The Effect of Different Concentrations of Anti-PD-1 and Anti-PD-L1 Antibodies on the Activity of Immune System Cells in Patients With Non-small Cell Lung Cancer

doi:10.21203/rs.3.rs-2581427/v1

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The Effect of Different Concentrations of Anti-PD-1 and Anti-PD-L1 Antibodies on the Activity of Immune System Cells in Patients With Non-small Cell Lung Cancer

https://doi.org/10.21203/rs.3.rs-2581427/v1

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published 06 Nov, 2023

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The last century was full of numerous scientific discoveries that allowed us to understand the operation and functioning of one of the most complex human systems, i.e. the immune system. One of the most important discoveries was the work of prof. James Alison and Prof. Tasuko Honjo on the development of anti-cancer therapy by inhibiting negative immune regulation (PD-1 and CTLA-4 molecules). Knowledge of the action of these molecules and their huge role in inhibiting the activity of the immune system, e.g. during the developing cancer, created the basis for the development of specific monoclonal antibodies, without which clinicians of many specialties cannot imagine modern cancer therapies. However, the side effects of the therapies used are still quite troublesome, as they can even lead to the death of the patient. In order to minimize the side effects, it would be necessary to reduce the dose, but still maintain the effective level of anticancer activity of the cells of the immune system. In the presented study, 24-hour cultures of PBMC isolated from peripheral blood and bronchoaspirate with various concentrations of nivolumab or atezolizumab were performed. The expression results of the individual activation markers on cultured cells were compared to the expression of these markers on cells not subjected to cell culture. The results of the research work may indicate that individualized dosage of anti-PD-1 and anti-PD-L1 antibodies may in the future contribute to the effective activation of the immune system cells while minimizing the side effects of the therapy.

Biological sciences/Cancer/Cancer therapy/Cancer immunotherapy

Biological sciences/Cancer/Cancer therapy

Biological sciences/Cancer/Lung cancer

Immune checkpoints are molecules present on the surface of many cells of the immune system, but also on the surface of other cells in the human body. Their action consists in ensuring the appropriate level of functioning of the immune system or inhibiting its excessive activity. It is important to remember that any activity of the immune system must be properly balanced and regulated. Too long arousal, as a result of i.a. current chronic inflammation, may result in excessive suppression of the immune system, while intensive and strong stimulation during acute infections may lead to too strong stimulation of the immune system cells and directing its activity against its own healthy tissues.

Currently, clinicians dealing with lung cancer patients cannot imagine modern therapies for this disease without the use of immunotherapy. The therapeutic possibilities of lung cancer have been enhanced by the use of anti-PD-1 and anti-PD-L1 monoclonal antibodies that block the inhibition pathway of the immune system. In the group of anti-PD-1 antibodies, nivolumab, pembrolizumab and cemiplimab should be mentioned, in the group of anti-PD-L1 antibodies - atezolizumab, durvalumab and avelumab. The effectiveness of both groups of antibodies has been proven in numerous clinical trials, which translated into numerous registrations of immunotherapeutics, not only in the indication of lung cancer. However, a large percentage of patients do not respond to anti-PD-L1/anti-PD-1 treatment, despite the presence of a predictive marker in the form of the PD-L1 molecule on the surface of cancer cells. Due to the presence of many mechanisms of cancer cells escaping from immune surveillance, the cancer very efficiently avoids recognition by the immune system ^1–3. In the presented study, immunological mechanisms of regulation of T lymphocyte activity in patients with non-small cell lung cancer as a result of stimulation with anti-PD-1 or anti-PD-L1 antibody were sought.

The study was conducted on a group of 15 patients with locally advanced or advanced lung adenocarcinoma. Six women and 9 men were qualified for the study, with an average age of 66 years. The patients had not previously received any systemic anticancer treatment, antibiotic therapy, drugs stimulating hematopoiesis, drugs affecting the activity of the immune system, and no autoimmune diseases were diagnosed in them. All methods were carried out in accordance with relevant guidelines and regulations. Informed consent was obtained from all subjects. The study protocol was approved by the Ethics Commitee of the Medical University of Lublin, Poland, under ethical permitt number KE-0254/318/2018).

• Isolation of peripheral blood mononuclear cells

Peripheral blood in the amount of 9 ml was collected into Monovette tubes with sodium heparin on the day of the planned bronchoscopy procedure. Bronchoaspirate in the amount of 15–20 ml was collected from the patient during bronchoscopy. Only patients diagnosed with lung adenocarcinoma without clinically significant molecular changes were qualified for the first line of anticancer treatment were included in the final statistical analysis.

