Journal of Cancer Research and Therapeutics

: 2022  |  Volume : 18  |  Issue : 3  |  Page : 704--711

Tim-3 and PD-1 blocking cannot restore the functional properties of natural killer cells in early clinical stages of chronic lymphocytic leukemia: An in vitro study

Mojgan Astaneh1, Hadiseh Rezazadeh1, Hadi Hossein-Nataj1, Ramin Shekarriz2, Ehsan Zaboli2, Mahdi Shabani3, Hossein Asgarian-Omran4,  
1 Department of Immunology, School of Medicine, Mazandaran University of Medical Sciences, Sari, Iran
2 Gastrointestinal Cancer Research Center, Non-Communicable Diseases Institute, Mazandaran University of Medical Sciences; Department of Hematology and Oncology, Imam Khomeini Hospital, Mazandaran University of Medical Sciences, Sari, Iran
3 Department of Immunology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran
4 Department of Immunology, School of Medicine, Mazandaran University of Medical Sciences; Gastrointestinal Cancer Research Center, Non-Communicable Diseases Institute, Mazandaran University of Medical Sciences; Immunogenetics Research Center, School of Medicine, Mazandaran University of Medical Sciences, Sari, Iran

Correspondence Address:


Background: Programmed death-1 (PD-1) and T-cell immunoglobulin- and mucin-domain-containing molecule-3 (Tim-3) are two major immune checkpoint receptors expressed on immune cells and their expression is related to the exhaustion phenotype. In the present in vitro study, blocking of PD-1 and Tim-3 molecules was performed on isolated natural killer (NK) cells from patients with chronic lymphocytic leukemia (CLL) to restore their functional properties. Materials and Methods: NK cells fraction was positively isolated from fresh peripheral blood of 18 CLL patients, treated with anti-PD-1 and anti-Tim-3 blocking monoclonal antibodies and co-cultured with K562 target cells to evaluate their apoptosis induction by Annexin V-PI method. Blocked NK cells were also incubated with anti-CD107a antibody to assess their degranulation properties by flow cytometry. The level of secreted tumor node factor-alpha (TNF-α) and interferon-gamma (IFN-γ) by NK cells was also measured by ELISA. Results: Our results showed similar functional properties in terms of degranulation and apoptosis of K562 target cells by isolated NK cells from CLL patients in PD-1/Tim-3 blocked and control groups. It was also shown that blocking of PD-1 and Tim-3 could not improve the production of pro-inflammatory TNF-α and IFN-γ cytokines by isolated NK cells from CLL patients. Conclusion: Altogether, our results indicated that pretreatment of NK cells with anti-PD-1 and anti-Tim-3 blocking antibodies in CLL patients at early clinical stages cannot improve their functional properties. Besides many other malignancies, the application of checkpoint inhibitors in CLL needs more investigations and complementary studies.

How to cite this article:
Astaneh M, Rezazadeh H, Hossein-Nataj H, Shekarriz R, Zaboli E, Shabani M, Asgarian-Omran H. Tim-3 and PD-1 blocking cannot restore the functional properties of natural killer cells in early clinical stages of chronic lymphocytic leukemia: An in vitro study.J Can Res Ther 2022;18:704-711

