Medicine, National Taiwan University, Taipei, Taiwan
  5. 5Department of Obstetrics and Gynecology, National Taiwan University Hospital Hsin-Chu Branch, Hsin‑Chu, Taiwan
  6. 6Department of Obstetrics and Gynecology, National Taiwan University Hospital Yun-Lin Branch, Yun‑Lin county, Taiwan
  7. 7Graduate Institute of Clinical Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan
  1. Correspondence to Dr Yu-Li Chen; uly1007{at}yahoo.com.tw

  • W-ZS and H-WL are joint first authors.

Abstract

Background Expression of immune checkpoints in the tumor microenvironment is one mechanism underlying paclitaxel (PTX) chemoresistance. This study aimed to investigate whether the addition of checkpoint blockade to PTX can improve the therapeutic efficacy against apparently disseminated intraperitoneal tumors.

Methods We analyzed the in vivo expression of various immune checkpoints in CD3+CD8+ cytotoxic T cells from tumor-bearing mice treated with or without PTX and validated the tumor-killing activities of selected checkpoint-expressing T-cell subpopulations ex vivo. The regulation of selected checkpoints was investigated in vitro. The therapeutic effects of inhibition of a targeted checkpoint pathway with antibodies added to PTX therapy were examined.

Results CD3+CD8+ T cells expressed with herpes virus entry mediator (HVEM), programmed cell death 1 (PD-1), and T-cell immunoglobulin domain and mucin domain 3 (TIM-3) in tumor-bearing hosts treated with PTX had effective tumoricidal activities. In addition to PTX and cytokines, B and T lymphocyte attenuator (BTLA) or homologous to lymphotoxin, exhibits inducible expression and competes with herpes simplex virus (HSV) glycoprotein D for binding to HVEM, a receptor expressed on T lymphocytes (LIGHT) interacting with HVEM can regulate the expression of PD-1 on CD3+CD8+ T cells. Interleukin (IL)-15 increased the percentage of HVEMhighgranzyme B (GZMB)+ cells among CD3+CD8+ T cells, which was suppressed by the BTLA/HVEM signal. LIGHT induced the percentage of HVEM+GZMB+ cells but not HVEMhighGZMB+ cells among CD3+CD8+ T cells. Expression of IL-15, BTLA, or LIGHT was detected in CD19+ B cells and regulated by damage-associated molecular patterns/Toll-like receptor interactions. In the tumor-bearing hosts treated with PTX, certain proportions of BTLA+ B or PD-1+ T lymphocytes were still noted. When dual inhibition of BTLA and PD-1 was added to PTX, the antitumor effects on intraperitoneally disseminated tumors can be significantly improved.

Conclusions Dual blockade of BTLA on B cells and PD-1 on cytotoxic T cells may have clinical potential for enhancing the efficacy of PTX in the treatment of tumors with intraperitoneal spread, including epithelial ovarian carcinomas.

  • B-Lymphocytes
  • CD8-Positive T-Lymphocytes
  • Combined Modality Therapy
  • Immune Checkpoint Inhibitors
  • Immunomodulation

Data availability statement

Data are available upon reasonable request.

http://creativecommons.org/licenses/by-nc/4.0/

This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See http://creativecommons.org/licenses/by-nc/4.0/.

https://doi.org/10.1136/jitc-2023-006694

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    WHAT IS ALREADY KNOWN ON THIS TOPIC

    • The immune response induced by chemotherapeutic agents, including paclitaxel (PTX), can be suppressed by immune checkpoints, which is one of the mechanisms underlying chemoresistance.

    WHAT THIS STUDY ADDS

    • The herpes virus entry mediator (HVEM)-related checkpoint pathway plays a role in the immune regulation of tumor-bearing hosts with PTX treatment.

    • Interleukin-15, B and T lymphocyte attenuator (BTLA), and LIGHT in CD19+ B cells are involved in the HVEM-related checkpoint pathway and regulated through damage-associated molecular patterns/Toll-like receptor interactions, modulating programmed cell death 1 (PD-1) expression on CD3+CD8+ T cells.

    • The addition of anti-BTLA antibody (Ab) and anti-PD-1 Ab to PTX chemotherapy can generate potent antitumor effects on intraperitoneally disseminated tumors.

    HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY

    • Application of this study has clinical potential for precisely combining immune checkpoint blockades with chemotherapy to tackle malignant diseases.

    Background

    Malignant cells express a variety of antigens that evoke tumor-associated immune responses.1 However, these tumor clones eventually evade the immunosurveillance and become clinically apparent after undergoing cancer immunoediting.2–4 One of the regulatory mechanisms of immune escape is immunoinhibitory checkpoints.2 These molecules can maintain self-tolerance and regulate immune responses to reduce tissue damage, but tumors can trigger certain checkpoint pathways to suppress the antitumor effects of antigen-specific T cells. However, the inhibitory signals can be blocked by antibodies (Abs) to rescue endogenous immune responses.5

    Chemotherapeutic agents are commonly employed to treat clinically apparent tumors with multiple metastatic sites. One of these agents is paclitaxel (PTX), which is essential in the treatment of several malignancies, including epithelial ovarian carcinomas (EOCs) characterized by intraperitoneal spread.6 7 In addition to increasing tumor cell apoptosis, PTX has immunomodulatory effects on effector T cells, dendritic cells (DCs), natural killer (NK) cells, regulatory T cells, and macrophages. Similar to PTX, carboplatin has high immunogenicity and induces apoptosis in ovarian cancer cells.8 In patients with high-grade serous ovarian cancer, the most common histology of EOCs, the initial response rates for platinum/PTX-based chemotherapy are 60.0–80.0%. However, most cases eventually have chemoresistant tumors with disease relapse. Expression of immune checkpoints in the tumor microenvironment (TME) is one of the mechanisms underlying chemoresistance.9

    We previously demonstrated that PTX combined with immune checkpoint blockade (ICB) can enhance antitumor effects. However, the treatment was started 3 days after intraperitoneal challenge with 1×105 WF-3/Luc cells/mouse, at which time no visible tumors could be observed in the hosts.10 In the current study, mice were challenged with 5×105 cells/mouse and underwent treatment 14 days after the tumor challenge. The intraperitoneal tumor profile and treatment time point were very similar to the clinical scenario. We analyzed in vivo the expression of various immune checkpoints in CD3+CD8+ T cells from tumor-bearing hosts treated with or without PTX. These checkpoints included adenosine A2A receptor (A2AR), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), herpes virus entry mediator (HVEM), programmed cell death 1 (PD-1), and T-cell immunoglobulin domain and mucin domain 3 (TIM-3). The potential checkpoint-expressing T-cell subpopulations from tumor-bearing mice with PTX were validated ex vivo. Regulation of the selected immune checkpoints and HVEM-related pathway were then examined in vitro. We also investigated the therapeutic effects when inhibition of HVEM pathway-related molecules was added to PTX therapy.

    Methods

    Mice

    Female C57BL/6J mice were kept in the animal facilities of College of Medicine, National Taiwan University. They were used in experiments at 6–8 weeks old.

    Cell line

    WF-3/Luc tumor cells were generated in the animal model.10 11 Intraperitoneal spread of tumors with ascites was observed. Malignant cells, various tumor-associated cells (TACs), such as tumor-associated lymphocytes (TALs), and several regulatory factors, including cytokines, could be identified in the ascites, which was considered part of the TME to investigate host immunological features.12 13

    In vivo tumor treatment

    PTX (Sigma-Aldrich), anti-B and T lymphocyte attenuator (BTLA) Ab (clone 6A6, Bio X cell), and anti-PD-1 Ab (clone RMP1-14, Bio X cell) were administered intraperitoneally. Dose determination was performed.10 14 PTX was applied at 6 mg/kg and 100 µg of anti-BTLA or anti-PD-1 Ab was used in each dose. Mice were challenged with 5×105 tumor cells on day 0. For the PTX alone group, daily PTX was started on day 14. For the PTX and ICB group, PTX was used daily from day 14 and ICB (anti-BTLA Ab, anti-PD-1 Ab, or both) was applied twice a week from day 16. The immune profiles and tumor volumes of the tumor-bearing mice with or without treatment were analyzed on day 35. Splenocytes were prepared, as well as the supernatants and TACs of ascites, and tumor bioluminescence was detected.10 11 15 The remaining animals were maintained until day 100 or death for survival analyses.

