Interferon-α and thymosin-α1 plus tislelizumab enhance CD8⁺ T cell cytotoxicity toward pancreatic ductal adenocarcinoma

Summary

The strong immunosuppression and immune evasion of pancreatic ductal adenocarcinoma (PDAC) result in poor efficacy of immune checkpoint blockade. In this study, the PD-1 level on CD8⁺ T cells in the peripheral blood of patients with PDAC was significantly greater than that in the peripheral blood of healthy individuals. To enhance the anticancer activity of adoptive CD8⁺ T cells toward PDAC, interferon-α (IFN-α) and thymosin-α1 (Tα1) plus tislelizumab were preclinically explored. Compared with those of tislelizumab monotherapy, the proliferation and cytokine secretion of CD8⁺ T cells and the cytotoxic activity toward PDAC cells were significantly greater with the combination treatment of IFN-α and Tα1 plus tislelizumab. Moreover, the growth of PDAC tumors was inhibited by CD8⁺ T cells with high efficacy under the combination treatment. Thus, IFN-α and Tα1 plus tislelizumab enhance the anticancer activity of CD8⁺ T cells toward PDAC, representing an alternative strategy for improving cancer immunotherapy.

Graphical abstract

Highlights

• •High level of PD-1 on CD8⁺ T cells is associated with the malignant transformation of PDAC
• •IFN-α and Tα1 plus tislelizumab enhance the proliferation and cytokine secretion of CD8⁺ T cells
• •CD8⁺ T cells treated with IFN-α and Tα1 plus tislelizumab inhibits PDAC tumor growth

Immunology; Cancer

Introduction

Pancreatic cancer (PC) is the third leading cause of death in patients with malignant tumors. With the increase in mortality, PC may rank as the second leading cause of cancer death in 2030.^1,2 Moreover, the five-year overall survival rate of PC is currently 13%.³ In addition to the difficulty in early diagnosis, rapid metastasis also makes PC a fatal disease,⁴ leading to a 5-year overall survival rate of only 3% for patients with PC with distant metastasis.³ Although surgical resection remains the most effective treatment for early-diagnosed and locally resectable PC, the prognosis of patients with PC postsurgery is still poor, with a 5-year overall survival rate of only 20%.⁵ Pancreatic ductal adenocarcinoma (PDAC) is the most common pathological type of PC, accounting for approximately 80% of all pathological types.⁶ However, nearly 80% of patients with PDAC who present with locally advanced or metastatic characteristics are not suitable for therapeutic surgery, making systemic therapy the mainstay treatment for PDAC.⁷ The development of therapeutic methods for treating PDAC is urgently needed.

Programmed cell death protein 1 (PD-1) is an apoptosis-associated gene that is expressed mainly on activated T cells, B cells, and NK cells.^8,9,10 The interaction between programmed cell death ligand 1 (PD-L1) on cancer cells and PD-1 on immune cells blocks the cytotoxic immune response.^11,12,13 Thus, PD-1/PD-L1 signaling is a key factor in sustaining immunosuppressive function, and neutralizing PD-1 is a useful method for improving the anticancer effect of T cells.^14,15 A variety of PD-1-blocking antibodies, such as nivolumab, pembrolizumab, cemiplimab, and dostarlimab, have been approved by the US Food and Drug Administration (FDA) for the treatment of more than 20 tumor types.^16,17 Tislelizumab is a PD-1 monoclonal IgG4 antibody developed by BeiGene in China and is approved primarily for patients with relapsed or refractory classical Hodgkin’s lymphoma after second-line chemotherapy and multiple types of solid tumors.¹⁸ Considering the poor efficacy of PC in surgical treatment and chemotherapy, immunotherapy has become a new option for PC treatment. However, most PDACs have strong immunosuppressive networks, limiting the ability of the immune system to actively eradicate disease.¹⁹ The strong immunosuppression and immune evasion of PDAC greatly limit the therapeutic efficacy of PD-1 inhibitor monotherapy.

Interferons (IFNs) are a family of cytokines widely expressed by host cells in response to viral infection and injury stress.²⁰ By acting on host cells, including CD8⁺ T cells, dendritic cells (DCs) and natural killer (NK) cells, IFN-α contributes to anticancer immunity.²¹ Combination treatments of PD-1/PD-L1 inhibitors and IFN-α are alternative options for improving the immunotherapy efficacy of cancer.²² Thymosin-α1 (Tα1) is a polypeptide hormone produced by thymic epithelial cells that may increase the number of T cells, support T cell differentiation and maturation, and alleviate T cell apoptosis.^23,24,25 In addition to acting as a cytokine for antiviral and immune enhancement, Tα1 is also used to treat immune deficiency diseases by promoting the reaction between DCs and antibodies, downregulating thymocyte apoptosis, and increasing cytokine and chemokine secretion.²⁶ A key advantage of Tα1 for clinical use is its recognized biosafety, as it has been demonstrated to be free of side effects and features a high dose tolerance.²⁷ Tα1 improves the cytotoxic response of T cells and plays an important role in combination therapy for malignant tumors. Gemcitabine combined with Tα1 inhibits the progression of NK/T lymphoma in vitro and in vivo.²⁸ Compared with monotherapy, combination treatment with Tα1 and IRX-2 is used in the hydrocortisone-mediated reduction recovery process, which more efficiently increases the T cell number.²⁹ In a melanoma lung metastasis model, tumor growth and the metastasis rate were attenuated when Tα1 was used together with an anti-PD-1 antibody.³⁰ However, whether IFN-α and Tα1 together can promote the response to PD-1 inhibitors is unclear.