How to cite this URL:
Astaneh M, Rezazadeh H, Hossein-Nataj H, Shekarriz R, Zaboli E, Shabani M, Asgarian-Omran H. Tim-3 and PD-1 blocking cannot restore the functional properties of natural killer cells in early clinical stages of chronic lymphocytic leukemia: An in vitro study. J Can Res Ther [serial online] 2022 [cited 2022 Sep 25 ];18:704-711
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Chronic lymphocytic leukemia (CLL) is a lymphoproliferative disorder of mature B lymphocytes accumulating in peripheral blood, bone marrow, and lymphoid organs.[1] CLL is the most common leukemia among adults in western countries with annual incidence rate of 4–6/100,000 people.[2] The standard first-line therapy for CLL patients depends on some factors; patients in early clinical stages should be watched and wait until they become symptomatic. In symptomatic or advanced stage, depending on TP53 dysfunction, mutation status of immunoglobulin heavy chain variable region and del(17p), various treatments including ibrutinib, venetoclax, idelalisib, obinutuzumab, rituximab, and chemoimmunotherapy are prescribed.[3] Since the overall treatment is not completely efficient, some CLL patients may relapse and show disease progression. The reason can be attributed to the escape of the CLL leukemic cells from the immune surveillance mechanisms of the host immune system.[4] Recent reports indicated that like cytotoxic T-cells, natural killer (NK) cells can become functionally impaired and exhausted in some chronic conditions such as viral infections and malignancies.[4],[5],[6] The exhaustion status in NK cells is defined as the loss of killing capabilities and cytokine secretion. These characteristics are accomplished through direct cytotoxicity of virus-infected and tumor cells as well as with IFN-γ and TNF-α production by NKs which the exhaustion processes can impair their immunosurveillance and cytotoxic activities.[7],[8] One major characteristic of the exhausted phenotype is the upregulation of inhibitory receptors called immune checkpoints molecules, such as cytotoxic T lymphocyte antigen-4 (CTLA-4), programmed death-1 (PD-1), and T-cell immunoglobulin- and mucin-domain-containing molecule-3 (Tim-3).[9],[10],[11],[12],[13] In recent years, the blockade of inhibitory receptors by monoclonal antibodies to reactivate the exhausted cells shows promising benefits as a new trend in therapeutic strategies for a variety of cancers.[14],[15],[16],[17],[18],[19],[20],[21] A few of these checkpoint inhibitors such as ipilimumab (anti-CTLA-4), pembrolizumab and nivolumab (anti-PD-1), and durvalumab (anti-PD-L1) have been approved by Food and Drug Administration for some cancers.[14],[15],[16],[20],[21] Among inhibitory receptors of NK cells, Tim-3, PD-1, KIR3DL-1, KIR2DL-3, and NKG2A are more in focus. PD-1 over-expression on NK cells and its role in the downregulation of killing potentials are reported in some chronic conditions.[22],[23],[24] It was demonstrated that CT-011, a monoclonal antibody against PD-1 molecule, could enhance the NK cells trafficking, immune complex formation, and killing of multiple myeloma (MM) cells.[9] In follicular lymphoma, a humanized monoclonal anti-PD-1 antibody in combination with rituximab was shown to enhance the expression of activating receptors on NK cells resulting improvement in cytotoxicity.[25] Besides PD-1, Tim-3 has also been introduced as an exhaustion marker of NK cells in some chronic conditions.[9],[10],[11],[25],[26],[27] In chronic hepatitis B, elevated expression of Tim-3 expression was shown on NK cells and blockade with anti-Tim-3 lead to an increase in NK cell's cytotoxicity and IFN-γ secretion.[11] Furthermore, upregulation of Tim-3 inhibitory receptor and downregulation of NKp-30 activating receptor were also demonstrated in CLL patients.[10] Besides promising results in other malignancies, there are no approved checkpoint inhibitors for CLL. In the present study, we targeted to block two major inhibitory receptors, PD-1 and Tim-3, to restore the function of NK cells in CLL patients.

 Materials and Methods


For separation of NK cells, anti-CD56 microbeads, columns, and filters were purchased from Miltenyi Biotec (Bergisch Gladbach, Germany). For purity analysis of NK cells, anti-CD56 monoclonal antibody (TULY 56)-FITC and anti-CD3 monoclonal antibody (UCHT1)-PE together with their relevant mouse IgG1-kappa isotype controls were purchased from eBioscience (California, United States). For stimulation of NK cells, human interleukin-2 (IL-2) recombinant protein and cell stimulation cocktail (×500) were purchased from eBioscience (California, United States). Blocking antibodies, anti- PD-1 (EH12-27) and anti- Tim-3 (F38-2E2) and their corresponding purified IgG1-kappa isotype controls were from BioLegend (California, United States). Anti-CD107a (LAMP-1) monoclonal antibody (eBioH4A3)-PE and Annexin V-FITC Apoptosis Detection Kit with PI were purchased from eBioscience (California, United States). For cytokines measurements, human IFN-γ and TNF-α ELISA kits were obtained from eBioscience (California, United States).

Cell line

K562 cell line (a human erythroleukemia cell line [acute myeloid leukemia-M6 {AML}]) was purchased from Pasteur Institute of Iran. Before NK cells cytotoxicity experiments, K562 cells were cultured in complete RPMI-1640 media (Biosera, Nuaille, France) supplemented with 10% fetal bovine serum (Biosera, Nuaille, France), 2 mM L-glutamine, 100 IU/ml penicillin, and 100 μg/ml streptomycin.