    Surface and intracellular staining of splenocytes and TACs analyzed by flow cytometry, t-distributed stochastic neighbor embedding, and PhenoGraph

    Surface and intracellular markers were stained with various Abs (online supplemental table 1) as described previously.10 11 CD69 and CD103 were considered markers of resident memory T cells.16 In addition to tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ), granzyme B (GZMB), perforin and CD107a were used as markers of the killing ability of cytotoxic T lymphocytes.17 18 Data were collected by the BD FACSLyric Flow Cytometry System and analyzed using FlowJo V.10.8. For application of the t-distributed stochastic neighbor embedding (t-SNE) module, downsampling function of intra-individual and inter-individual measurements was employed before data concatenation.19 For t-SNE reduction, the input signals were set to 1000 iterations, 40 perplexity, 0.5 Theta. On the t-SNE map, CD3+CD8+ T cells were clustered by CD69, CD103, A2AR, CTLA-4, HVEM, PD-1, and TIM-3 expressions using PhenoGraph V.3.0.

    Supplemental material

    Certain clusters were determined to further investigate the expression of these markers in tumor-bearing mice with or without PTX treatment by two criteria. First, the percentages of selected clusters of CD3+CD8+ T cells were significantly different between the two groups. Second, the difference with a gap ≥5.0% was required. Clusters with a percentage ≥10.0% in CD3+CD8+ T cells were employed to evaluate the expression of these markers in tumor-bearing mice treated with PTX, anti-PD-1 Ab and anti-BTLA Ab.

    Enzyme-linked immunosorbent assays

    Direct ELISAs of interleukin (IL)-2, IL-4, IL-6, IL-10, IL-12, IL-15, TNF-α, and IFN-γ (R&D systems) were performed using the supernatants of cultured splenocytes (2×106 cells) and ascites.15 ELISAs of IL-15/IL-15 receptor alpha (IL-15Rα) complex (Invitrogen) and IL-15 were performed using supernatants of cultured B lymphocytes (2×106 cells) with lipopolysaccharide (LPS) (0.5 µg/mL, Sigma-Aldrich) or CpG-oligodeoxynucleotide (CpG-ODN) (1 µg/mL, InvivoGen).

    Sorting of CD3+CD8+ T lymphocytes, subgroups of CD3+CD8+ T lymphocytes, and CD19+ B lymphocytes by flow cytometry

    CD3+CD8+ T lymphocytes and CD3−CD19+ B lymphocytes were obtained from the splenocytes of naïve mice. On day 35, the HVEM+ and HVEM+PD-1+TIM-3+ CD3+CD8+ T lymphocytes were collected from the splenocytes of tumor-bearing mice receiving PTX.

    Ex vivo tumoricidal activity of subgroups of CD3+CD8+ T lymphocytes

    Splenocytes, HVEM+CD3+CD8+ T lymphocytes, and HVEM+PD-1+TIM-3+CD3+CD8+ T lymphocytes from tumor-bearing mice receiving PTX were co-cultured with WF-3/Luc tumor cells at 50:1 in a 96-well plate (1×104 cells/well) for 24 hours. The luciferase activity of tumor cells was measured.11

    In vitro effects of various molecules on splenocytes, CD3+CD8+ T lymphocytes, or CD19+ B lymphocytes

    For the impact of PTX on various molecules, splenocytes (5×105 cells) were treated with phosphate buffered saline (PBS) or PTX (1 µM) for 48 hours and the expression of HVEM, PD-1, TIM-3, CD69, or CD103 was evaluated. For the regulation of IL-15 or LIGHT (homologous to lymphotoxin, exhibits inducible expression and competes with HSV glycoprotein D for binding to HVEM, a receptor expressed on T lymphocytes), splenocytes (5×105 cells) were co-cultured with PBS, WF-3/Luc tumor cells (1×105 cells), or WF-3/Luc tumor cells (1×105 cells) and PTX (1 µM) for 48 hours and the expression of IL-15 or LIGHT was analyzed. Percentages of CD11c+, CD19+, or NK1.1+ cells in the IL-15-secreting or LIGHT-expressing splenocytes were evaluated.

    PBS, phorbol myristate acetate (PMA, 100 ng/mL) and ionomycin (1 µg/mL), PTX (1 µM), or various recombinant proteins (online supplemental table 2), including IL-2, IL-6, IL-10, IL-15, TNF-α, IFN-γ, IL-15/IL-15Rα complex, BTLA, and LIGHT, were loaded with sorted T lymphocytes (5×105 cells) for 24 hours. Expression of HVEM, PD-1, TIM-3, CD69, CD103, GZMB, perforin, TNF-α, IFN-γ, or CD107a was analyzed. Sorted B lymphocytes (5×105 cells) were treated with PBS, PTX (1 µM), LPS (0.5 µg/mL), or CpG-ODN (1 µg/mL) for 24 hours. Expression of IL-15, BTLA, LIGHT, or CD11c was evaluated. B cells were treated with PBS, LPS or CpG-ODN for 24, 48, and 72 hours for detecting IL-15/IL-15Rα complex. Marker expression was detected by flow cytometry.

    Supplemental material

    Western blot analysis

    Sorted CD3+CD8+ T cells (1×106/well) were prepared with serum-free media and seeded in a 24-well plate for 6 hours. These T cells were treated with PBS and recombinant proteins, including IL-15, BTLA, and LIGHT (online supplemental table 2) for analyzing signaling pathways and harvested after 20 min of incubation. Twenty micrograms of each cell lysate were resolved by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE) (10.0% gel), transferred onto a PVDF/nylon membrane (Millipore), and probed with various Abs (online supplemental table 3). The membrane was then probed with horseradish peroxidase (HRP)-conjugated secondary Ab. The specific bands were visualized with an ECL Western blot system (GE Healthcare).11 To detect the expression of HVEM, PD-1, and GZMB in CD3+CD8+ T lymphocytes, IL-15, BTLA, and LIGHT (online supplemental table 2) were incubated with sorted T cells for 48 hours. LPS (0.5 µg/mL) and CpG-ODN (1 µg/mL) were incubated with sorted B cells for 24 and 48 hours to detect the expression of IL-15 and IL-15/IL-15Rα complex, respectively.

    Supplemental material

    In vitro tumoricidal activity of splenocytes receiving various treatment modalities

    WF-3/Luc cells (5×105 cells/well) were seeded in a 6-well plate overnight. Before splenocytes (2×106 cells/well) were co-cultured with WF-3/Luc cells, tumor medium was replaced with CTL medium. PTX (1 µM) was added and incubated with cells for 24 hours. For combinational treatment, anti-PD-1 Ab (100 µg/mL), anti-BTLA Ab (100 µg/mL), or both were administered and incubated with cells for another 24 hours. The luciferase activity of tumor cells was measured.11 The expression of GZMB or perforin in CD3+CD8+ T cells was analyzed by flow cytometry.

    Statistical analyses

    All statistical analyses were performed in SPSS for Windows V.15.0 (SPSS, Chicago, Illinois, USA). The in vivo, ex vivo, and in vitro data were expressed as mean±SE, which represented at least two different experiments. The results of luminescence, ELISA, and flow cytometry were examined by Kruskal-Wallis test. In the survival analyses, event time distributions were evaluated by a logrank test. P value<0.05 was defined as significant.