The tumor microenvironment (TME) of PDAC is composed mainly of stromal cells and extracellular matrix components.³¹ The main stromal cells responsible for PDAC progression are pancreatic stellate cells (PSCs), myelogenous suppressor cells (MDSCs), regulatory T cells (Tregs), and tumor-associated macrophages (TAMs), which secrete extracellular components, including growth factors, transforming growth factor β (TGF-β), the extracellular matrix (ECM), and matrix metalloproteinases (MMPs).³² The unique genomic landscape of PDAC formed by carcinogenic drivers promotes immunosuppression from the early stage of tumorigenesis to destroy adaptive T cell immunity³³; thus, among malignances, immune checkpoint blockade (ICB) therapy for PDAC is generally ineffective. An increased PD-1 level results in T cell failure, which is characterized by decreased T cell effector function and proliferation.³⁴ Although monotherapy with PD-1 inhibitors is hindered by immune evasion, combination immunotherapy mobilizes the therapeutic potential of CD8⁺ T cells and ICBs, which are PD-1 inhibitors, in patients with PDAC.³⁵ In the present study, a combination treatment consisting of IFN-α and Tα1 plus tislelizumab was designed to enhance the anticancer activity of adoptive CD8⁺ T cells for PDAC treatment. The findings of this study may provide alternative strategies for improving the immune therapeutic efficacy in patients with PDAC.

Results

High levels of programmed cell death protein 1 on CD8⁺ T cells in the peripheral blood indicate strong immunosuppression in the pancreatic ductal adenocarcinoma microenvironment

To determine the regulatory effect of PD-1 on CD8⁺ T cells in PDAC, peripheral blood samples derived from 40 patients with PDAC and 36 healthy individuals were collected to isolate CD8⁺ T cells. Through flow cytometry, the surface PD-1 level on CD8⁺ T cells in the peripheral blood of patients with PDAC was found to be significantly greater than that in healthy individuals (36.75 ± 6.29 versus 20.45 ± 4.43, p < 0.0001) (Figures 1A and B). Thus, high levels of PD-1 on CD8⁺ T cells may be closely associated with the development and progression of PDAC. Statistically, the protein level of PD-1 on CD8⁺ T cells in peripheral blood was significantly greater in patients with PDAC with undifferentiation and poor differentiation than in those with moderate and high differentiation (39.75 ± 5.13 versus 33.09 ± 5.70, p < 0.001) (Figure 1C), which was also significantly greater in patients with PDAC at stages III and IV than in those at stages I and II (40.37 ± 5.05 versus 34.59 ± 5.97, p < 0.01) (Figure 1D). These results suggest that the PD-1 level on CD8⁺ T cells in the peripheral blood may be positively associated with the TNM stage and negatively associated with the differentiation status of patients with PDAC, and that a high level of PD-1 on CD8⁺ T cells in the peripheral blood may indicate the progression and metastasis of PDAC. We determined the relationships between PD-1 levels on CD8⁺ T cells and the clinicopathological characteristics of patients with PDAC via chi-square analysis. A high level of PD-1 on CD8⁺ T cells might suggest undifferentiated and poorly differentiated (p = 0.024), elevated node (N) stages (p = 0.011), and enhanced tumor node metastasis (TNM) stages (p = 0.028) of PDAC (Table 1). We suggest that PD-1 expression on CD8⁺ T cells may reflect the level of tumor immunosuppression, with higher levels of PD-1 indicating greater immunosuppression in higher-grade tumors.

Figure 1.

Increased PD-1 expression on CD8⁺ T cells in the peripheral blood of patients with PDAC

(A) Flow cytometry analysis of PD-1 levels on CD8⁺ T cells in the peripheral blood of healthy individuals and patients with PDAC.

(B) Scatterplots showing the significant difference in PD-1 levels on CD8⁺ T cells in the peripheral blood between healthy individuals and patients with PDAC.

(C and D) Scatterplots show the significant difference in PD-1 expression on CD8⁺ T cells in the peripheral blood of patients with PDAC with different levels of differentiation (C) and TNM stages (D).

The data were analyzed by two-sided Student’s t tests and are presented as the means ± SDs. ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001.

Table 1.

Correlation between the PD-1 level on CD8⁺ T cells in the peripheral blood of patients with PDAC and the clinicopathological characteristics

Clinicopathological characteristics	Cases	PD-1 level		p
		Low	High
Age (years)
<55	16	9	7	0.70
≥55	24	12	12
Sex
Male	23	13	10	0.55
Female	17	8	9
Differentiation
Moderate and high differentiations	18	13	5	0.024
Undifferentiation and poor differentiation	22	8	14
Tumor site
Head of pancreas	27	14	13	0.91
Body and tail of pancreas	13	7	6
N stage
N0	19	14	5	0.011
N1	21	7	14
TNM stage
I–II	24	16	8	0.028
III–V	16	5	11

Interferon-α and thymosin-α1 facilitate the ability of tislelizumab to decrease programmed cell death protein 1 levels on CD8⁺ T cells

To determine whether anti-PD-1 antibodies can enhance the immune function of T cells in PDAC, CD8⁺ T cells with high levels of surface PD-1 derived from the peripheral blood of patients with PDAC were isolated and incubated with different concentrations of tislelizumab. After cultivation for 7 days, the PD-1 level on CD8⁺ T cells derived from peripheral blood gradually decreased with increasing concentrations of tislelizumab and the positive control pembrolizumab but did not change with negative control IgG4 treatment (Figure 2A). Thus, tislelizumab has an excellent effect on decreasing PD-1 levels on active CD8⁺ T cells.

