Reprogrammed CD8+ T-Lymphocytes Isolated from Bone Marrow Have Anticancer Potential in Lung Cancer

CD8+ T-lymphocytes play a key role in antitumor immune response. Patients with lung cancer often suffer from T-lymphocyte dysfunction and low T-cell counts. The exhaustion of effector T-lymphocytes largely limits the effectiveness of therapy. In this study, reprogrammed T-lymphocytes used MEK inhibitors and PD-1 blockers to increase their antitumor activity. Antitumor effects of reprogrammed T-lymphocytes were shown in vitro and in vivo in the Lewis lung carcinoma model. The population of T- lymphocytes with persistent expression of CCR7 was formed as a result of reprogramming. Reprogrammed T-lymphocytes were resistant to apoptosis and characterized by high cytotoxicity against Lewis lung carcinoma (LLC) cells in vitro. Administration of reprogrammed T-lymphocytes to C57BL/6 mice with LLC reduced the number of lung metastases. The antitumor effect resulted from the elimination of tumor cells and cancer stem cells, and the effect of therapy on cytotoxic T-lymphocyte counts. Thus, reprogramming of T-lymphocytes using MEK inhibitors is a promising approach for targeted therapy of lung cancer.


Introduction
Lung cancer is the leading cause of cancer-related deaths among men and women [1]. Major advances have been made using targeted therapies and checkpoint inhibitors, which improve the survival of patients with lung cancer [2]. However, some forms of lung cancer remain resistant to treatment.
Cytotoxic CD8 + T-lymphocytes are the most powerful effector cells in immune response, which play a key role in antitumor immunity. CD8 + T-lymphocytes induce tumor cell death by producing cytotoxic molecules such as perforin and granzymes [3]. In addition, interferon-γ secreted by CD8 + T-lymphocytes enhance antitumor properties of other immune cells [4]. However, T-lymphocytes become dysfunctional in the tumor microenvironment. In addition, CD8 + T-lymphocyte counts are significantly lower in patients with lung cancer compared with healthy individuals. High levels of CD8 + T cells after lung cancer therapy may be a predictor of better survival rates in patients [5].
Tumor and tumor microenvironments suppress proliferation of CD8 + T-lymphocytes and decrease their numbers [6]. Interestingly, during interaction with the tumor, CD8 + D. Goldberg Research Institute of Pharmacology and Regenerative Medicine (protocol No. 189092021). The animals were maintained in accordance with the European Convention for the Protection of Vertebrates (Strasbourg, 1986); Principles on Good Laboratory Practice (OECD, ENV/ MC/ CUEM (98)17, 1997).

Lewis Lung Carcinoma Cell Line
The Lewis lung carcinoma (LLC) cell line of C57BL strain was used in experiments in vivo and in vitro (400263 CLS Cell Lines. Service, GmbH, Köln, Germany). LLC cells were established from the spontaneous lung adenocarcinomas that occur in C57BL/6 mice.

Lewis Lung Carcinoma Cell Culture
The LLC cells were plated at a seeding density of 3 × 10 5 cells/1 cm 2 in the T-25 flask. The cells were maintained in RPMI 1640 medium supplemented with 2 mM L-glutamine and 10% fetal bovine serum (FBS, Sigma-Aldrich, St. Louis, MO, USA) at 37 • C in a humidified atmosphere containing 5% CO 2 . The culture medium was changed 2-3 times per week.

Orthotopic Model of Lewis Lung Carcinoma
Under sterile conditions, mice fur was removed on the left side of the chest above the lower line of the ribs and just below the lower border of the scapula. Each animal was injected with 50 µL suspension of LLC cells (1.5 × 10 6 cells) by an insulin syringe with 30 G needle to a depth of 5 mm into the left lung between the 6th and 7th rib [17,18]. All manipulations were accomplished with isoflurane inhalation using an inhalation anesthesia machine (UGO BASIELE, model 21050, Comerio, Italy). After 7 days of LLC cell inoculation, the animals were euthanized.

Study Design
The study design is shown in Figure 1. In the first stage, we carried out reprogramming of CD8 + T-lymphocytes derived from the bone marrow. Next, we evaluated migration of reprogrammed CFSE-labeled CD8 + T-lymphocytes into the lungs of the recipient mice. The cells were injected into the tail vein. In the third stage, we assessed cytotoxic activity of reprogrammed CD8 + T-lymphocytes in the LLC cell culture in vitro. Additionally, we studied apoptosis of reprogrammed CD8 + T-lymphocytes. Finally, the antitumor and antimetastatic activity of reprogrammed CD8 + T-lymphocytes was evaluated in vivo.

Isolation of Mononuclear Cells
Mononuclear cells from blood, lungs, and bone marrow were isolated as described earlier [19,20].