    Results

    HVEM+PD-1+TIM-3+ cytotoxic T-cell subpopulation had potent antitumor abilities in PTX-treated tumor-bearing hosts

    The intraperitoneally disseminated tumor-bearing mice started PTX treatment on day 14 (figure 1A). On day 35, the mice undergoing PTX treatment exhibited less luminescence than those not being treated (figure 1B,C). The PTX-treated mice could not survive more than 55 days after the tumor challenge. In splenic lymphocytes, the percentage of CD3+CD8+ T cells from mice treated with PTX was higher than that of mice not treated with PTX; however, in TALs from ascites, the percentages between the two groups were not different (figure 1D). Levels of IL-2, IL-6, IL-10, IL-15, TNF-α, and IFN-γ in spleens or ascites of mice were obviously noted. These cytokines were higher in mice treated with PTX than in those without PTX (figure 1E). Distribution of CD3+CD8+ T cells was different on the t-SNE map between the spleens and ascites of mice with or without PTX treatment (figure 1F). The locations and densities of A2AR, CTLA-4, HVEM, PD-1, TIM-3, CD69, and CD103 on CD3+CD8+ T cells were different on the raw t-SNE map (figure 1G). Eleven clusters of CD3+CD8+ T cells were categorized by these markers (figure 1H).

    Figure 1
    Figure 1

    Distribution of CD3+CD8+ T-cell subgroups in disseminated tumor-bearing hosts treated with or without PTX. (A) Representative luminescence image of tumor volumes (left), tumor-associated ascites and disseminated gross tumors (middle and right) of mice 14 days after WF-3/Luc tumor (5×105 tumor cells/mouse on day 0) challenge. On day 14, daily PTX (6 mg/kg) was started to treat these mice. (B) Representative luminescence images of tumor-bearing mice treated with or without PTX (five mice in each group). (C) Luminal analyses of tumor volumes in tumor-bearing mice. The mice with PTX treatment exhibited less luminescence than those without PTX treatment. (D) Percentages of CD3+CD8+ T cells in lymphocytes. In splenic lymphocytes, the percentage of CD3+CD8+ T cells from tumor-bearing mice with PTX treatment was higher than that of mice without treatment. In TALs of ascites, the percentages of CD3+CD8+ T cells between the two groups did not show differences. (E) Heat map of various cytokines in spleens and ascites. Expression levels of IL-2, IL-6, IL-10, IL-15, TNF-α, and IFN-γ in spleens or ascites of tumor-bearing mice with or without PTX treatment were obviously observed. These cytokines were higher in mice treated with PTX than in those without PTX treatment. (F) Distribution of CD3+CD8+ T cells in lymphocytes of spleens or TALs of ascites on the t-SNE map. Note: left: raw t-SNE map with presentation of CD3+CD8+ T cells in spleens and ascites of tumor-bearing mice treated with or without PTX; middle: t-SNE map with presentation of CD3+CD8+ T cells in spleens of tumor-bearing mice treated with or without PTX; right: t-SNE map with presentation of CD3+CD8+ T cells in ascites of tumor-bearing mice treated with or without PTX. Distribution regions of CD3+CD8+ T cells were different between the spleens and ascites of mice with or without PTX treatment. (G) Surface markers on CD3+CD8+ T cells on the raw t-SNE map. The locations and densities of A2AR, CTLA-4, HVEM, PD-1, TIM-3, CD69, or CD103 on CD3+CD8+ T cells were different. (H) Clusters of CD3+CD8+ T cells on the raw t-SNE map. CD3+CD8+ T cells were clustered by A2AR, CTLA-4, HVEM, PD-1, TIM-3, CD69, and CD103 expressions using PhenoGraph. Eleven clusters of CD3+CD8+ T cells could be categorized. (I) Percentages of 11 clusters of CD3+CD8+ T cells in spleens. The percentages of cluster 1, 2, 3, 4, 5, 6, 7, 8, 9, or 11 of CD3+CD8+ T cells between with and without PTX treatment groups had a significant difference. The difference with a gap ≥5.0% was identified in cluster 1 (12.0%), 2 (12.1%), 3 (7.6%), 4 (9.4%), 5 (7.5%), or 6 (19.3%). Note: # as significant differences with a gap ≥5.0%. (J) Percentages of 11 clusters of CD3+CD8+ T cells in ascites. The percentages of cluster 1, 3, 4, 6, 7, 8, 9, 10, or 11 of CD3+CD8+ T cells between with and without PTX treatment groups had a significant difference. The difference with a gap ≥5.0% was identified in cluster 3 (22.1%), 4 (7.1%), 7 (22.0%), 8 (10.7%), or 9 (24.3%). Note: # as significant differences with a gap ≥5.0%. (*p<0.05, **p<0.01, ***p<0.001, by Kruskal-Wallis test). A2AR, A2A receptor; CTLA-4, cytotoxic T-lymphocyte-associated protein 4; HVEM, herpes virus entry mediator; IFN-γ, interferon-gamma; IL, interleukin; PD-1, programmed cell death 1; PTX, paclitaxel; TALs, tumor-associated lymphocytes; TIM-3, T-cell immunoglobulin domain and mucin domain 3; TNF-α, tumor necrosis factor-alpha; t-SNE, t-distributed stochastic neighbor embedding.

    In spleens, the percentages of cluster 1, 2, 3, 4, 5, 6, 7, 8, 9, or 11 of CD3+CD8+ T cells between with and without PTX treatment groups were significantly different. A gap ≥5.0% was identified in clusters 1, 2, 3, 4, 5, and 6 (figure 1I). In ascites, the percentages of cluster 1, 3, 4, 6, 7, 8, 9, 10, or 11 of CD3+CD8+ T cells were significantly different between with and without PTX treatment groups. A gap ≥5.0% was also observed in clusters 3, 4, 7, 8, and 9 (figure 1J). Therefore, clusters 1, 2, 3, 4, 5, and 6 in spleens or clusters 3, 4, 7, 8, and 9 in ascites were included to investigate the expression of surface markers.

    Among the clusters from spleens, clusters 1, 2, and 5 were observed more in mice without PTX, but clusters 3, 4, and 6 were observed in the PTX group. Alteration of HVEM, PD-1, TIM-3, CD69, and CD103 was noted in the clusters (figure 2A,B). Among the clusters from ascites, clusters 7 and 9 were observed more in mice not treated with PTX, whereas clusters 3, 4, and 8 were observed in the PTX group. Alteration of the same markers was identified in these clusters (figure 2C,D), which was more apparent than in the spleen. Accordingly, the relationships between GZMB and these markers or those among various checkpoints in mice receiving PTX were evaluated using CD3+CD8+ T cells from the ascites.

    Figure 2
    Figure 2

    HVEM+PD-1+TIM-3+CD8+ T cells could have potent antitumor abilities. (A) Distribution of selected clusters of CD3+CD8+ T cells from spleens. Clusters 1, 2, and 5 were observed more in tumor-bearing mice without PTX treatment (left); however, clusters 3, 4, and 6 were observed in PTX treatment group (right). (B) Expression of surface markers in selected clusters of CD3+CD8+ T cells from spleens. Among these six selected clusters, alteration of HVEM, PD-1, TIM-3, CD69, and CD103 was detected. (C) Distribution of selected clusters of CD3+CD8+ T cells from ascites. Clusters 7 and 9 were observed more in tumor-bearing mice without PTX treatment (left), but clusters 3, 4, and 8 were noted in PTX treatment group (right). (D) Expression of surface markers in selected clusters of CD3+CD8+ T cells from ascites. Among these five selected clusters, alteration of HVEM, PD-1, TIM-3, CD69, and CD103 was identified, which was more apparent than in the spleen. (E) Representative flow cytometric analyses of relationships between GZMB and respective selected surface markers in tumor-bearing mice treated with PTX using CD3+CD8+ T cells from ascites. (F) Bar figures of percentages of GZMB expression in various subgroups of CD3+CD8+ T cells from ascites in tumor-bearing mice treated with PTX. More than 50.0% of CD3+CD8+ T cells had GZMB expression. GZMB could be detected in more than 80.0% of PD-1+ or TIM-3+ CD3+CD8+ T cells or more than 50.0% of HVEM+, CD69+, or CD103+ CD3+CD8+ T cells (n=5). (G) Representative flow cytometric analyses of relationships among HVEM, PD-1 and TIM-3 in tumor-bearing mice treated with PTX using CD3+CD8+ T cells from ascites. (H) Bar figures of percentages of checkpoint co-expression on various subgroups of CD3+CD8+ T cells from ascites in tumor-bearing mice treated with PTX. Almost all of the PD-1+ or TIM-3+ CD3+CD8+ T cells had HVEM co-expression. Only a portion of the HVEM-expressing CD3+CD8+ T cells had PD-1 or TIM-3 co-expression. More than 80.0% of TIM-3+CD3+CD8+ T cells co-expressed PD-1 (n=5). (I) Representative luminescence figures of ex vivo tumor killing abilities of isolated immune cells from spleens of mice receiving PTX treatment (1×104 cells/well, respective immune cells/WF-3/Luc cells=50:1). On day 35, the HVEM+ and HVEM+PD-1+TIM-3+ CD3+CD8+ T lymphocytes were collected from the splenocytes of tumor-bearing mice receiving PTX. The immune cells were co-cultured with tumor cells for 24 hours. (J) Quantification of luminescence of ex vivo tumor killing abilities of isolated immune cells from spleens of mice receiving PTX treatment. Compared with the luminescence of WF-3/Luc cells co-cultured with splenocytes or HVEM+CD3+CD8+ T cells, the least luminal activity was detected in WF-3/Luc cells with HVEM+PD-1+TIM-3+CD3+CD8+ T cells (n=5). (K) Numbers of GZMB-expressing CD3+CD8+ T cells/3×104 lymphocytes. In spleens, the number of GZMB-expressing CD3+CD8+ T cells from tumor-bearing mice with PTX treatment (238.6±23.8/3x104 lymphocytes) was greater than that of mice without treatment (146.2±13.4/3x104 lymphocytes). In ascites, the number of GZMB-expressing CD3+CD8+ T cells of mice with PTX treatment (1685.6±77.8/3x104 lymphocytes) was not different from that of mice without treatment (1951.6±390.5/3x104 lymphocytes) (five mice in each group) (*p<0.05, **p<0.01, by Kruskal-Wallis test). A2AR, A2A receptor; CTLA-4, cytotoxic T-lymphocyte-associated protein 4; GZMB, granzyme B; HVEM, herpes virus entry mediator; PD-1, programmed cell death 1; PTX, paclitaxel; TIM-3, T-cell immunoglobulin domain and mucin domain 3.