Peripheral blood mononuclear cells (PBMC) were generated by layering blood diluted with PBS without calcium and magnesium ions (1:1 ratio) on Lymphoprep. The isolated PBMCs were rinsed twice with a buffered saline solution, counted in a Neubauer chamber, and their viability was assessed using trypan blue. The bronchospirate was filtered through sterile gauze to remove conglomerates of exfoliated epithelial cells and mucus strands. Then, it was centrifuged for 5 min at 2000 rpm.

• Conducting short-term cell cultures

The PBMC and bronchoaspirate fraction was cultured for 24 hours in three 6-well plates with RPMI 1640 medium (PAA Laboratories, USA) supplemented with antibiotics (1% penicillin-streptomycin-neomycin, Sigma Aldrich, USA) at 37ºC and 5% CO2 in different concentrations of nivolumab (5µg/ml, 10 µg/ml, 20 µg/ml culture) (Bristol-Myers Squibb, USA) or atezolizumab (150 µg/ml, 300 µg/ml, 600 µg/ml culture) (Roche, France). The scheme of the cultures carried out is shown in Fig. 1. On the day of completion of the culture, the cells were recovered from the culture well and subjected to immunophenotyping.

The fraction of control cells from peripheral blood and bronchoaspirate, not intended for culture, was aliquoted into cytometric tubes and incubated with a panel of monoclonal antibodies for 30 min. at 4°C. Then, the cells were washed from the remains of unbound antibodies with PBS buffer without Ca²⁺ and Mg²⁺ ions (centrifugation parameters: 2000 rpm/5 min), and a detailed analysis of the cell immunophenotype was performed in a flow cytometer.

In turn, cells subjected to short-term cultures with individual anti-PD-1 or anti-PD-L1 antibodies, after 24-hour incubation, were transferred from each culture well with a sterile Pasteur pipette to falcons with a capacity of 15 ml. The cell suspension was then centrifuged for 5 min at 2000 rpm, the supernatant was pooled and the resulting cell suspension was incubated with a panel of monoclonal antibodies for 30 min at 4°C. Then, the cells were washed from the remains of unbound antibodies with PBS buffer without Ca²⁺ and Mg²⁺ ions (centrifugation parameters: 2000 rpm/5 min), and a detailed analysis of the cell immunophenotype was performed in a flow cytometer.

Monoclonal antibodies conjugated with appropriate fluorochromes were used for cytometric analysis: anti-CD4-FITC, anti-CD274-FITC, anti-CD14-FITC, anti-CD8-PE, anti-CD14-PE, anti-CD25-APC, anti-CD69-APC, anti-CD95-APC, anti-CD279-APC (Becton Dickinson, USA).

Isolated cells from peripheral blood and bronchoaspirate were divided into 2 parts, the first of which was intended for the control immunophenotype analysis on the day of material collection (so-called control cells), and the second part was intended for establishing short-term cell cultures.

• Evaluation of the expression of intracellular markers

In the case of evaluation of intracellular expression of selected markers, in the first stage, control cells (from peripheral blood and bronchoaspirate) and cells subjected to short-term cultures were incubated with a panel of surface antibodies for 30 min at 4^oC. Then, the cells were washed from the remains of unbound antibodies with PBS buffer without Ca²⁺ and Mg²⁺ ions (2000 rpm/5 min) and the permabilization procedure was performed with the Fixation/Permablization Kit (Becton Dickinson, USA) consisting in incubation of cells with FixPerm buffer (incubation at 4^oC/40 minutes). The cells were then washed again in PermWash buffer (2000 rpm/5 min). Then, a set of antibodies for the evaluation of selected intracellular markers was added to the cells and incubated at 4^oC for 40 minutes. The final stage is washing the cells from the remains of unbound antibodies with PBS buffer without Ca²⁺ and Mg²⁺ ions (anti-CD4-FITC/anti-CD152-PE/anti-FoxP3-AF647 and anti-CD14-FITC/anti-B7-H4-PE/anti -IL10-APC), centrifugation parameters: 2000 rpm/for 5 min and analysis of the immunophenotype in a flow cytometer.

1. Evaluation of the percentage of helper, cytotoxic T lymphocytes and monocytes isolated from peripheral blood and bronchoaspirate, not stimulated with anti-PD-1 and anti-PD-L1 antibodies (control cells)

• Th or Tc lymphocytes expressed CD25⁺, CD69⁺ or CD95⁺

In the group of CD4⁺/CD25⁺ and CD4⁺/CD69⁺ lymphocytes, a higher percentage of these cells was observed in bronchoaspirate compared to cells isolated from peripheral blood (p = 0.003, p = 0.037, respectively). A similar observation was found in the percentage of Tc cells (CD25⁺ and CD69⁺) (p = 0.004 and p = 0.013, respectively) (Figs. 2A, 2B). The percentages of Th and Tc lymphocytes (CD95⁺) did not differ significantly between the tested materials (Fig. 2C).