Figure 2.

Regulatory effects of tislelizumab, IFN-α, and Tα1 on the surface PD-1 expression of CD8⁺ T cells

(A) Statistical analysis of surface PD-1 expression on CD8⁺ T cells after incubation with different concentrations of hIgG4, pembrolizumab, or tislelizumab for 24 h, as assayed by flow cytometry.

(B) Flow cytometry analysis of PD-1 levels on CD8⁺ T cells after incubation with different concentrations of tislelizumab in the presence or absence of IFN-α and Tα1.

(C) Statistical analysis of PD-1 levels in CD8⁺ T cells, as examined by flow cytometry in (B).

The data were analyzed by one-way ANOVA and are presented as the means ± SDs of four biological replicates. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.

To test the influence of IFN-α and Tα1 on PD-1 expression in PDAC, activated CD8⁺ T cells were cultivated in culture medium supplemented with 1000 U/ml of IFN-α and 10 μg/mL of Tα1 on Day 1, and PD-1 levels on CD8⁺ T cells were examined on Day 7 by flow cytometry (Figure 2B). Compared with that of the blank control, the PD-1 level on CD8⁺ T cells was significantly decreased by treatment with IFN-α and Tα1 (16.30 ± 0.82 versus 22.68 ± 1.86, p < 0.0001) (Figures 2B and 2C). To maximize the suppression of PD-1 expression, IFN-α and Tα1 were added to the culture medium of active CD8⁺ T cells along with tislelizumab (mAb). The PD-1 levels on CD8⁺ T cells in the group treated with 1 μg/mL mAb + IFN-α + Tα1 were significantly lower than those in the groups treated with 1 μg/mL mAb (3.37 ± 0.34 versus 12.93 ± 1.24, p < 0.01) and IFN-α + Tα1 (3.37 ± 0.34 versus 16.30 ± 0.82, p < 0.0001), and similar results were observed in the groups treated with 10 μg/mL mAb (Figure 2C). Although the PD-1 level decreased with increasing concentrations of tislelizumab, no significant difference was detected between the 1 μg/mL mAb + IFN-α + Tα1 and 10 μg/mL mAb + IFN-α + Tα1 groups (3.37 ± 0.34 versus 3.17 ± 0.44, p > 0.05) (Figure 3C). These findings suggest that IFN-α and Tα1 together improve the inhibitory effect of tislelizumab on the immune checkpoint PD-1 of CD8⁺ T cells and that IFN-α and Tα1 together may play a role in inhibiting PD-1 expression independent of tislelizumab. IFN-α and Tα1 enhance the function of tislelizumab and may reduce the dosage of tislelizumab used in the clinic. We therefore used a dosage of 1 μg/mL of tislelizumab to conduct the subsequent experiments.

Figure 3.

Influence of tislelizumab, IFN-α, and Tα1 on the surface PD-1 expression of CD8⁺ T cells

(A) Statistical analysis of the number of CD8⁺ T cells at different times within 14 days after incubation with 1 μg/mL of tislelizumab with or without IFN-α and Tα1 treatment, as determined by a CCK8 assay.

(B–D) Statistical analysis of the levels of the secreted cytokines IL-2 (B), TNF-α (C), and IFN-γ (D) in suspensions of cultured CD8⁺ T cells after incubation with 1 μg/mL of tislelizumab with or without the addition of IFN-α and Tα1 for 14 days, as assayed by ELISA.

The data were analyzed by one-way ANOVA and are presented as the means ± SDs of four or five biological replicates. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.

Interferon-α and thymosin-α1 plus tislelizumab promote the proliferation and cytokine secretion of CD8⁺ T cells

We further evaluated the influence of IFN-α and Tα1 plus tislelizumab on the phenotypic characteristics of CD8⁺ T cells. With the extension of culture and treatment time, the numbers of CD8⁺ T cells in all groups increased continuously, and three main results were observed in the present study (Figure 3A): (1) CD8⁺ T cell proliferation was significantly promoted by stimulation with IFN-α and Tα1 from the seventh day, which was maintained to the fourteenth day thereafter; (2) CD8⁺ T cell proliferation was not influenced by monotherapy with 1 μg/mL tislelizumab; and (3) CD8⁺ T cell proliferation under tislelizumab monotherapy was activated by the addition of IFN-α and Tα1. Moreover, the secretion of proinflammatory factors, including IL-2, TNFα, and IFN-γ, on the fourteenth day after treatment was similar to that of CD8⁺ T cell proliferation (Figures 3B–3D). Specifically, the cytokine secretion of CD8⁺ T cells was not influenced by monotherapy with 1 μg/mL of tislelizumab, whereas that of CD8⁺ T cells in culture medium supplemented with 1 μg/mL of tislelizumab was significantly increased with the addition of IFN-α and Tα1 (Figures 3B–3D). These findings suggest that the combination of IFN-α and Tα1 plus tislelizumab may be a promising method for enhancing the active phenotypes of CD8⁺ T cells.