    More than 50.0% of CD3+CD8+ T cells expressed GZMB. When the CD3+CD8+ T cells were subgrouped, GZMB could be detected in more than 80.0% of PD-1+ or TIM-3+ CD3+CD8+ T cells or more than 50.0% of HVEM+, CD69+, or CD103+ CD3+CD8+ T cells (figure 2E,F). Among the three checkpoints, almost all of the PD-1+ or TIM-3+ CD3+CD8+ T cells had HVEM co-expression. Only a portion of the HVEM+CD3+CD8+ T cells had PD-1 or TIM-3 co-expression. More than 80.0% of TIM-3+CD3+CD8+ T cells co-expressed PD-1 (figure 2G,H). Ex vivo tumor killing effects of these checkpoint-expressed subpopulations from spleens of mice receiving PTX treatment were evaluated. Compared with the luminescence of WF-3/Luc cells with splenocytes or HVEM+CD3+CD8+ T cells, the least luminal activity was detected in WF-3/Luc cells with HVEM+PD-1+TIM-3+CD3+CD8+ T cells (figure 2I,J).

    The number of GZMB-expressing CD3+CD8+ T cells in spleens from mice treated with PTX was greater than that of untreated mice. However, the numbers of GZMB-expressing CD3+CD8+ T cells in ascites were not different between the two groups (figure 2K). Thus, targeting HVEM+PD-1+TIM-3+CD3+CD8+ T cells may have the potential to increase the number of GZMB-expressing CD3+CD8+ T cells in ascites and improve the therapeutic effects of PTX.

    Regulation of PD-1 or HVEM on CD3+CD8+ T lymphocytes

    In vitro expression of PD-1, CD69, or CD103 on CD3+CD8+ T cells in splenocytes treated with PTX was significantly higher than in splenocytes not treated with PTX; however, the expression of HVEM or TIM-3 was similar between these two groups (figure 3A,B). When sorted CD3+CD8+ T cells treated in vitro with or without PTX were analyzed, HVEM was detected on nearly all CD3+CD8+ T cells. Expression of PD-1, CD69, or CD103 on sorted CD3+CD8+ T cells was significantly higher with PTX treatment than without PTX; however, expression of HVEM or TIM-3 was similar between the two groups (figure 3C,D).

    Figure 3
    Figure 3

    Direct or indirect regulation of PD-1 or HVEMhigh by PTX. (A) Representative flow cytometric analyses of in vitro expression of various surface markers on CD3+CD8+ T cells in splenocytes treated with or without PTX. Splenocytes (5×105 cells) were treated with PBS or PTX (1 µM) for 48 hours and the expression of various surface markers on CD3+CD8+ T cells was evaluated. (B) Bar figures of in vitro expression of various surface markers on CD3+CD8+ T cells in splenocytes with or without PTX treatment. Expression of PD-1, CD69, or CD103 on CD3+CD8+ T cells in splenocytes treated with PTX was significantly higher than in splenocytes without PTX treatment. Expression of HVEM or TIM-3 on CD3+CD8+ T cells in splenocytes was not different between these two groups (n=5). (C) Representative flow cytometric analyses of expression of various surface markers on sorted CD3+CD8+ T cells treated in vitro with or without PTX. PBS or PTX (1 µM) was loaded with sorted T lymphocytes (5×105 cells) for 24 hours. The expression of various surface markers was analyzed. (D) Bar figures of in vitro expression of various surface markers on sorted CD3+CD8+ T cells with or without PTX treatment. HVEM was detected on nearly all CD3+CD8+ T cells. Expression of PD-1, CD69, or CD103 on CD3+CD8+ T cells was significantly higher with PTX treatment than without PTX treatment. Expression of HVEM or TIM-3 on CD3+CD8+ T cells was not different between the two groups (n=5). (E) Representative flow cytometric analyses of in vitro expression of various surface markers on sorted CD3+CD8+ T cells treated with or without several cytokines. PBS or several cytokines were loaded with sorted T lymphocytes (5×105 cells) for 24 hours. The expression of various surface markers was analyzed. (F) Bar figures of in vitro expression of various surface markers on sorted CD3+CD8+ T cells treated with or without several cytokines. Compared with sorted CD3+CD8+ T cells with PBS treatment, the expression of PD-1 was indistinguishable on CD3+CD8+ T cells with IL-10 treatment; however, lower expression of PD-1 was identified on CD3+CD8+ T cells treated with IL-2, IL-6, or IL-15. Similar results were observed in the analyses of CD69 or CD103 expression. Expression of HVEM, or TIM-3 on CD3+CD8+ T cells was not different among these groups (n=5). (G) Bar figures of the percentages of HVEMhighCD3+CD8+ T cells in sorted CD3+CD8+ T cells in vitro treated with different modalities. A higher percentage of HVEMhighCD3+CD8+ T cells was detected in the IL-2, IL-6, or IL-15 group (n=5). (H) Representative flow cytometric analyses of relationships between GZMB and HVEMhighCD3+CD8+ T cells in sorted CD3+CD8+ T cells in vitro treated with different modalities. PBS, PMA (100 ng/mL) and ionomycin (1 µg/mL), PTX (1 µM), or several cytokines were loaded with sorted T lymphocytes (5×105 cells) for 24 hours. T cells with PMA and ionomycin, TNF-α, or IFN-γ were used as positive control. Relationship between GZMB and HVEMhighCD3+CD8+ T cells was evaluated. Il-2 or IL-15 could induce more GZMB expression in HVEMhighCD3+CD8+ T cells. The percentage of HVEMhighCD3+CD8+ T cells with GZMB expression was higher in the IL-15 group than in the IL-2 group. (*p<0.05, **p<0.01, by Kruskal-Wallis test). GZMB, granzyme B; HVEM, herpes virus entry mediator; IFN-γ, interferon-gamma; IL, interleukin; PBS, phosphate buffered saline; PD-1, programmed cell death 1; PMA, phorbol myristate acetate; PTX, paclitaxel; TIM-3, T-cell immunoglobulin domain and mucin domain 3; TNF-α, tumor necrosis factor-alpha.