• Th or Tc lymphocytes expressed PD-1 and monocytes expressed PD-L1

The percentage of both CD4⁺ and CD8⁺ T cells expressing the PD-1 molecule was almost 3-fold significantly higher in the population of cells isolated from bronchoaspirate compared to cells isolated from blood (p = 0.009 and p = 0.005, respectively) (Fig. 2D). A significantly higher percentage of monocytes expressing the PD-L1 molecule was isolated from bronchoaspirate than from peripheral blood (p = 0.012) (Fig. 2E).

• Monocytes expressed B7-H4 and IL-10

The percentage of monocytes expressing the B7-H4 molecule was more than 17 times higher among cells obtained from bronchoaspirate than among cells isolated from peripheral blood. There were no significant differences in the percentage of B7-H4⁺/IL-10⁺ monocytes between the materials tested (Fig. 2F).

• Th lymphocytes expressed CTLA-4 and FoxP3 protein

A significantly higher percentage of helper T cells expressing the CTLA-4 molecule was observed in peripheral blood compared to bronchoaspirate, while a non-significantly higher percentage of CD4⁺/CTLA-4⁺/FoxP3⁺ cells was found in bronchoaspirate (Fig. 2G).

2. Evaluation of the percentage of helper and cytotoxic T lymphocytes and monocytes isolated from peripheral blood stimulated with anti-PD-1 antibody

• Th or Tc lymphocytes expressed CD25⁺, CD69⁺ or CD95⁺

In the group of Th lymphocytes, a significant increase in the percentage of cells expressing the CD25 molecule was observed in the culture with the addition of nivolumab at 5 µg/ml and 20 µg/ml compared to unstimulated culture (p = 0.004 and p = 0.004, respectively). Similarly, in the CD8⁺ T cell group, an increase in the percentage of CD25-positive cells was observed at all nivolumab concentrations compared to unstimulated culture, with the increase being statistically significant at the nivolumab concentration of 10 µg/ml culture (p = 0.032) (Fig. 3A).

The percentage of Th cells expressing the CD69 molecule increased at all concentrations of nivolumab compared to unstimulated culture, with the increase being statistically significant at nivolumab concentrations of 10 µg/ml culture (p = 0.033) and 20 µg/ml culture (p = 0.016) and at a concentration of 20µg/ml of culture compared to the lowest concentration of nivolumab used (p = 0.049). In the group of CD8⁺ T cells, a significant increase in the percentage of cells expressing the CD69 molecule was observed at each concentration of nivolumab (5 µg/ml; 10 µg/ml; 20 µg/ml culture) compared to the control culture (p = 0.017; p = 0.006, p = 0.004) (Fig. 3B).

The percentage of Th cells expressing the CD95 molecule was higher at all concentrations of nivolumab compared to the unstimulated culture, with a significant result obtained at the concentration of 10µg/ml and 20µg/ml (p = 0.01, p = 0.004, respectively). In the Tc cell population, there was a non-significant increase in the percentage of CD95-positive cells in culture stimulated with 5 µg nivolumab and a significant increase after stimulation with 10 µg nivolumab (p = 0.016) compared to unstimulated culture, with a significant decrease in the percentage stimulated with 20 µg nivolumab vs. lowest concentration used (p = 0.033) (Fig. 3C).

• Th or Tc lymphocytes expressed PD-1 and monocytes expressed PD-L1

In the group of Th lymphocytes, a statistically significant increase in the percentage of cells expressing the PD-1 molecule was observed at each concentration of nivolumab (5µg/ml, 10µg/ml and 20µg/ml) compared to unstimulated culture (p = 0.007; p = 0.004; p = 0.01). Among cytotoxic T cells, the percentage of cells expressing the PD-1 molecule increased at all concentrations of nivolumab, with the increase being statistically significant at 10µg/ml (p = 0.003) compared to control culture and compared to the lowest concentration used (p = 0.008) and at 20µg/ml (p = 0.006) compared to unstimulated culture (Fig. 3D).

In the monocyte population, compared to the unstimulated culture, the percentage of CD14⁺ PD-L1⁺ cells increased non-significantly after stimulation with 5µg/ml nivolumab, and then decreased with increasing nivolumab concentration and was non-significantly lower than in control cultures (Fig. 3E).