Interferon-α and thymosin-α1combined with tislelizumab enhance the anticancer activity of adoptive CD8⁺ T cells toward pancreatic ductal adenocarcinoma

Next, we explored the application potential of IFN-α and Tα1 plus tislelizumab in promoting the anticancer activity of CD8⁺ T cells toward PDAC. After coculture of PDAC patient-derived CD8⁺ T cells and PDAC cells with different E:T ratios, the cytotoxic effects of CD8⁺ T cells on PDAC cells generally increased with increasing E:T ratios (Figures 4A–4C). Moreover, treatment with IFN-α and Tα1 plus 1 μg/mL of tislelizumab resulted in the highest cytotoxic efficacy of CD8⁺ T cells in killing the PDAC cell lines PANC-1, BXPC-3, and MIAPaCa-2 in all groups (Figures 4A–4C). The cytotoxic efficacy of CD8⁺ T cells toward PDAC cells in the group treated with 1 μg/mL of tislelizumab was significantly enhanced with the addition of IFN-α and Tα1, whereas monotherapy with 1 μg/mL of tislelizumab might not have a stable effect on sustaining the cytotoxic efficacy of CD8⁺ T cells (Figures 4A–4C). These findings suggest that combination treatment with IFN-α and Tα1 plus tislelizumab improves the cytotoxic activity of active CD8⁺ T cells toward PDAC cells in vitro.

Figure 4.

Influence of tislelizumab and IFN-α and Tα1 on the cytotoxic activity of CD8⁺ T cells toward PDAC cells

(A–C) Statistical analysis of lysed PANC-1 (A), BXPC-3 (B), and MIAPaCa-2 (C) cells after coculture with CD8⁺ T cells, as determined by a CCK8 assay. The isolated CD8⁺ T cells were treated with 1 μg/mL of tislelizumab in the presence or absence of IFN-α and Tα1 for 7 days and then separated and cocultured with PDAC cells for 48 h.

The data were analyzed by one-way ANOVA and are presented as the means ± SDs of five biological replicates. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001.

Furthermore, BxPC3 cells were subcutaneously injected into NCG mice to form tumors. After tumor formation by BxPC3 cells for 10 days, human-derived CD8⁺ T cells stimulated with IFN-α and Tα1 or/and tislelizumab for 7 days were administered to the mice via tail vein injection every 4 days. Thirty-one days after cancer cell injection, changes in tumor size in the mice revealed that IFN-α and Tα1 promoted the anticancer activity of CD8⁺ T cells (Figures 5A and 5B). Notably, the inhibitory effect of CD8⁺ T cells on tumor growth after treatment with tislelizumab was further enhanced by IFN-α and Tα1, which resulted in the highest therapeutic efficacy in this study (Figures 5A and 5B). Further study revealed the highest level of CD8 protein in tumor tissues after combination treatment with IFN-α and Tα1 plus tislelizumab (Figure 5C), suggesting that the greatest number of infiltrating CD8⁺ T cells was present. Thus, combination treatment with IFN-α and Tα1 plus tislelizumab may disrupt the immunosuppressive microenvironment in adoptive CD8⁺ T cell therapy, improving the anticancer activity of CD8⁺ T cells in PDAC therapy.

Figure 5.

Anticancer activity of tislelizumab in CD8⁺ T cells in vivo after treatment with IFN-α and Tα1

(A) Statistical analysis of the growth curve of tumors formed by BxPC-3 cells within 31 days. After BxPC3 cells formed tumors, CD8⁺ T cells treated with 1 μg/mL of tislelizumab with or without IFN-α and Tα1 for 7 days were administered to the mice via tail vein injection every 4 days.

(B) Image of tumors formed by BxPC3 cells in (A) for 31 days.

(C) Protein level of CD8 in tumor specimens derived from 3 representative mice, as examined by Western blotting.

The data were analyzed by one-way ANOVA and are presented as the means ± SDs of three or six biological replicates. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗∗p < 0.0001.

Discussion

Cancer immunotherapies, including ICBs and adoptive cell therapy, aim to manipulate the immune system to recognize and attack cancer cells.³⁶ CTLA-4 and PD-1 are immune inhibitory receptors that exist in activated T cells and function as immune checkpoint targets for some solid tumors. The inhibition of these receptors or their corresponding ligands can enhance the immune response of activated T cells to tumors.³⁷ The overall response rate of patients with solid tumors to PD-1 blockade is approximately 20–30%.³⁸ Compared with chemotherapy, immune-based therapy may improve the survival rate of patients with pancreatic cancer. In our study, the PD-1 level on the surface of CD8⁺ T cells in the peripheral blood of patients with PDAC was significantly greater than that in healthy individuals, which increased with increasing disease severity: (1) the PD-1 level on CD8⁺ T cells in patients with advanced disease was significantly greater than that in patients with early disease; (2) the PD-1 level on CD8⁺ T cells in patients with undifferentiated and poorly differentiated disease was significantly greater than that in patients with moderate or high differentiation; and (3) the PD-1 level on CD8⁺ T cells in patients with lymph node metastasis was significantly greater than that in patients without lymph node metastasis. The PD-1 level on CD8⁺ T cells is closely associated with the development and progression of PDAC and may be used as a diagnostic marker. However, therapies with single agents, including PD-1 antibodies, can eliminate immune suppression but do not activate the immune system, leading to disappointing results in PC therapy.³⁹