    Compared with sorted CD3+CD8+ T cells treated with PBS, the expression of PD-1 was indistinguishable on CD3+CD8+ T cells treated with IL-10, but lower expression of PD-1 was observed on CD3+CD8+ T cells treated with IL-2, IL-6, or IL-15. Similar results were noted in the analyses of CD69 or CD103 expression. Expression of HVEM or TIM-3 on sorted CD3+CD8+ T cells was not significantly different (figure 3E,F). When flow cytometric analysis of HVEM expression on sorted CD3+CD8+ T cells treated with or without various cytokines (figure 3E) was carried out, an alteration of the percentages of HVEMhighCD3+CD8+ T cells was identified. A higher percentage of HVEMhighCD3+CD8+ T cells was observed in the IL-2, IL-6, or IL-15 group (figure 3G). IL-2 or IL-15 could induce more GZMB expression in HVEMhighCD3+CD8+ T cells (figure 3H). The percentage of HVEMhighCD3+CD8+ T cells with GZMB expression was higher in the IL-15 group than the IL-2 group. Thus, HVEMhigh expression on CD3+CD8+ T cells could be regulated by IL-15.

    The significance of BTLA/HVEM/LIGHT pathway is still under investigation; therefore, the modulatory impacts of BTLA and LIGHT remain to be elucidated. In vitro, IL-15 could stimulate HVEMhigh expression on sorted CD3+CD8+ T cells, whereas LIGHT seemed to have no effect and BTLA inhibited it. BTLA suppressed HVEMhigh expression induced by IL-15 (figure 4A,B). In vitro, BTLA or LIGHT could promote PD-1 expression on sorted CD3+CD8+ T cells, but IL-15 inhibited it. IL-15 also inhibited PD-1 expression induced by BTLA but partially suppressed LIGHT-induced PD-1 expression (figure 4C,D).

    Figure 4
    Figure 4

    Impacts of IL-15, BTLA and LIGHT on sorted CD3+CD8+ T lymphocytes. (A) Representative flow cytometric analyses of in vitro impacts of various modalities on HVEMhigh expression on sorted CD3+CD8+ T cells. Various modalities were loaded with sorted T lymphocytes (5×105 cells) for 24 hours. The expression of HVEMhigh was analyzed. (B) Bar figures of in vitro HVEMhigh expression on sorted CD3+CD8+ T cells with various modalities. IL-15 could stimulate HVEMhigh expression on sorted CD3+CD8+ T cells, whereas LIGHT seemed to have no effects, and BTLA inhibited it. BTLA could suppress HVEMhigh expression induced by IL-15 (n=5). (C) Representative flow cytometric analyses of in vitro impacts of various modalities on PD-1 expression on sorted CD3+CD8+ T cells. Various modalities were loaded with sorted T lymphocytes (5×105 cells) for 24 hours. The expression of PD-1 was analyzed. (D) Bar figures of in vitro PD-1 expression on sorted CD3+CD8+ T cells with various modalities. BTLA, or LIGHT could increase PD-1 expression on sorted CD3+CD8+ T cells, but IL-15 inhibited it. IL-15 inhibits PD-1 expression induced by BTLA but might partially suppress PD-1 expression induced by LIGHT (n=5). (E) Representative flow cytometric analyses of in vitro relationship between HVEM and GZMB in CD3+CD8+ T cells treated with different modalities. Different modalities were loaded with sorted T lymphocytes (5×105 cells) for 24 hours. Relationship between HVEM and GZMB was evaluated. IL-15 induced GZMB expression in HVEMhighCD3+CD8+ T cells. LIGHT stimulated GZMB expression in other HVEM+CD3+CD8+ T cells but not in HVEMhighCD3+CD8+ T cells. (F) Bar figures of percentages of HVEMhighGZMB+ cells in sorted CD3+CD8+ T cells with various modalities. IL-15-contained strategies could in vitro increase the percentages of HVEMhighGZMB+ cells in CD3+CD8+ T cells, which was mainly caused by the stimulatory effect of IL-15. BTLA partially suppressed the stimulatory effect of IL-15, but LIGHT had no impact (n=5). (G) Bar figures of percentages of HVEM+GZMB+ cells in sorted CD3+CD8+ T cells with various modalities. In vitro, IL-15 combined with LIGHT could synergistically induce the highest percentage of HVEM+GZMB+ cells in CD3+CD8+ T cells (n=5). (H) Expression levels of HVEM, PD-1 and GZMB in sorted CD3+CD8+ T cells treated with IL-15, BTLA or LIGHT recombinant proteins. CD3+CD8+ T cells (1×106) were treated with PBS and different recombinant proteins and harvested after 48 hours of incubation for western blot analysis. (I) Analyses of signaling pathways in sorted CD3+CD8+ T cells treated with IL-15, BTLA or LIGHT recombinant proteins. CD3+CD8+ T cells (1×106) were prepared with serum-free media for 6 hours, then treated with PBS and different recombinant proteins and harvested after 20 min of incubation for western blot analysis. (*p<0.05, **p<0.01, by Kruskal-Wallis test). BTLA, B and T lymphocyte attenuator; GZMB, granzyme B; HVEM, herpes virus entry mediator; IL, interleukin; PBS, phosphate buffered saline; PD-1, programmed cell death 1.

    The relationship between HVEM and GZMB in CD3+CD8+ T cells undergoing different modalities was investigated in vitro. IL-15, IL-15 and BTLA, or IL-15 and LIGHT could increase the percentages of HVEMhighGZMB+ cells in CD3+CD8+ T cells, which was mainly caused by the stimulatory effect of IL-15. BTLA partially suppressed the stimulatory effect of IL-15, but LIGHT had no impact (figure 4E,F). LIGHT stimulated GZMB expression in other HVEM+CD3+CD8+ T cells but not in HVEMhighCD3+CD8+ T cells. Therefore, IL-15 combined with LIGHT had synergistic effects on inducing the highest percentage of HVEM+GZMB+ cells in CD3+CD8+ T cells (figure 4E,G).

    The impacts of IL-15, BTLA, or LIGHT on the expression level of HVEM, PD-1, and GZMB in CD3+CD8+ T cells were analyzed by Western blotting. The expression of HVEM was stimulated by IL-15 but inhibited by BTLA (figure 4H). BTLA or LIGHT upregulated the expression of PD-1, but IL-15 inhibited it. IL-15 or LIGHT could elevate the expression of GZMB. We further elucidated the possible signal transduction pathways in CD3+CD8+ T cells treated with IL-15, BTLA, or LIGHT recombinant protein (figure 4I). IL-15 mainly stimulated the phosphorylation of STAT3, STAT5, and ERK. BTLA or LIGHT upregulated the phosphorylation of STAT1, JNK, IKKα/β, and p65. The phosphorylation status of AKT was not altered by LIGHT, but inhibited by BTLA. Therefore, expression of HVEM or PD-1 on CD3+CD8+ T cells was regulated by IL-15, BTLA, or LIGHT.

    Regulation and impact of IL-15 from CD19+ B cells

    To investigate the mechanisms regulating IL-15, distribution of the molecule was examined. Increased expression of IL-15 was detected in splenocytes co-cultured with tumor cells alone or tumor cells and PTX. Expression of IL-15 was higher in splenocytes treated with tumor cells and PTX (55.6±4.1%) than tumor cells alone (48.8±3.9%; figure 5A,B). Figure 5C presents the representative flow cytometric analyses of CD11c+, CD19+, or NK1.1+ cells in IL-15-secreting splenocytes induced by tumor cells and PTX. The percentage of CD19+ cells (80.8±4.4%) was highest in the IL-15-secreting splenocytes (figure 5D).