• Monocytes expressed B7-H4 and IL-10

In the monocyte population, the percentage of cells expressing the B7-H4 molecule decreased in the cultures stimulated with each concentration of nivolumab tested compared to the control culture, with the decrease being significant only at the concentration of 10µg/ml nivolumab (p = 0.037). The percentage of CD14- and B7-H4-positive cells with intracellular expression of IL-10 did not change statistically significantly at the concentration of 5µg/ml compared to the unstimulated culture, while in the cultures stimulated with 10µg and 20µg nivolumab/ml the percentage of these cells decreased significantly relative to the culture control (Fig. 3F).

• Th lymphocytes expressed CTLA-4 and FoxP3 protein

The percentage of helper T cells expressing the CTLA-4 molecule decreased with increasing concentration of nivolumab compared to unstimulated culture, with statistical significance only reached in the culture with 20µg nivolumab per milliliter of culture (p = 0.047). Analyzing the intracellular expression of the FoxP3 protein, in the group of helper T cells expressing the CTLA-4 molecule, a non-significant increase in the percentage of CD4⁺/CTLA-4⁺/FoxP3⁺ cells was observed in the culture stimulated with nivolumab at a concentration of 5µg/ml of culture compared to the unstimulated culture, followed by of these cells began to decrease non-significantly with increasing concentration of nivolumab (10µg/ml and 20µg/ml) (Fig. 3G).

3. Evaluation of the percentage of helper and cytotoxic T lymphocytes and monocytes isolated from peripheral blood stimulated with anti-PD-L1 antibody

• Th or Tc lymphocytes expressed CD25⁺, CD69⁺ or CD95⁺

In the group of Th cells, the percentage of cells expressing the CD25 molecule significantly increased after the use of atezolizumab at a concentration of 150 µg/ml culture (p = 0.008), 300 µg/ml (p = 0.013) and 600 µg/ml (p = 0.016) compared to unstimulated cultures. In the Tc cell group, the percentage of CD25-positive cells was non-significantly increased at each concentration of atezolizumab compared to unstimulated culture (Fig. 4A). In the group of Th cells, a significant increase in the percentage of CD69-positive cells was observed after the use of anti-PD-L1 antibody (150µg/ml - p = 0.009, 300µg/ml- p = 0.026 and 600µg/ml p = 0.008) and among the lymphocyte population Tc, (respectively: 150µg/ml - p = 0.003, 300µg/ml - p = 0.041; 600µg/ml - p = 0.003) compared to the control culture (Fig. 4B). Among Th lymphocytes, a significant increase in the percentage of cells expressing the CD95 molecule was observed at all concentrations of atezolizumab compared to the control culture (150 µg - p = 0.041; 300 µg - p = 0.021; 600 µg - p = 0.026). In the group of cytotoxic T lymphocytes, the percentage of cells expressing the CD95 molecule, compared to the control culture, increased significantly at all concentrations of atezolizumab: 150, 300 and 600µg/ml (respectively: p = 0.013, p = 0.010, p = 0.003) (Fig. 4C).

• Th or Tc lymphocytes expressed PD-1 and monocytes expressed PD-L1

In the group of CD4⁺ T lymphocytes, a significant increase in the percentage of cells expressing the PD-1 molecule was observed at each of the concentrations of atezolizumab compared to the control culture (150µg - p = 0.021; 300µg - p = 0.004; 600µg - p = 0.026). Among the cytotoxic T cells, a significant increase in the percentage of PD-1-positive cells was observed in each of the concentrations of atezolizumab used (respectively: 150 µg - p = 0.004, 300 µg - p = 0.006 and 600 µg - p = 0.006) compared to the control culture (Fig. 4D). In the group of monocytes, after the use of atezolizumab at a concentration of 150µg/ml, there was a non-significant decrease in the percentage of cells expressing the PD-L1 molecule compared to the control culture. Comparing the percentage of analyzed cells in cultures stimulated with 150µg and 300µg atezolizumab, a significant (p = 0.041) decrease in the percentage of analyzed cells was observed at the concentration of 600µg/ml (Fig. 4E).

• Monocytes expressed B7-H4 and IL-10

Among monocytes, a significant decrease in the percentage of cells expressing the B7-H4 molecule was observed compared to the culture not stimulated with atezolizumab at a concentration of 150 µg/ml (p = 0.037) and 300 µg/ml (p = 0.022), while at a concentration of 600 µg/ml this decrease was statistically insignificant. In the group of monocytes expressing the B7-H4 molecule on their surface, a non-significant decrease in the percentage of cells with intracellular expression of IL-10 at 150µg/ml and 300µg/ml was observed, followed by a non-significant increase in this percentage at the concentration of 600µg atezolizumab compared to the control culture (Fig. 4F).