Although immune checkpoint inhibitors alone do not achieve high efficacy, the combination of immune checkpoint inhibitors and traditional treatments achieves good results in treating malignant tumors.⁴⁰ For example, lactate dehydrogenase inhibition and IL-21 synergistically promote CD8⁺ T cell stemness and anticancer immunity,⁴¹ and ICBs have been widely used in combination with chemotherapy for gastric, lung, and breast cancers.^42,43,44 Type-I IFNs not only stimulate and activate CD8⁺ T cells by dendritic cells (DCs) but also inhibit regulatory T cells (Tregs).^45,46 As an immune adjuvant, IFN-α is used in combination therapy for a variety of malignant tumors. The combined use of IFN-α enhances the efficacy of chemotherapeutic agents in renal cell carcinoma.⁴⁷ Through the activation of STAT3 signaling, PD-L1 levels on cancer cells may also be increased by IFN-α. Fortunately, treatment with tislelizumab provides a way to block PD-1 expressed on CD8⁺ T cells, suppressing immunosuppressive PD-1/PD-L1 signaling and thereby the exhaustion of T cells. Tα1 has immunoregulatory and direct effects on cancer cells or virus-infected cells and is widely used alone or in combination with other therapies.⁴⁸ A study has shown that the combination of Tα1 and IFN-α or IL-2 has high biological activity in defending against viral infection and treating malignant tumors.⁴⁹ For highly immunosuppressed malignancies, restoring the activity of CD8⁺ T cells and enhancing the immunotherapy efficacy of PD-1 blockade are equally important, and combination treatment may effectively improve the anticancer activity of CD8⁺ T cells.⁵⁰ In this study, the potential of combination therapy with IFN-α and Tα1 plus tislelizumab for the treatment of PDAC was explored. Compared with that of tislelizumab monotherapy, the PD-1 level on CD8⁺ T cells was much lower after combination treatment with Tα1 and IFN-α plus tislelizumab. Interestingly, the inhibitory effects of IFN-α and Tα1 on PD-1 expression on CD8⁺ T cells were independent of the tislelizumab concentration. Thus, the combination of IFN-α and Tα1 may reduce the dosage of tislelizumab used in the clinic. Moreover, combination treatment with IFN-α and Tα1 plus tislelizumab maximized the proliferation and cytokine secretion of CD8⁺ T cells, thus enhancing the anticancer effect of CD8⁺ T cells toward PDAC. According to previous reports, we speculate that Tα1 and IFN-α may function mainly by recruiting CD8⁺ T cell infiltration and immune response in the PDAC microenvironment. IFN-α recruits cytotoxic CD8⁺ T cell infiltration mainly by inducing cancer cells to secrete the chemokines represented by CCL4.⁵¹ However, IFN-α treatment can also increase PD-1 expression via activating the IFNAR1-JAK1-STAT3 signaling pathway, inhibiting the proliferation and cytotoxic capacity of CD8⁺ T cells.⁵¹ As a complementary of IFN-α, Tα1 mainly enhances the infiltration ability of CD8⁺ cytotoxic T lymphocytes, improving immunosenescence and decreasing immune exhaustion.^52,53,54 Moreover, the combination of PD-1 blockade decreases the upregulated PD-1 level by IFN-α,⁵¹ highlighting the necessary of tislelizumab application for restoring and enhancing tumor antigen-specific T cell responses. Therefore, this study may provide a strategy for improving immunotherapy efficacy and overcoming immune resistance in patients with PDAC.

Immunotherapy for malignant tumors remains a reliable and unmet field. The emergence of immune resistance in cancer has led to a focus on combination treatment strategies. Through this study, we can conclude the following: (1) PD-1 expression on CD8⁺ T cells may reflect the level of tumor immunosuppression, with a higher level of PD-1 indicating greater immunosuppression in higher-grade tumors. Thus, a high level of PD-1 on CD8⁺ T cells may be an important indicator for the clinical diagnosis of PDAC. (2) Adoptive therapy with CD8⁺ T cells is highly effective at suppressing PDAC, thus overcoming the limitations of the immunosuppressive microenvironment. (3) Combination treatment with IFN-α and Tα1 plus tislelizumab may be an alternative strategy for enhancing the therapeutic efficacy of adoptive CD8⁺ T cells. The combination of IFN-α and Tα1 plus tislelizumab not only reduces PD-1 levels on active CD8⁺ T cells but also promotes the proliferation and cytokine secretion of CD8⁺ T cells, thus enhancing the anticancer activity of adoptive CD8⁺ T cells. Therefore, the introduction of IFN-α and Tα1 may facilitate the use of tislelizumab to prevent immune suppression in patients with PDAC and activate the anticancer immune microenvironment. This study highlights the application potential of CD8⁺ T cells in PDAC therapy and reveals the application potential of combination treatment with IFN-α and Tα1 plus tislelizumab for improving the therapeutic efficacy of adoptive immunity.