    Figure 5
    Figure 5

    Regulation of IL-15 expression through TLR4 and TLR9 pathway activation. (A) Representative flow cytometric analyses of in vitro impacts of various manipulations on IL-15 expression in splenocytes. Splenocytes (5×105 cells) were co-cultured with PBS, WF-3/Luc tumor cells (1×105 cells), or WF-3/Luc tumor cells (1×105 cells) and PTX (1 µM) for 48 hours and the expression of IL-15 was analyzed. (B) Bar figures of IL-15 expression in splenocytes with various manipulations. When splenocytes were in vitro co-cultured with tumor cells alone or tumor cells and PTX, increased expression of IL-15 was detected in splenocytes. Expression of IL-15 was higher in splenocytes treated with tumor cells and PTX than tumor cells alone (n=5). (C) Representative flow cytometric analyses of CD11c+, CD19+, or NK1.1+ cells in IL-15-secreting splenocytes induced by tumor cells and PTX. Splenocytes (5×105 cells) were co-cultured with WF-3/Luc tumor cells (1×105 cells) and PTX (1 µM) for 48 hours and percentages of CD11c+, CD19+, or NK1.1+ cells in the IL-15-secreting splenocytes were evaluated. (D) Bar figures of expression of CD11c+, CD19+, or NK1.1+ cells in IL-15-secreting splenocytes induced by tumor cells and PTX. The percentage of CD19+ cells was highest in these IL-15-secreting splenocytes (n=5). (E) Representative flow cytometric analyses of in vitro impact of PTX, LPS, or CpG-ODN on IL-15 expression in sorted CD19+ B cells. Sorted B lymphocytes (5×105 cells) were treated with PBS, PTX (1 µM), LPS (0.5 µg/mL), or CpG-ODN (1 µg/mL) for 24 hours. The expression of IL-15 was evaluated. (F) Bar figures of IL-15 expression in sorted CD19+ B cells in vitro treated with PTX, LPS, or CpG-ODN. PTX could not increase IL-15 expression in sorted CD19+ B cells, but LPS or CpG-ODN could (n=5). (G) Representative flow cytometric analyses of in vitro impact of PTX, LPS, or CpG-ODN on BTLA expression on sorted CD19+ B cells. Sorted B lymphocytes (5×105 cells) were treated with PBS, PTX (1 µM), LPS (0.5 µg/mL), or CpG-ODN (1 µg/mL) for 24 hours. The expression of BTLA was analyzed. (H) Bar figures of BTLA expression on sorted CD19+ B cells in vitro treated with PTX, LPS, or CpG-ODN. PTX inhibited BTLA expression; however, LPS or CpG-ODN could increase it on sorted CD19+ B cells (n=5). (I) Representative flow cytometric analyses of relationship between BTLA and IL-15 in sorted CD19+ B cells in vitro treated with PTX, LPS, or CpG-ODN. Sorted B lymphocytes (5×105 cells) were treated with PBS, PTX (1 µM), LPS (0.5 µg/mL), or CpG-ODN (1 µg/mL) for 24 hours. Relationship between BTLA and IL-15 was evaluated. BTLA was detected on more than 50.0% of IL-15-expressing CD19+ B cells with LPS or CpG-ODN treatment. (J) Bar figures of percentages of BTLA+IL-15+ cells in sorted CD19+ B cells in vitro treated with PTX, LPS, or CpG-ODN. The percentage of BTLA+IL-15+ cells was higher in sorted CD19+ B cells treated with LPS or CpG-ODN than those with PTX treatment (n=5). (*p<0.05, **p<0.01, by Kruskal-Wallis test). BTLA, B and T lymphocyte attenuator; CpG-ODN, CpG-oligodeoxynucleotide; IL, interleukin; LPS, lipopolysaccharide; PBS, phosphate buffered saline; PTX, paclitaxel; TLR, Toll-like receptor.

    Killing tumor cells with chemotherapeutic agents can enhance the release of damage-associated molecular patterns (DAMPs). Toll-like receptors (TLRs) are the major DAMP receptors.20 PTX could not increase IL-15 expression in sorted CD19+ B cells, but LPS (for TLR4 pathway) or CpG-ODN (for TLR9 pathway) could (figure 5E,F). PTX inhibited BTLA expression; however, LPS or CpG-ODN could increase it on sorted CD19+ B cells (figure 5G,H). BTLA was present on more than 50.0% of IL-15-expressing CD19+ B cells treated with LPS or CpG-ODN (figure 5I). The percentage of BTLA+IL-15+ cells was higher in sorted CD19+ B cells treated with LPS or CpG-ODN than those treated with PTX (figure 5J).

    The secretion of IL-15 from B cells in the LPS or CpG-ODN group was higher than that in the PBS group, which was similar to the regulation of IL-15/IL-15Rα complex secretion (figure 6A,B). Compared with the PBS group, the percentage of IL-15/IL-15Rα complex-expressing B cells was higher in the LPS or CpG-ODN group (figure 6C,D). With the treatment of LPS or CpG-ODN, significant expression of IL-15 and IL-15/IL-15Rα complex was detected in B lymphocytes by western blotting (figure 6E). When the sorted CD3+CD8+ T lymphocytes were in vitro treated with IL-15 or IL-15/IL-15Rα complex, percentage of GZMB, perforin, TNF-α, IFN-γ, or CD107a-expressing CD3+CD8+ T cells in IL-15 or IL-15/IL-15Rα complex group was higher than that in PBS group (figure 6F,G). Therefore, IL-15 and IL-15/IL-15Rα complexes produced by B cells via TLR pathways could activate CD3+CD8+ T cells.

    Figure 6
    Figure 6

    Regulation of IL-15 and IL-15/IL-15Rα complex derived from B cells and their effects on CD3+CD8+ T cells. (A) Secretion of IL-15 from sorted CD19+ B cells in vitro treated with LPS or CpG-ODN. Supernatants of cultured B lymphocytes (2×106 cells) with LPS (0.5 µg/mL) or CpG-ODN (1 µg/mL) for 24, 48, and 72 hours were prepared for measuring IL-15 levels. Compared with PBS group, concentration of IL-15 was higher in LPS or CpG-ODN group at indicated time (n=5). (B) Secretion of IL-15/IL-15Rα complex from sorted CD19+ B cells in vitro treated with LPS or CpG-ODN. Supernatants of cultured B lymphocytes (2×106 cells) with LPS (0.5 µg/mL) or CpG-ODN (1 µg/mL) for 24, 48, and 72 hours were prepared for measuring IL-15/IL-15Rα complex levels. Compared with PBS group, concentration of IL-15/IL-15Rα complex was higher in LPS or CpG-ODN group at indicated time (n=5). (C) Representative flow cytometric analyses of in vitro impact of LPS or CpG-ODN on IL-15/IL-15Rα complex expression on sorted CD19+ B cells (72 hours). (D) Bar figures of IL-15/IL-15Rα complex expression on sorted CD19+ B cells in vitro treated with LPS or CpG-ODN. Sorted B lymphocytes (5×105 cells) were treated with LPS (0.5 µg/mL) or CpG-ODN (1 µg/mL) for 24, 48, and 72 hours. Expression of IL-15/IL-15Rα complex on B lymphocytes was analyzed. Compared with PBS group, percentage of IL-15/IL-15Rα complex-expressing B lymphocytes was higher in LPS or CpG-ODN group at indicated time (n=5). (E) Expression levels of IL-15 and IL-15/IL-15Rα complex in sorted CD19+ B cells treated with LPS or CpG-ODN. With the treatment of LPS (0.5 µg/mL) or CpG-ODN (1 µg/mL), significant expression of IL-15 (24 hours) and IL-15/IL-15Rα complex (48 hours) could be detected in B lymphocytes by western blotting. (F) Representative flow cytometric analyses of GZMB expression in sorted CD3+CD8+ T cells in vitro treated with IL-15 or IL-15/IL-15Rα complex. IL-15 (30 ng/mL) or IL-15/IL-15Rα complex (100 ng/mL) was loaded with sorted T lymphocytes (5×105 cells) for 24 hours. T cells with PMA (100 ng/mL) and ionomycin (1 µg/mL) were used as positive control. GZMB expression in CD3+CD8+ T cells was evaluated. (G) Bar figures of expression of various cytotoxic markers in sorted CD3+CD8+ T cells in vitro treated with IL-15 or IL-15/IL-15Rα complex. Compared with PBS group, percentage of GZMB, perforin, TNF-α, IFN-γ, or CD107a-expressing CD3+CD8+ T lymphocytes was higher in IL-15 or IL-15/IL-15Rα complex group (n=5). (*p<0.05, **p<0.01, by Kruskal-Wallis test). CpG-ODN, CpG-oligodeoxynucleotide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GZMB, granzyme B; HVEM, herpes virus entry mediator; IFN-γ, interferon-gamma; IL, interleukin; IL-15Rα, IL-15 receptor alpha; LPS, lipopolysaccharide; PBS, phosphate buffered saline; PMA, phorbol myristate acetate; TNF-α, tumor necrosis factor-alpha.