• Th lymphocytes expressed CTLA-4 and FoxP3 protein

In the group of helper T cells, a significant decrease in the percentage of cells expressing the CTLA-4 molecule was observed in the cultures stimulated with 150 µg (p = 0.008) and 300 µg atezolizumab (p = 0.041) compared to the control culture. In the group of CTLA-4-positive helper T cells, the percentage of cells with intracellular expression of FoxP3 protein increased non-significantly at the lowest concentration of atezolizumab used (150µg/ml) compared to the control culture, then decreased at the next concentration (300µg/ml) and then increased at a concentration of 600µg atezolizumab (Fig. 4G).

4. Evaluation of the percentage of helper and cytotoxic T lymphocytes and monocytes isolated from bronchoaspirate stimulated with anti-PD-1 antibody

• Th or Tc lymphocytes expressed CD25⁺, CD69⁺ or CD95⁺

In the population of Th lymphocytes, a non-significant decrease in the percentage of cells expressing the CD25 molecule was observed at each of the anti-PD-1 concentrations used compared to unstimulated culture. In the Tc cell group, the percentage of cells expressing the CD25 molecule was non-significantly lower at all nivolumab concentrations used than in the control culture (Fig. 5A). In the Th lymphocyte population, a non-significant increase in the percentage of CD69-positive cells was observed at the lowest concentration of nivolumab (5µg/ml) compared to the control culture. Then, at the concentration of 10µg/ml, the percentage decreased insignificantly, and after application of 20µg nivolumab in culture, the percentage of CD4⁺/CD69⁺ cells increased insignificantly compared to unstimulated culture. In the Tc cell group, the percentage of CD69-positive cells increased non-significantly at the lowest concentration of nivolumab 5µg/mL relative to the control culture, then decreased at the next concentration (10µg/mL) compared to the control culture, and increased again non-significantly at the highest (20µg/ml) of nivolumab relative to the control culture (Fig. 5B). In the group of Th lymphocytes, a decrease in the percentage of cells expressing the CD95 molecule was observed at all concentrations of nivolumab compared to the control culture. Similarly, in the group of CD8⁺ T cells, a non-significant decrease in the percentage of cells expressing the CD95 molecule was observed with increasing nivolumab concentration compared to unstimulated culture (Fig. 5C).

• Th or Tc lymphocytes expressed PD-1 and monocytes expressed PD-L1

Among the helper T cells, a decrease in the percentage of cells expressing the PD-1 molecule was observed at all concentrations of nivolumab compared to the control culture, and it was statistically significant at the concentration of nivolumab 20µg/ml (p = 0.049) compared to the control culture. In the group of Tc cells, the percentage of cells expressing the PD-1 molecule was lower compared to the control culture, but significantly lower at the concentration of nivolumab 20µg/ml (p = 0.036) (Fig. 5D). At each of the concentrations of nivolumab used, the percentage of monocytes expressing the PD-L1 molecule was non-significantly lower compared to the control culture, with this value being the lowest at the highest concentration used (Fig. 5E).

• Monocytes expressed B7-H4 and IL-10

In the monocyte group, a non-significant increase in the percentage of cells expressing the B7-H4 molecule was observed with increasing concentration of nivolumab (5µg/ml, 10µg/ml, 20µg/ml) compared to the control culture. The expression of the B7-H4 molecule on the surface of CD14-positive cells was non-significantly decreased with increasing concentration of nivolumab compared to control culture. In the CD14-positive cell population, no significant differences in the percentage of cells expressing the B7-H4 molecule and intracellular IL-10 expression were observed at all concentrations of nivolumab compared to the control culture (Fig. 5F).

• Th lymphocytes expressed CTLA-4 and FoxP3 protein

In the helper T cell group, the percentage of cells expressing the CD152 (CTLA-4) molecule was non-significantly increased at each concentration (5µg/ml, 10µg/ml and 20µg/ml culture) of nivolumab compared to the culture not stimulated with anti-PD-1 antibody. Among the CD4-positive T cells, the percentage of helper cells expressing the CTLA-4 molecule and intracellular FoxP3 protein expression was not significantly changed compared to the unstimulated culture (Fig. 5G).