Limitations of the study

The sample size of patients with PDAC who met the inclusion requirements was limited. A larger sample size and a multicenter cooperative study may improve the credibility of the study. In addition, treatment with IFN-α and Tα1 plus tislelizumab has been verified only in in vitro experiments and animal models; whether it can guide treatment needs to be confirmed in clinical trials. Detailed mechanism of combination treatment with IFN-α and Tα1 plus tislelizumab in PDAC may need to be further explored.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Chaohui Zuo (zuochaohui@vip.sina.com).

Materials availability

The datasets used and/or analyzed during the present study are available from the corresponding author upon reasonable request.

Data and code availability

• •Date: Data reported in this article will be shared by the lead contact upon request.
• •Code: This article does not report original code.
• •All other requests: Any additional information required to reanalyze the data reported will be shared by the lead contact upon request.

Acknowledgments

This study was supported by the National Natural Science Foundation of China (82170192 to C.Z. and 82303190 to R.D.), the Science and technology innovation of Hunan Province (2023SK4060 to C.Z.), and the Changsha City Third Science and Technology Project of China (KQ2004130 to C.Z.).

Author contributions

C.Z. conceived and designed the experiments. S.D., R.D., J.W., and Q.H. carried out the main experiments and analyzed the data. S.D., B.X., and L.D. collected the data. R.D., J.Z., H.Z., and W.T. helped design the experiments. D.S. and C.Z. wrote the article, R.D. and M.P. performed language correction, and R.D. and C.Z. contributed to funding acquisition. All the authors have read and approved the final version of the article.

Declaration of interests

The authors declare that they have no conflicts of interest related to this study.

STAR★Methods

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Mouse anti-CD8-β (5F2)	Santa Cruz Biotechnology	Cat# sc-19994; RRID: AB_627210
Mouse anti-GAPDH	Merck Millipore	Cat# MAB374; RRID: AB_2107445
Goat anti-mouse IgG (HRP-linked)	Merck Millipore	Cat# AP124P; RRID: AB_90456
PE Mouse IgG1, κ Isotype Ctrl Antibody	BioLegend	Cat# 400112; RRID: AB_2847829
PE anti-human CD279 (PD-1)	BioLegend	Cat# 329906, RRID: AB_940481
InVivoSIM anti-human PD-1 (Tislelizumab Biosimilar)	Bio X Cell	Cat# SIM0038
InVivoSIM anti-human PD-1 (Pembrolizumab Biosimilar)	Bio X Cell	Cat# SIM0010
RecombiMAb human IgG4 (S228P/R409K) isotype control, anti-hen egg lysozyme	Bio X Cell	Cat# CP183
GMP Ultra-LEAFTM Purified anti-human CD3 SF	BioLegend	Cat# 317353; RRID: AB_2894461
Ultra-LEAF™ Purified anti-human CD28 (Superagonistic) Antibody	BioLegend	Cat# 377803; RRID: AB_2910434
Biological samples
Peripheral blood of PDAC patients	Hunan Cancer Hospital	N/A
Peripheral blood of healthy individuals	Hunan Cancer Hospital	N/A
Formed tumors in NCG mice	This paper	N/A
Chemicals, peptides, and recombinant proteins
Proteinase inhibitor cocktail	TOPSCIENCE	Cat# L1100
RIPA buffer	Thermo Fisher Scientific	Cat# 89900
IFN-α	MCE	Cat# HY-P7023
Tα1	MCE	Cat# HY-P0091
Cell staining buffer	BioLegend	Cat# 420201
CCK8	Dojindo	Cat# CK04
Critical commercial assays
SuperSignal® West Pico Chemiluminescent Substrate	Thermo Fisher Scientific	Cat# 34578
MojoSort™ Human CD8 Naive T cell Isolation Kit	BioLegend	Cat# 480045
IFN-γ ELISA Kit	Sino Biological	Cat# SEK13222
TNFα ELISA Kit	Sino Biological	Cat# KIT10602
IL-2 ELISA Kit	Sino Biological	Cat# KIT11848
Experimental models: Cell lines
PANC-1	Cell Resource Center of Shanghai Academy of Health Sciences, Chinese Academy of Sciences	RRID: CVCL_0480
BxPC3	Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd.	RRID: CVCL_0186
MIA PaCa-2	Wuhan Boster Biotechnology Co., Ltd.	RRID: CVCL_0428
Human CD8+ T cell	Peripheral blood	N/A
Experimental models: Organisms/strains
NCG mouse	GemPharmatech	RRID: IMSR_GPT:T001475
Software and algorithms
SPSS 26.0 software	IBM Corporation	https://www.ibm.com/support/pages/downloading-ibm-spss-statistics-26-end-support-30-sep-2025
GraphPad Prism 8.3 software	GraphPad	https://www.graphpad.com/
ImageJ software	ImageJ	https://imagej.en.softonic.com/mac
Other
DMEM	Thermo Fisher Scientific	Cat# C11965500BT
RPMI1640 medium	Thermo Fisher Scientific	Cat# C11875500BT
Penicillin-Streptomycin	Thermo Fisher Scientific	Cat# 15140122
FBS	Thermo Fisher Scientific	Cat# 10270-106

Experimental model and study participant details

Ethics approval and consent to participate

Research involving human participants was approved by the Ethics Committee of Hunan Cancer Hospital (No. KYJJ-2021-073). The procedures conducted in studies involving human participants were in accordance with the ethical standards of the institutions and/or the National Research Council and with the 1964 Declaration of Helsinki and subsequent amendments or similar ethical standards, and written informed consent was obtained from all participants. The protocols for the animal experiments were approved by the Animal Care and Experiment Committee of Hunan Cancer Hospital (No. 2021-057).