    Therapeutic effect of disseminated tumors was improved by PTX combined with dual inhibition of BTLA and PD-1

    Putative mechanisms from our overall analyses are illustrated in figure 7A. However, certain proportions of BTLA-expressing or PD-1-expressing immune cells remained in the lymphocytes of tumor-bearing mice treated with PTX (figure 7B). In splenic lymphocytes, the percentages varied between PD-1-expressing CD3+CD8+ T cells (10.0±1.2%) and BTLA-expressing CD19+ B cells (42.2±5.8%). The percentages of these two cells were similar in TALs from ascites (PD-1-expressing CD3+CD8+ T cells, 50.7±6.6%; BTLA-expressing CD19+ B cells, 48.2±4.3%). Therefore, the preclinical potential of PTX combined with anti-PD-1 Ab, anti-BTLA Ab, or both should be considered to elevate the therapeutic effects of intraperitoneally disseminated tumors.

    Figure 7
    Figure 7

    Therapeutic effects of PTX combined with dual inhibition of BTLA and PD-1 on disseminated tumors. (A) Schematic diagram showing possible regulation in this study. (B) Distribution of BTLA-expressing or PD-1-expressing cells in the lymphocytes of tumor-bearing mice with PTX treatment. In splenic lymphocytes, the percentages varied between PD-1-expressing CD3+CD8+ T cells and BTLA-expressing CD19+ B cells. The percentages of these two cells were similar in TALs from ascites (five mice in the analysis). (C) Representative figures of in vitro tumoricidal activities of various treatment modalities. Splenocytes (2×106 cells/well) were co-cultured with WF-3/Luc cells (5×105 cells/well). PTX (1 µM) was added and incubated with cells for 24 hours. For combinational treatment, anti-PD-1 Ab (100 µg/mL), anti-BTLA Ab (100 µg/mL), or both was administered and incubated with cells for another 24 hours. (D) Quantification of luminescence of in vitro tumor killing abilities of various treatment modalities. When tumor cells were co-cultured with splenocytes, PTX, anti-PD-1 Ab, and anti-BTLA Ab, the least luminal activity of WF-3/Luc cells was detected (n=5). (E) Bar figures of GZMB or perforin expression in CD3+CD8+ T cells in vitro treated with various modalities. Among these groups, when tumor cells were treated with splenocytes, PTX, anti-PD-1 Ab, and anti-BTLA Ab, the highest percentages of GZMB or perforin expression was identified in CD3+CD8+ T cells (n=5). (F) Diagrammatic representation of different in vivo treatment protocols using PTX alone or combined with different ICBs. (G) Representative luminescence images of tumor-bearing mice undergoing PTX alone or combined with different ICBs (five mice in each group). (H) Luminal analyses of tumor volumes in tumor-bearing mice undergoing PTX alone or combined with different ICBs. Among these treatment strategies, mice undergoing PTX, anti-PD-1 Ab, and anti-BTLA Ab (group D) exhibited the lowest luminescence. (I) Survival analysis of tumor-bearing mice undergoing PTX alone or combined with different ICBs. None of the mice in group B could survive 75 days after the tumor challenge. Sixty percent of mice in group C and all mice in group D were alive 100 days after the tumor challenge. (J) Representative flow cytometric analyses of percentages of CD3+CD8+ T cells in splenic lymphocytes (five mice in each group). (K) Bar figures of percentages of CD3+CD8+ T cells in splenic lymphocytes. The percentage of CD3+CD8+ T cells was higher in splenic lymphocytes from mice in group D than the mice in other groups. (L) Bar figures of numbers of GZMB or perforin-expressing CD3+CD8+ T cells / 3×104 splenic lymphocytes. The numbers of GZMB or perforin-expressing CD3+CD8+ T cells were greater in tumor-bearing mice treated with PTX and different ICBs than PTX alone. (M) Bar figures of numbers of GZMB or perforin-expressing CD3+CD8+ T cells / 3×104 TALs of ascites. The numbers of GZMB or perforin-expressing CD3+CD8+ T cells were greater in mice treated with PTX and different ICBs than PTX alone. (*p<0.05, **p<0.01, by a logrank test for survival analysis and Kruskal-Wallis test for other analyses). Ab, antibody; BTLA, B and T lymphocyte attenuator; DAMPs, damage-associated molecular patterns; GZMB, granzyme B; HVEM, herpes virus entry mediator; ICBs, immune checkpoint blockades; IFN-γ, interferon-gamma; IL, interleukin; IL-15Rα, IL-15 receptor alpha; PBS, phosphate buffered saline; PD-1, programmed cell death 1; PD-L1, programmed death-ligand 1; PTX, paclitaxel; TALs, tumor-associated lymphocytes; TLR, Toll-like receptor; TNF-α, tumor necrosis factor-alpha.

    When tumor cells were co-cultured with splenocytes, PTX, anti-PD-1 Ab, and anti-BTLA Ab, the least luminal activity of WF-3/Luc cells was detected in vitro (figure 7C,D). In this group, the highest percentages of GZMB or perforin expression were identified in CD3+CD8+ T cells (figure 7E). The therapeutic efficacy of PTX alone or combined with ICB against disseminated tumors was investigated in vivo using various protocols (figure 7F). Among these treatment groups, mice in group D exhibited the lowest luminescence (figure 7G,H). None of the mice in group B could survive 75 days after the tumor challenge. Sixty percent of mice in group C and all mice in group D were alive 100 days after the tumor challenge (figure 7I).

    The percentage of CD3+CD8+ T cells was higher in splenic lymphocytes from mice in group D than the mice in other groups (figure 7J,K). The numbers of GZMB or perforin-expressing CD3+CD8+ T cells were greater in lymphocytes of spleens (figure 7L) or ascites (figure 7M) from mice treated with PTX and ICB than PTX alone. In addition to HVEM, TIM-3, CD69, and CD103, alteration of A2AR and CTLA-4 was observed among the selected clusters of CD3+CD8+ T cells from spleens and ascites in mice undergoing PTX, anti-PD-1 Ab and anti-BTLA Ab (group D) (online supplemental figure 1).

    Supplemental material

    Discussion

    In this study, the effective HVEM+PD-1+TIM-3+cytotoxic T-cell subpopulation was identified in tumor-bearing hosts treated with PTX. In addition to PTX and PTX treatment-associated cytokines, the molecules BTLA or LIGHT interacting with HVEM could regulate the expression of PD-1 on CD3+CD8+ T cells. IL-15 increased the percentage of HVEMhighGZMB+ cells in CD3+CD8+ T cells, which was suppressed by the BTLA/HVEM signal. LIGHT increased the percentage of HVEM+GZMB+ cells but not HVEMhighGZMB+ cells in CD3+CD8+ T cells. Expression of IL-15, BTLA, or LIGHT was detected in CD19+ B cells and regulated through DAMP/TLR interactions. In the tumor-bearing hosts receiving PTX, certain proportions of BTLA-expressing or PD-1-expressing immune cells remained in the lymphocytes. When BTLA/HVEM and PD-1/programmed death-ligand 1 (PD-L1) pathways were targeted via dual inhibition of BTLA and PD-1, the therapeutic effects of PTX on tumors with intraperitoneal spread significantly improved.

    The challenging obstacle in the immune escape cascade is the immunosuppressive TME.21 Tumors can induce certain inhibitory checkpoint pathways as a mechanism of immune resistance, particularly against the cytotoxic effects of T cells.5 Inhibition of the PD-1/PD-L1 signaling pathway has been clinically approved for the treatment of multiple types of tumors.22 However, not all cancer sites, including EOC, respond well to blockade of the PD-1/PD-L1 pathway alone.3 23 This experience indicates that additional immunoregulatory mechanisms exist in the development of different tumors. Therefore, therapies for these diseases with poor responsiveness to blocking PD-1/PD-L1 signaling may be improved by modulating other checkpoint pathways.