5. Evaluation of the percentage of helper and cytotoxic T lymphocytes and monocytes isolated from bronchoaspirate stimulated with anti-PD-L1 antibody

• Th or Tc lymphocytes expressed CD25⁺, CD69⁺ or CD95⁺

In the helper T cell group, a statistically significant decrease in the percentage of CD4⁺/CD25⁺ cells (p = 0.016) compared to 300 µg/ml was observed in cultures stimulated with atezolizumab at a concentration of 600 µg/ml. At the other concentrations of atezolizumab (150µl and 300µg/ml), a non-significant decrease and an increase in the percentage of cells expressing the CD25 molecule were observed, respectively, compared to the control culture. In the group of CD8⁺ T cells, only cultures stimulated with anti-PD-L1 antibody at a concentration of 300 µg/ml showed a non-significant increase in the percentage of CD8⁺/CD25⁺ cells compared to the control culture. In the remaining concentrations of this antibody, the percentage of analyzed cells was insignificantly lower than in control cultures (Fig. 6A). Both the helper (CD4-positive) and cytotoxic (CD8-positive) T cells group showed a non-significant increase in the percentage of cells expressing the CD69 molecule at all atezolizumab concentrations compared to the control culture (Fig. 6B). The percentage of CD95-positive Th cells was non-significantly higher compared to the control culture only in cultures stimulated with anti-PD-L1 antibody at a concentration of 600 µg/ml. At the remaining concentrations of anti-PD-L1 antibody, the percentage of these cells was non-significantly lower than in the control culture. The percentage of CD95-positive Tc cells was non-significantly lower at each concentration of atezolizumab used compared to the control culture (Fig. 6C).

• Th or Tc lymphocytes expressed PD-1 and monocytes expressed PD-L1

Among the helper T cell population, the percentage of cells expressing the PD-1 surface molecule was non-significantly lower at all atezolizumab concentrations compared to the control culture. In the group of cytotoxic T cells, a non-significant decrease in the percentage of cells expressing the PD-1 molecule was observed (Fig. 6D). In the monocyte group, a non-significant decrease in the percentage of cells expressing the PD-L1 molecule was observed (Fig. 6E).

• Th lymphocytes expressed CTLA-4 and FoxP3 protein

Among the T helper cell population, the percentage of cells expressing the CTLA-4 molecule was non-significantly higher at all concentrations of atezolizumab used compared to the control culture. In the group of double-positive CD4⁺/CTLA-4⁺ lymphocytes, the percentage of cells expressing intracellular FoxP3 protein was non-significantly higher at all atezolizumab concentrations compared to the control culture (Fig. 6F).

• Monocytes expressed B7-H4 and IL-10

In the monocyte population, a non-significant increase in the percentage of cells expressing the B7-H4 molecule was observed at all concentrations of atezolizumab compared to control culture. In the monocyte group, the percentage of cells simultaneously expressing the B7-H4 molecule and intracellularly expressing IL-10 after stimulation with each concentration of atezolizumab was not significantly changed compared to the control culture (Fig. 6G).

Tables listing all percentages of analyzed cells isolated from peripheral blood and bronchoaspirate (with standard deviations) stimulated with nivolumab or atezolizumab are a supplement to the article (at the end of the article).

In this study, an attempt was made to analyze in vitro changes in the immunophenotype and T lymphocyte activity after the use of anti-PD-1 and anti-PD-L1 antibodies. The study proved that checkpoint inhibitors (nivolumab and atezolizumab) have an impact on the blockade of the PD-1/PD-L1 connection, but also on many functions of immune cells manifested by variable expression of markers analyzed in the study on the surface of T lymphocytes or monocytes, which indicate the current state of the immune system.

The CD25 molecule forms a receptor for interleukin 2, it appears on activated T cells (it is not occured on naive T cells)⁴. In this study, helper and cytotoxic T lymphocytes expressing the CD25 molecule were analyzed and an almost 10-times higher percentage of CD4⁺/CD25⁺ and CD8⁺/CD25⁺ T cells was observed in the bronchoaspirate than in the peripheral blood, which may indicate increased inflammation and increased cell activation in the bronchoaspirate. around the tumor. In addition, immunohistochemical analysis confirmed that the presence of activated T lymphocytes has a beneficial effect on the prognosis for patients with NSCLC⁵.

The CD69 molecule is widely known as an early marker of leukocyte activation and its main function is to stimulate the proliferation of T lymphocytes in various tissues. Despite this, it turned out that it is also involved in inhibiting the activation of T lymphocytes in the tumor microenvironment⁶. In the presented study, after using different concentrations of nivolumab or atezolizumab, the percentage of CD4-positive and CD8-positive cells expressing the CD69 molecule increased in cultures of cells isolated from peripheral blood, while the expression of this molecule significantly decreased with the increase in the concentration of anti-PD-1 or anti-PD-L1 antibodies compared to control culture. Such relationships were not observed in cultures of cells isolated from bronchoaspirate. This may indicate a slight response of these cells to stimulation with anti-PD-1 or anti-PD-L1 antibodies and confirm the results of the cited studies on the inhibition of T lymphocyte activation in the tumor microenvironment.