Human samples

Blood samples were obtained from 18 untreated CLL patients admitted to the oncology ward of the hospital. Disease diagnosis was done by two special oncologists based on patients' clinical symptoms, white blood cell count, and immunophenotyping analysis according to the WHO criteria.[28] Clinical stage of CLL patients was determined according to the Rai staging system. Exclusion criteria were consumption of chemotherapeutic agents, autoimmunity, other hematologic or nonhematologic malignancies, and chronic viral infections. All patients provided written informed consent under an approved protocol of the ethics committee. Major clinical and paraclinical findings of CLL patients are listed in [Table 1].{Table 1}

Isolation of NK cells

Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll density gradient centrifugation (Ficoll-Paque PLUS, Biosera). NK cell fraction was positively isolated by magnetic-activated cell sorting (MACS) assay using CD56 microbeads, according to the manufacturer's protocol (Miltenyi, Germany). Briefly, PBMCs were washed twice by MACS buffer, the MS column was assembled on a magnetic stand, and cells were rinsed into the column. CD56+ cells were attached to the column and other unbound cells were passed through the column. After washing, CD56+ cells were eluted and applied for subsequent experiments. The purity of CD56+ cells was defined by flow cytometry using anti-CD3 and anti-CD56 monoclonal antibodies, which was more than 90%.

Blocking of programmed death-1 and Tim-3 receptors on NK cells

To block the inhibitory PD-1 and Tim-3 receptors on NK cells, the purified cells were categorized into four groups including blocked with anti-Tim-3 alone, blocked with anti-PD-1 alone, double blocked with anti-Tim-3 and anti-PD-1, and unblocked using corresponding isotype control antibodies. A total of 105 NK cells were incubated with 10 μg/ml of blocking anti-Tim-3, anti-PD-1, and their corresponding isotype control antibodies in complete RPMI-1640 medium for 2 h in the dark at 4°C.

Degranulation assay

CD107a degranulation assay was used to evaluate the degranulation of perforin/granzyme contained granules of NK cells. The isolated NK cells, initially blocked with anti-PD-1 and anti-Tim-3 monoclonal antibodies, were stimulated with 1 ng/ml of human IL-2 recombinant protein and PMA/ionomycin cell stimulation cocktail for 30 min at 37°C with 5% CO2 and then co-cultured with K562 cells as their target at an effector: target ratio of 1:1 in complete RPMI-1640 medium. Overnight incubation was performed with 5 μl of anti-CD107a-PE monoclonal antibody and its irrelevant isotype control at 37°C with 5% CO2 (eBioscience, USA). The translocation of CD107a on the surface of NK cells was monitored on the Partec PAS flow cytometer system (Partec GmBH, Munster, Germany) using FlowMax software (Partec GmBH, Munster, Germany). For CD107a analysis, NK cells were gated, and then, the protein expression was determined.

Cytotoxicity assay

Isolated NK cells were blocked and stimulated as mentioned above and then were co-cultured with K562 cells at an effector: target ratio (1:1) overnight at 37°C and 5% CO2. The apoptosis rate of K562 cells was assessed using Annexin V-PI Apoptosis Detection Kit according to the manufacturer's protocol (eBioscience, USA). Briefly, cells were washed twice with binding buffer and then were treated with 5 μl of Annexin V-FITC. After 15 min of incubation at room temperature, cells were washed and 5 μl of PI was added. The apoptosis of K562 cells was assessed on the Partec PAS flow cytometer system (Partec GmBH, Munster, Germany) following gating on the K562 cells population and was analyzed using FlowMax software.

Cytokines production

To evaluate the effects of PD-1 and Tim-3 blocking on the cytokine production activity of the NK cells, isolated cells were blocked with anti-PD-1 and anti-Tim-3 and then stimulated with 1 ng/ml of human IL-2 recombinant protein and PMA/Ionomycin cell stimulation cocktail for 30 min at 37°C with 5% CO2. After that, the cells were co-cultured with K562 cells as their target at an effector: target ratio of 1:1 in complete RPMI-1640 medium. Following overnight incubation, the supernatants were collected and the concentration of human IFN-γ and TNF-α cytokines was measured by ELISA according to the manufacture's protocol (eBioscience, USA). The detection sensitivity for both kits was 4 pg/ml.

Statistical analysis

Statistical analyses were performed with GraphPad Prism 6 (San Diego, California, USA) and SPSS 20 softwares (IBM Corp., Armonk, N.Y., USA). All data are expressed as mean ± standard deviation. Normality distribution was checked with Kolmogorov–Smirnov test. Nonparametric Mann–Whitney U and Kruskal–Wallis tests were appropriately used to calculate the mean difference between two or more groups. P < 0.05 was considered statistically significant.