    In this study, the expression of HVEM on CD3+CD8+ T cells was much higher than the expression of PD-1 on CD3+CD8+ T cells, indicating that the HVEM-related checkpoint pathway may be another candidate for T-cell regulation (figure 3D). The possible modulatory mechanism is that the HVEM network delivers bidirectional signals regulating T-cell activation depending on the ligand that is engaged. The binding of BTLA to HVEM has a co-inhibitory effect, but the interaction between LIGHT and HVEM has a costimulatory role.24 This study also showed that IL-15 has stimulatory effects on the percentage of HVEMhighGZMB+ cells among CD3+CD8+ T cells, which was suppressed by the BTLA/HVEM signal. IL-15 and LIGHT could synergistically induce HVEM+GZMB+ cells in CD3+CD8+ T cells through different signaling pathways (figure 4E–I).

    The secretion of detectable IL-15 protein appears to be restricted mostly to monocytes, DCs, epithelial cells, bone marrow stromal cells, and fibroblasts.25–27 In this study, IL-15 expression levels were low in naïve splenocytes but induced in splenocytes co-cultured with tumor cells or tumor cells and PTX. The IL-15 level was higher in splenocytes with tumor cells and PTX than tumor cells alone. In splenocytes with tumor cells and PTX, IL-15 proteins were mainly identified in CD19+ B cells, which could be modulated by DAMP/TLR interactions (figure 5A–F). Similar to previous studies,28 29 we found that IL-15 had a regulatory effect on cytotoxic T cells through the phosphorylation of STAT3, STAT5, and ERK (figure 4H,I). In addition, IL-15/IL-15Rα complexes could be induced from B cells through TLR pathways. IL-15 and IL-15/IL-15Rα complexes could activate CD3+CD8+ T cells (figure 6). In the previous report, transfection with a chimeric construct linking IL-15 to IL-15Rα can elevate the CD8+ T-cell function for adoptive T-cell immunotherapy.30

    LIGHT is primarily noted on inflammatory effector cells, including DCs, NK cells, macrophages, neutrophils, innate lymphoid cells, NK T cells and activated effectors, and CD4+ and CD8+ memory T cells, suggesting a role in acute inflammatory and adaptive immune responses.31–33 The highest LIGHT expression in the current study was induced on splenocytes treated with tumor cells and PTX. CD11c expression on B cells has been reported.34 In our analysis, regulation of LIGHT expression was observed in the CD11c+CD19+ B cell subpopulation through the interaction between DAMPs and the TLR signaling pathways (online supplemental figure 2A-I). Cytotoxic T cells were modulated by LIGHT through the phosphorylation of STAT1, JNK, IKKα/β, and p65 (figure 4H,I).

    Supplemental material

    In our previous and other studies, BTLA was expressed on CD19+ B cells.10 35 This study showed that expression of the inhibitory checkpoint on B cells is regulated by DAMP/TLR interactions (figure 5G,H). BTLA co-expressed with IL-15 (figure 5J) or LIGHT (online supplemental figure 2M) on B cells could also be influenced by the same pathways. BTLA generated an inhibitory effect on cytotoxic T cells stimulated by IL-15 (figure 4F). Another report showed that LIGHT binding to HVEM enhances the opportunity for BTLA to bind HVEM, which can suppress LIGHT/HVEM signaling-mediated T-cell activation.36 BTLA regulated cytotoxic T cells through upregulated phosphorylation of STAT1, JNK, IKKα/β, and p65 but downregulated phosphorylation of AKT (figure 4H,I). In principle, CD8+ T cells could be activated by B cells directly through cross-presentation or indirectly through CD4+ T-cell activation and subsequent actions of the CD4+ T cells.37 Our study implies that, during tumorigenesis, interactions between B cells and cytotoxic T cells may be involved in the cytokine and immune checkpoint mechanisms.

    Clinically apparent tumors, including intraperitoneally disseminated tumors, have the ability to evade immune elimination. Chemotherapy is commonly applied to treat these malignancies. This strategy not only possesses cytotoxic abilities, but also modulates antitumor T-cell responses by reinforcing tumor antigenicity, inducing immunogenic cell death, disrupting immune suppressive signals, and enhancing the effector T-cell response.38–41 In the tumor-bearing hosts treated with PTX, our analysis showed that immune checkpoints could be expressed individually or co-expressed on cytotoxic T cells. The tumoricidal activities of these checkpoint-expressing subpopulations isolated from hosts treated with PTX were effective ex vitro (figure 2G–J). When the expression of various cytotoxic markers in HVEM+PD-1+TIM-3+ CD3+CD8+ T cells was further investigated, more than 50.0% of these cells expressing GZMB, TNF-α, IFN-γ or CD107a were observed indeed (online supplemental figure 3). However, the tumor-killing abilities were possibly suppressed by interactions with immune checkpoints in the TME. These cytotoxic T cells, especially those expressing multiple immune checkpoints, have been thought of as exhausted in TME and associated with disease progression.42 43

    Supplemental material

    In addition to the HVEM-related checkpoint pathway, IL-15, BTLA, and LIGHT can modulate PD-1 expression on CD3+CD8+ T cells (figure 4). Certain proportions of BTLA-expressing or PD-1-expressing immune cells remained in the lymphocytes of tumor-bearing hosts receiving PTX (figure 7B). The possibility of overcoming immune inhibition by dual inhibition of BTLA and PD-1 may be higher than with single blockade, which can enhance the efficacy of PTX. In our animal model, the numbers of GZMB or perforin-expressing CD3+CD8+ T cells were greater in spleens and ascites from mice treated with PTX and ICB than mice treated with PTX alone. The mice (group D) receiving chemotherapy combined with dual inhibition of BTLA and PD-1 had the lowest peritoneal tumor volumes and best survival outcomes (figure 7F–M). Therefore, ICB can repolarize the TME to improve the proimmune effects of cancer treatment modalities, including chemotherapy.21

    Conclusions

    In conclusion, the HVEM-related checkpoint pathway plays a role in the immune regulation of PTX-treated tumor-bearing hosts. IL-15, BTLA, and LIGHT are involved in this pathway and expressed in CD19+ B cells, modulating PD-1 expression on CD3+CD8+ T cells. Certain proportions of BTLA-expressing or PD-1-expressing immune cells remained in the lymphocytes of tumor-bearing hosts treated with PTX. The addition of anti-BTLA Ab and anti-PD-1 Ab to PTX could generate potent antitumor effects in an animal model. Therefore, dual inhibition of BTLA and PD-1 combined with PTX may have clinical potential for the treatment of intraperitoneally disseminated tumors, including EOC.

    Supplemental material

    Data availability statement

    Data are available upon reasonable request.

    Ethics statements

    Patient consent for publication

    Ethics approval

    Not applicable.

    Acknowledgments

    This work was also supported by (1) Instrumentation Center, College of Science, National Taiwan University, (2) Laboratory Animal Center, College of Medicine, National Taiwan University, (3) the 3rd and 7th core laboratory facilities of Department of Medical Research, National Taiwan University Hospital, and (4) the service provided by the Flow Cytometric Analyzing and Sorting Core Facility at National Taiwan University Hospital.

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    Supplementary materials

    Footnotes

    • W-ZS and H-WL contributed equally.

    • Contributors W-ZS and H-WL designed and performed all experiments and contributed to the data analyses and paper writing. W-YC performed experiments and analyzed data in the animal tumor model. C-LC performed experiments involving the regulation of immune molecules in sorted lymphocytes. Y-LL and JC participated in the experimental design and t-SNE data analyses. Y-LC designed the experiments, analyzed the data, wrote the paper, and supervised the project. W-FC supervised the project. Y-LC acts as the guarantor.

    • Funding This study was supported by grants from the National Science and Technology Council of Taiwan (109-2314-B-002-119-MY3, 110-2314-B-002-058-MY3, and 110-2314-B-002-079-MY3) and National Taiwan University Hospital (110-N4926 and 112-S0122).

    • Competing interests None declared.

    • Provenance and peer review Not commissioned; externally peer reviewed.

    • Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.