The CD95 molecule (Fas receptor) on the surface of T lymphocytes is primarily a marker of apoptosis of the cell that will undergo this process after binding to the FasL ligand ⁷. The results of the study indicate that with the increase in the concentration of nivolumab, with which the cells isolated from bronchoaspirate were stimulated, the percentage of helper and cytotoxic T cells expressing the CD95 molecule decreased and the expression intensity of this molecule decreased. In the case of stimulation of the studied cell population with atezolizumab, at the initial concentration (150µl/ml) the percentage of analyzed T lymphocytes decreased, then increased with increasing concentration, to reach a value comparable to the initial value in the control culture at the highest concentration (600µl/ml). This may indicate the inhibition of apoptosis of helper and cytotoxic T lymphocytes in the tumor microenvironment after the use of atezolizumab, which may significantly increase the infiltration of tumor tissue by active cells of the immune system. On the other hand, in cultures of cells isolated from peripheral blood, the opposite situation was observed, when the percentage of tested lymphocytes increased after both nivolumab and atezolizumab, while the expression of the CD95 molecule decreased. This situation may indicate the influence of anti-PD-1 or anti-PD-L1 antibodies on the stimulation of early apoptosis of circulating helper and cytotoxic T lymphocytes.

According to Richards et al. in lung cancer, CD8-positive lymphocytes express the CD95L molecule, which induces apoptosis in CD95-positive cells. The authors indicate that the anticancer response to immune checkpoint inhibitors may depend on the sensitivity of T lymphocytes to apoptosis, i.e. on the expression of the CD95 molecule on their surface ⁸.

It would seem that the functions of PD-1/PD-L1 molecules on the surface of cells of the immune system have been thoroughly investigated, but many studies have been carried out on too small groups of patients and they do not allow to clearly define all situations in which these molecules are involved.

Studies conducted over the last few years indicate that cytotoxic T lymphocytes are mainly responsible for the direct destruction of cancer cells. Many cells present in the tumor microenvironment cooperate directly or indirectly with CD8-positive T lymphocytes as pro- or anti-cancer cells (including dendritic cells, natural killer cells and tumor-associated macrophages). In order for CD8-positive T cells to begin their cytotoxic function, dendritic cells must present them with a tumor antigen in the context of MHC class I molecules. During this time, helper T cells secrete cytokines that directly support the differentiation and activation of cytotoxic T cells. In addition, another indirect mechanism of CD8⁺ T lymphocyte support is the secretion by NK cells and T helper cells of chemokines affecting the maturation and chemotaxis of other innate response cells, including macrophages and dendritic cells ^9,10.

Overexpression of the PD-1 molecule on cytotoxic T lymphocytes and its stimulation by specific ligands contributes to TCR dysfunction and, consequently, to blocking the activity of these cells. Immunotherapy with the use of anti-PD-1 antibodies inhibits the extinction of the activity of these cells, leading to the re-activation of their functions. However, recent studies have shown that reactivated T cells are more likely to be from a group of freshly tumor-infiltrating cells, as they are less regenerative than originally thought ¹¹.

A group of researchers led by Dr. Mazzaschi tried to answer the question of whether the analysis of the tumor microenvironment can predict the response to immunotherapy in NSCLC patients. An interesting phenomenon in the analyzed study was also the fact that in the glandular subtype an almost two-fold increase in the number of CD3-positive T lymphocytes and a three-fold decrease in the number of CD4-positive lymphocytes was observed compared to the squamous subtype. On the other hand, no changes in the number of cytotoxic CD8-positive T lymphocytes were observed when comparing both histological types of lung cancer. In turn, regulatory T lymphocytes expressing CD57 and CD25 and FoxP3 markers were the least numerous population, their percentage did not exceed 2% of all lymphocytes. However, an almost 3-fold higher percentage of T lymphocytes expressing CD57 and PD-1 was observed in the squamous subtype than in the glandular subtype. With regard to the correlation between the tumor stage and the number of infiltrating T cells with the expression of the PD-1 molecule, the researchers observed a two-fold lower number of these cells in stage I compared to stage II and III disease. In addition, the researchers took into account the presence of PD-L1 on cancer cells and PD-1 on T cells in the tumor microenvironment and correlated it with the clinical effect of immunotherapy. It turned out that patients with tumors with high expression of PD-L1 and a low percentage of T lymphocytes (subtype III – PD-L1⁺/TILs⁻) achieved longer overall survival times than patients with subtype II–PD-L1⁻/TILs⁻ (16, 2 months vs. 6.1 months) ¹².