Clinical samples

From June 2020 to January 2022, peripheral blood samples from 40 patients who were diagnosed with PDAC at Hunan Cancer Hospital were collected for this study before surgery, and peripheral blood samples from 36 healthy volunteers were used as controls. The inclusion criteria were as follows: (1) PDAC was first diagnosed and surgically treated; (2) no anticancer treatment, such as chemoradiotherapy or immunotherapy, was conducted before surgery; (3) PDAC was diagnosed in the postoperative pathological report; (4) no history of infection, inflammation or autoimmune diseases; and (5) complete medical records were available. There were 23 males and 17 females among the 40 patients with PDAC; the average age was 57.7 years, with a range from 37 to 78 years, and the median age was 58 years. According to the staging criteria specified by the AJCC in 2017, 24 patients were in stages I-II, and 16 patients were in stages III-IV. Two patients with stage IV PDAC with only a single site of liver metastasis were included in this study. The histological classifications were as follows: 22 cases presented undifferentiation and poor differentiation, and 18 cases presented moderate and high differentiation. The lymph node metastasis classifications were as follows: 19 patients had no lymph node metastasis, and 21 patients had lymph node metastasis. Among the 36 healthy volunteers, 21 were male, and 15 were female; the average age of the patients was 58.6 years, with a range from 34 to 74 years, and the median age was 60 years. This study involving human participants was approved by the Ethics Committee of Hunan Cancer Hospital (Innovative Research Team of Basic and Clinical Application of Gastrointestinal Malignancies, No. KYJJ-2021-073). Human blood samples were collected and used with informed consent and written consent from all patients, families, or healthy donors.

Cell lines

Human CD8⁺ T cells were isolated from the peripheral blood of healthy individuals and patients with PDAC. The human in situ pancreatic adenocarcinoma cell line BxPC-3 (RRID: CVCL_0186) was purchased from Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd. (China), the human pancreatic cancer cell line MIA PaCa-2 (RRID: CVCL_0428) was purchased from Wuhan Boster Biotechnology Co., Ltd. (China), and the human pancreatic cancer cell line PANC-1 (RRID: CVCL_0480) was provided by the Cell Resource Center of the Shanghai Academy of Health Sciences, Chinese Academy of Sciences (China).

Mice

Six-week-old female immune-deficient NCG mice (GemPharmatech, China) were used for this study. The mice were fed under standard conditions (25°C and 50% humidity) with free access to sterile feed and water in a pathogen-free environment with a 12 h light/dark cycle at the animal care facility of Hunan University. The protocol used for the animal experiments was approved by the Animal Care and Experiment Committee of Hunan Cancer Hospital (No. 2021-057).

Method details

Isolation of CD8⁺ T cells

CD8⁺ T cells were isolated using a MojoSort Human CD8⁺ T cell Isolation Kit (Cat# 480045, BioLegend, USA). Heparin anticoagulant tubes were used to collect fasting fresh peripheral venous blood from patients with PDAC or healthy donors in the morning, and CD8⁺ T cell magnetic bead sorting was performed as follows. The MojoSort Buffer in the kit was diluted to 1× with ultrapure water before the start and was sterilized by filtration with a 0.22 μm needle filter and placed in ice-water bath. A sterile flow tube (5 mL) was used to collect 1 mL of blood sample, which was mixed with 50 μL of magnetic beads after gentle blowing and incubated in an ice-water bath for 15 min. After mixing 2 mL of 1× MojoSort Buffer with the blood samples, the tube was placed in the sorter and allowed to stand for 5 min. The suspension containing CD8⁺ T cells was carefully poured out and collected, and the sample tube was further washed with 3 mL of 1× MojoSort Buffer two times using the same method. The isolated CD8⁺ T cells were collected by centrifugation at 350 × g at room temperature for 5 min, and 200 μL of RPMI-1640 medium (Cat# C11875500BT, Thermo Fisher Scientific) was added for cell resuspension. The proportion of CD8⁺ T cells and the PD-1 level on CD8⁺ T cells were examined by flow cytometry.

Cell culture

The isolated CD8⁺ T cells at a density of 1×10⁶ per mL were seeded into coated cell culture flasks and cultivated in RPMI-1640 medium supplemented with 10% (v/v) FBS (Cat# 10270-106, Thermo Fisher Scientific, USA) and penicillin‒streptomycin (Cat# 15140122, Thermo Fisher Scientific). Anti-CD3 antibody (Cat# 317353, BioLegend) at a final concentration of 2 μg/mL and anti-CD28 antibody (Cat# 377803, BioLegend) at a final concentration of 2 μg/mL were added to the culture medium to activate the CD8⁺ T cells.

BxPC-3 cells were cultured in RPMI-1640 medium supplemented with 10% (v/v) FBS and penicillin‒streptomycin at 37°C in a cell incubator with 5% CO₂, and PANC-1 and MIA PaCa-2 cells were cultured in DMEM (Cat# C11965500BT, Thermo Fisher Scientific) supplemented with 10% (v/v) FBS and penicillin‒streptomycin at 37°C in a cell incubator with 5% CO₂.