Tim-3 and programmed death-1 blockade did not improve the degranulation properties of NK cells in chronic lymphocytic leukemia patients

To investigate the effects of Tim-3 and PD-1 blockade on the degranulation properties of NK cells in CLL patients, MACS-isolated NK cells were blocked with anti-PD-1 and anti-Tim-3, stimulated with hrIL-2 and PMA/ionomycin, and then co-cultured with K562 target cells. The expression of CD107a on NK cells was measured by flow cytometry. As represented in [Figure 1], the obtained results showed no statistically significant difference among the blocked and control groups (P > 0.05).{Figure 1}

Blockade of Tim-3 and programmed death-1 on NK cells could not improve the apoptosis of K562 cells in chronic lymphocytic leukemia patients

Following no improvements of NK cells degranulation properties after PD-1 and Tim-3 blocking, we aimed to compare the cytotoxic activity of blocked and unblocked NK cells against K562 cell line. NK cells from CLL patients were blocked, stimulated and co-cultured with K562. In flow cytometry analysis, the K562 target cells were gated and the apoptosis was monitored by Annexin V/PI apoptosis detection system. As shown in [Figure 2], either single or combined blocking with anti-Tim-3 and anti-PD-1 antibodies did not improve the killing activity of isolated NK cells against K562 in CLL patients (P > 0.05).{Figure 2}

Pro-inflammatory cytokine production by the NK cells from chronic lymphocytic leukemia patients was not altered by blocking with anti-Tim-3 and anti-programmed death-1 antibodies

Following the observations that PD-1 and Tim-3 blockade had no enhancing effects on degranulation and cytotoxic characteristics of NK cells in CLL patients, in our final experiments, we assessed the NK cells function in the production of pro-inflammatory cytokines. Considering the fact that the production of TNF-α and IFN-γ decrease during the exhaustion process of NK cells, we sought to investigate whether the blockade of Tim-3 and PD-1 can reverse the exhaustion effects on the NK cells' cytokine secretion. We used the ELISA method to measure the concentration of TNF-α and IFN-γ cytokines secreted from the NK cells after blocking with anti-Tim-3 and anti-PD-1 antibodies. Our results showed no significant difference in the concentration of secreted TNF-α [Figure 3]a and IFN-γ [Figure 3]b among blocked and control groups (P > 0.05).{Figure 3}


T-cells exhaustion has been widely studied during recent years in many types of chronic infections and tumors. Besides T-cells, little is known about the status of NK cell exhaustion in the context of cancers, especially in hematopoietic malignancies. Exhaustion properties of NK cells can be conferred to the low functional properties and upregulation of some surface immune checkpoint inhibitory molecules, such as Tim-3 and PD-1. Recently, blockade of these checkpoint inhibitory molecules showed promising benefits in restoring the exhausted characteristics of NK cells in some cancers, including metastatic melanoma, non-small cell lung carcinoma, and MM.[14],[27],[29] In the case of CLL, previous studies have shown the upregulation of Tim-3 together with lower expression of NKp30 activating receptor[10] and increased expression of PD-1 along with decreased expression of NKG2D activating receptor on the surface of peripheral blood NK cells.[30] In the present study, we explored whether the blockade of Tim-3 and/or PD-1 would restore the exhausted functions of NK cells in CLL patients.

PD-1 and Tim-3 blocking have been examined in various malignancies to investigate their blocking effects on restoration the functional properties of killer cells. Single blockade of Tim-3 signaling with anti-Tim-3 antibody showed improvements in cytotoxicity and cytokine production of peripheral blood NK cells in patients with lung adenocarcinoma and advanced melanoma.[13],[27] It was shown that PD-1 blockade is effective in enhancing the apoptosis of a specialized melanoma cell line RPMI8226 by killer cells.[29] Improvements in apoptosis were also demonstrated by blocking of Tim-3 and PD-1 on cytokine-induced killer cells isolated from the bone marrow of AML patients, which co-cultured with myeloid leukemia cell line U937, but the blockade showed a failure in the apoptosis of lymphoid leukemia cell line Raji.[31] Besides these enhancing effects, the results of our current study showed that single blockade of Tim-3 or PD-1 and even double blockade of both molecules on isolated NK cells could not enhance the degranulation properties of NK cells in CLL patients. Moreover, PD-1 and Tim-3 blocking caused no significant improvements in the apoptosis of K562 cells by peripheral blood NK cells. These results are concordant with our previous study regarding no improvements in the functional characteristics of peripheral blood CD8+ T-cells of CLL patients following blockade with Tim-3 and/or PD-1 molecules.[32] In MM, blockade of PD-1 alone was shown to enhance the translocation of CD107a molecule on the surface of expanded NK cells rather than resting NK cells. Anti-CD16 antibody was applied to expand NK cells for 21 days and then it has been demonstrated that the amount of PD-1 was low on the resting NK cells and gradually increased through the expansion process. It was demonstrated that the blockade of these upregulated PD-1 molecules by anti-PD-1 antibody could enhance the degranulation capacity of expanded NK cells compared with resting NK cells.[29] Hence, we can postulate that the low amount of PD-1 on the surface of peripheral blood NK cells in CLL may be the reason why the blockade strategy did not work as we expected. Treatment with anti-CD16 stimulatory antibody seems to be a good approach in increasing the amount of PD-1 on NK cells in CLL. In advanced melanoma, it has been shown that blocking of Tim-3 could partially reverse the degranulation of NK cells. This finding was correlated with an increasing in the expression of IL-2 receptor on the surface of NK cells and their hyper-responsiveness to IL-2 stimulation. It was suggested that the enhanced responsiveness may be a reason for a partial reversal of NK cells' degranulation.[27] In our study, NK cells were stimulated with 1 ng/ml of human IL-2 recombinant protein which might interfere with the blocking of inhibitory molecules. Indeed, overnight incubation with human IL-2 may have such great stimulatory effects on NK cells by making them saturated and over-reactive in killing of their targets and finally masking the enhancing effects of immune checkpoint inhibitors.