Jin et al. correlated the presence of tumor-infiltrating T cells with the expression of PD-L1 on the surface of tumor cells and observed that a high percentage of TILs also had a high expression of PD-L1 on tumor cells. The authors conclude that the induction of high PD-L1 expression is certainly one of the mechanisms of defense of cancer cells against the activity of the immune system. At the same time, it is also a predictive marker of the response of such patients to immunotherapy ¹³.

Gros et al. observed that CD8-positive T cells expressing TCR specific for melanoma antigens were present in the CD8+/PD-1 + lymphocyte fraction, but not in the fraction of cells without the PD-1 molecule. This may suggest that these cells are ready to recognize cancer antigens, and only require unlocking their cytotoxic activity ^14,15.

Literature data indicate that the B7-H4 transmembrane molecule has the function of inhibiting helper and cytotoxic T lymphocytes ^16,17. An attempt was made to correlate the expression of B7-H4 on cancer cells with clinical observations of patients treated with nivolumab in order to define this molecule as a potential prognostic factor in NSCLC. In the studies of Genova et al., the expression of the B7-H4 molecule was determined by immunohistochemical staining, where only 37% of the collected tumors showed the expression of this molecule. It was observed that the presence of the B7-H4 molecule on cancer cells was significantly associated with shorter PFS and OS ¹⁸. In the analyzed project, the B7-H4 molecule was determined on monocytes isolated from bronchoaspirate stimulated with nivolumab in in vitro cultures, where with increasing concentration of this antibody (5µl/ml, 10µl/ml and 20µl/ml) the percentage of analyzed cells increased, while B7-H4 expression on monocytes decreased. This may indicate that the anti-PD-1 antibody not only affects the reactivation of the function of the innate response cells, but also affects the expression of immunosuppressive markers on the surface of the innate response cells.

The tumor microenvironment has a huge and undeniable impact on the activity of the cells of the immune system. The division of tumor types in terms of the presence of the immune system into the following types is well described in the literature: hot - with strong infiltration of cancer cells by the inactive immune system; the cold type - with the lack of elements of the immune system in the tumor tissue, and the infiltrating type, in which the immune system marginally tries to penetrate the tumor tissue, but the strong immunosuppressive microenvironment does not allow it to do so^19,20. Moreover, it should be borne in mind that effective anti-tumor defense requires the cooperation of both the specific response and active non-specific response cells. In the presented study, no significant effect of the applied antibodies on the activity of monocytes isolated from bronchoaspirate was observed. This may indicate that a single-point approach to immunotherapy - aimed only at stimulating T lymphocyte activity - may not be sufficient to achieve a clinical effect. A comprehensive approach to immunotherapy, in which the activity of T lymphocytes is reactivated and at the same time the activity of non-specific response cells is stimulated, seems to be an interesting approach in the modern treatment of cancer.

In conclusion, a comprehensive analysis of changes in the percentages of T lymphocytes and monocytes examined allows us to draw two significant conclusions:

In the bronchoaspirate of patients with non-small cell lung cancer, compared to peripheral blood, there is a higher percentage of T helper and cytotoxic PD-1 lymphocytes - positive. Also in the bronchoaspirate, PD-L1 monocytes are present - positive, which may indicate the immunosuppression of the non-specific response in this compartment compared to peripheral blood.

Both anti-PD-1 and anti-PD-L1 antibody stimulation had a more significant effect on the activation of the specific response of peripheral blood mononuclear cells compared to cells in bronchoaspirate, which may be due to their functional extinction in bronchoaspirate. This material may be a model of the influence of the neoplastic environment on the immune system in the lungs.

CD25-positive and CD69-positive helper and cytotoxic T cells are present in the bronchoaspirate of patients with NSCLC, but these cells seem unable to form an immune synapse due to the low expression of the CD28 molecule.

A decrease in the expression of the PD-1 molecule on the surface of the cells of the specific response was observed on mononuclear cells of peripheral blood and bronchoaspirate after stimulation with both anti-PD-1 and anti-PD-L1 antibodies, regardless of the concentration of antibodies used. This indicates the possibility of restoring T lymphocyte function with the use of a minimal dose of anti-PD-1 or anti-PD-L1 antibodies.

All data generated or analysed during this study are included in this published article and its supplementary information files.

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No competing interests reported.

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The Effect of Different Concentrations of Anti-PD-1 and Anti-PD-L1 Antibodies on the Activity of Immune System Cells in Patients With Non-small Cell Lung Cancer

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1. Evaluation of the percentage of helper, cytotoxic T lymphocytes and monocytes isolated from peripheral blood and bronchoaspirate, not stimulated with anti-PD-1 and anti-PD-L1 antibodies (control cells)

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