Flow cytometry analysis of PD-1 levels on CD8⁺ T cells

After being treated with IFN-α (Cat# HY-P7023, MCE, USA), Tα1 (Cat# HY-P0091, MCE), tislelizumab (Cat# SIM0038, Bio X Cell, USA), pembrolizumab (Cat# SIM0010, Bio X Cell), or IgG4 (Cat# CP183, Bio X Cell), the isolated CD8⁺ T cells were resuspended in 100 μL of Cell Staining Buffer (Cat# 420201, BioLegend, USA), and PE-conjugated anti-human CD279 (PD-1) antibody (Cat# 329906, BioLegend) was added to the tubes to mix with the cell suspensions. After incubation at room temperature for 20 min in the dark, the cells were resuspended in 2 mL of cell staining buffer and then centrifuged at 350 × g for 5 min at room temperature. The cells were then resuspended in 200 μL of cell staining buffer and were then subjected to flow cytometry analysis using a BD FACSAria II flow cytometer (BD Bioscience, USA) within 1 h. The data were analyzed using FlowJo 10.6.2 software (FlowJo, USA).

ELISA

After in vitro cultivation for 7 days, the CD8⁺ T cells in each group were separated by centrifugation at 350 × g at room temperature for 5 min and cultured with cytokine- and serum-free RPMI-1640 medium. After cultivation for 24 h, the cell supernatants were collected, and the protein levels of secreted IL-2, TNF-α, and IFN-γ were examined using ELISA kits (Sino Biological, China).

Examination of the cytotoxic activity of CD8⁺ T cells

A volume of 100 μL of RPMI-1640 supplemented with 10% (v/v) FBS and penicillin‒streptomycin containing 1×10⁴ PDAC cells was seeded in each well of a 96-well plate. After stimulation for 7 days in each group, the isolated CD8⁺ T cells were separated by centrifugation at 350 × g at room temperature for 5 min. After the cultivation of PDAC cells for 24 h, CD8⁺ T cells (effector cells) were cocultured with PDAC cells (target cells) at different ratios of effector/target cells (E:T), and wells containing only target or effector cells were used as controls. The culture plates were placed at 37°C in a cell incubator with 5% CO₂ and cultivated for 48 h. Then, 10 μL of CCK-8 reagent (Cat# CK04, Dojindo, China) without bubble formation was added to each well of a 96-well plate and incubated for 4 h at 37°C. The OD value at 450 nm was determined using a Synergy HTX microplate reader (BioTek Instruments, Inc., USA). The cytotoxic activity of CD8⁺ T cells was calculated according to the following formula: cytotoxic activity % = [1 - (OD effector and target cells - OD effector cells)/OD target cells] × 100%.

Animal experiments

The immunodeficient NCG mice were randomly assigned to four groups with an average of six mice per group. BxPC3 cells in the logarithmic growth phase were subjected to trypsin digestion, centrifuged at 250 × g at room temperature for 3 min, and resuspended in sterile PBS. After adjusting the cell density with sterile PBS, 200 μL of sterile PBS containing 5×10⁶ BxPC-3 cells were subcutaneously injected into each mouse. After BxPC3 cell injection for 10 days, 100 μL of sterile PBS containing CD8⁺ T cells treated with 1000 U/ml IFN-α and 10 μg/mL Tα1 or/and 1 μg/mL tislelelizumab for 7 days was injected into the tumors formed by tail vein injection every 4 days. The mice were sacrificed when they experienced sharp decreases in activity, water and diet intake. Thus, 31 days after BxPC3 cell injection, the mice were sacrificed by cervical dislocation immediately after isoflurane anesthesia (induction, 3%; maintenance, 1%). Tumor volumes were measured according to the desired time points and were calculated according to the following formula: tumor volume (mm³) = 1/2 × length × width.²

Western blot

The protocol used for immunoblotting was detailed in our previous research.¹² Cell lysates were extracted using RIPA buffer (Cat# 89900, Thermo Fisher Scientific) supplemented with proteinase inhibitor cocktail (Cat# L1100, TOPSCIENCE, China). Images of the protein bands were acquired using SuperSignal West Pico Chemiluminescent Substrate (Cat# 34578, Thermo Fisher Scientific) and analyzed using Image Lab 5.2 software (Bio-Rad, CA, USA). Mouse anti-CD8-β (Cat# sc19994, Santa Cruz, USA) was used to detect the human-derived CD8 antigen, mouse anti-GAPDH (Cat# MAB374, Merck Millipore, USA) was used to examine the internal control of human-derived GAPDH, and the secondary antibody goat anti-mouse IgG (HRP-linked) (Cat# AP124P, Merck Millipore) was used in this study.

Quantification and statistical analysis

Statistical analysis was performed using SPSS 26.0 software (IBM Corporation, USA), and figures were drawn using GraphPad Prism 8.3 software (GraphPad, USA). The high and low levels of PD-1 on CD8⁺ T cells were determined according to the median value. The significance of differences between two groups was analyzed by two-sided Student’s t tests, and the significance of differences between multiple groups was analyzed by one-way ANOVA. The data are presented as the means ± SDs of at least four biological replicates. Significance levels were set to ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, or not significant (ns).

Published: July 3, 2025