The amount of Tim-3 and/or PD-1 expression on NK cells may be associated with the clinical stage of malignancies. da Silva et al. have shown the correlation of Tim-3+-NK cells with the clinical stage of melanoma patients.[27] In another study, Xu et al. showed that Tim-3 expression increases during the progression of lung adenocarcinoma.[13] In the context of CLL, it was shown that the expression of Tim-3 correlates with poor prognostic factors in higher stages of CLL patients.[10] Taghiloo et al. have also shown a remarkable increase in the expression of PD-1/Tim-3 and their ligands PD-L1/Gal-9 at advanced clinical stages of CLL patients compared with early stages.[33],[34] Finally, in Richter syndrome which is a transformation of CLL characterized by aggressive clinical course and poor prognosis, there is a correlation between the disease stage and the amount of PD-1 expression.[35],[36] In the present study, most of the CLL patients were at low and intermediate clinical stages according to the Rai staging system. Therefore, it seems that the effect Tim-3 and PD-1 blockade on NK cells would be different in high-risk CLL patients at advanced clinical stages. In 2017, the first study was conducted to block PD-1 with pembrolizumab in CLL patients with Richter transformation and the results indicated selective efficacy and acceptable safety.[36] In a Phase II trial study, it was suggested that treatment with ibrutinib and anti-PD-1, nivolumab, has promising outcomes in patients with Richter syndrome but not relapsed/refractory CLL and follicular lymphoma.[37] In 2019, a Phase I/IIa clinical trial on high-risk patients with previously treated, relapsed, or refractory B-cell malignant diseases such as CLL showed an acceptable safety profile in combinational therapy of ibrutinib and nivolumab.[38] Altogether, these investigations confirm the importance of disease clinical stage in response to therapeutic strategies based on immune checkpoint inhibitors in CLL.

Regarding cytokines production by NK cells, IFN-γ and TNF-α are among major pro-inflammatory cytokines secreted from NK cells upon their activation against tumor cells. PD-1 and Tim-3 blocking in advanced melanoma reversed the downregulated secretion of IFN-γ and TNF-α by exhausted T-cells.[39],[40] Blocking of Tim-3 alone on NK cells of melanoma patients has shown to increase IFN-γ production,[27] and PD-1 blockade could enhance the IFN-γ secretion by NK cells of patients human lung adenocarcinoma.[13] There is no evidence of the effect Tim-3 and PD-1 blockade on NK cells of CLL patients. Here, we showed that Tim-3 and/or PD-1 blockade may not reverse the impaired IFN-γ and TNF-α secretion from NK cells of CLL patients.


Our present study is the first one to demonstrate the effect of Tim-3 and/or PD-1 blockade on restoration the functional properties of NK cells in CLL patients at early clinical stages. By considering the stage of our CLL patients, the result of this study may be useful in ongoing researches and developing new therapeutic approaches for CLL. Together with our previous data, it seems that immune checkpoint inhibition is not a suitable therapeutic choice for all clinical stages of CLL patients. Further experimental and clinical trial studies could more explain the status of immune checkpoint inhibition strategy in CLL patients.


The authors thank the patients and their families for their support, cooperation, and patience. We would like to thank the staff of the departments associated with the care and management of the patients. This study was financially supported by the grants from the National Institute for Medical Research Development (NIMAD, grant number: 962337), Mazandaran University of Medical Sciences (grant number: 10273) and Shahid Beheshti University of Medical Sciences (grant number: 15355).

Conflicts of interest

There are no conflicts of interest.


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