Magnetic Separation of CD8 + T-Lymphocytes
After isolation of mononuclear cells from the bone marrow, a magnetic separation was performed to enrich the cell fraction with CD8 + T-lymphocytes. The cell suspension was enriched with naive CD8 + T-lymphocytes (CD3 + CD8 + CD44 − CD62L + ) by magnetic separation. Enrichment was performed following a standard protocol using a mouse kit (EasySep TM Mouse Naive CD8 + T Cell Isolation Kit, as recommended by the manufacturer (StemCell Technologies, Vancouver, BC, Canada).
An antigen-presenting mix was prepared from LLC cell lysate by using a freeze-thaw cycle in 0.85% NaCl solution. The cycle was repeated five times in rapid succession from −70 • C to 37 • C, and then refrozen and stored at −70 • C before use ( Figure 1). After the final thawing, the lysate was stained by trypan blue (Sigma-Aldrich, St. Louis, MO, USA) [21]. The preparation of adjuvant (Freund's adjuvant) for the antigen-presenting mix was carried out according to the manufacturer's standard protocol (Sigma-Aldrich, St. Louis, MO, USA). Freund's adjuvant solution was mixed with the tumor cell lysate (3 × 10 4 /mL) at a 1:1 ratio to form a thick emulsion ( Figure S1b).
CD8 + T-lymphocytes were pre-incubated for 2 h before reprogramming. For reprogramming, 50 µL of an antigen-presenting mix with 1 µM MEK inhibitor was added to a flask with CD8 + T-lymphocytes of a given population (the concentration of CD8 + T-lymphocytes was 1 × 10 8 /mL, the volume of the medium in the flask was at least 5 mL). The resulting cell suspension was incubated for 48 h at 37 • C and 5% CO 2 . Reprogrammed CD8 + T-lymphocytes of a given phenotype were incubated for 2 h with human monoclonal antibody (MAT) nivolumab at a concentration 10 µg/mL in order to protect cells from the humoral action of LLC. At the end of the incubation cycle, suspensions were washed 2 times in the medium recommended for CD8 + T-lymphocytes ( Figure S1c). Immunophenotype and cytotoxicity of reprogrammed CD8 + T-lymphocytes were analyzed (Cytation 5).

CFSE Staining of CD8 + T-Lymphocytes
CFSE labeling of CD8 + T-lymphocytes isolated from bone marrow of donor mice was used to determine the content of CD8 + T-lymphocytes in the lungs of recipient mice. CFSE staining of CD8 + T-lymphocytes was performed according to the manufacturer's instruction (BD Biosciences, San Jose, CA, USA). All experimental samples, including controls, were analyzed using the same instrument settings. The cell population of intact control animals served as a negative control. The percentage of CFSE-positive cells in the resulting population was determined by analyzing data in flow-cytometry software, excluding doublets and other cell conglomerates from the analysis.

CD8 + T-Lymphocytes Injection
To evaluate the migration of CD8 + T-lymphocytes into the lungs of recipient mice from intact control and recipient mice with LLC, CFSE-labeled reprogrammed and nonreprogrammed CD8 + T-lymphocytes were injected intravenously at 1 × 10 6 cells/mouse in 0.1 mL PBS once.
To assess the antitumor and antimetastatic activity, CD8 + T-lymphocytes were administered intravenously to recipient mice with LLC at 1 × 10 6 cells/mouse in 0.1 mL of PBS on the 4th and 6th days of the experiment.

Detection of the CCR7 Expression, Cytotoxicity, and Apoptosis of CD8 + T-Lymphocytes In Vitro
Images of cells were obtained using the cell-imaging Cytation 5 (BioTek Instruments, Inc., Winooski, VT, USA) equipped with the following cubes: DAPI (blue), GFP (green), RFP (yellow).
To assess CCR7 expression, T-lymphocytes were stained with anti-CCR7 antibodies and polyclonal secondary antibody donkey anti-Rabbit IgG H&L Alexa Fluor ® 555 (all Abcam, Cambridge, MA, USA). Nuclei were additionally stained with Hoechst 34580 (blue); CD8 FITC (green) was used to CD8 + T-lymphocyte detection. The percentage of CD8+CCR7+ cells were determined as the ratio of cells counted in green and red channel to total cells counted using blue (DAPI) channel.
Cytotoxicity and apoptosis of CD8 + T-lymphocytes were studied in cell culture of LLC. After co-incubation, CD8 + T-lymphocytes and LLC cells were stained with Hoechst 34580 (for nuclear staining) and 7-AAD (for apoptotic cells detection). Preliminary CD8 + T-lymphocytes were stained with CFSE BD Horizon. Cytotoxicity of CD8 + T-lymphocytes in LLC culture was assessed by analyzing the ratio of cells counted in the blue and red channels to the total number of LLC (percentage of dead Hoechst + 7AAD + LLC). Determination of the percentage of dead T-lymphocytes Hoechst + CFSE + was made by the ratio of cells counted in blue and green channel to total cells.
All images were obtained with Cytation 5 (4× or 20× magnification) followed by cell analysis using Gen5™ data-analysis software (BioTek, Instruments, Friedrichshall, Germany). Prior to the analysis, images were preprocessed to align the background.

Histological Examination of the Lungs
Lung preparations were fixed in 10% neutral buffered formalin, passed through increasing concentrations of alcohol to xylene and embedded in paraffin wax according to the standard method, then sectioned into 5 µm-thick slices, and stained with hematoxylin and eosin [20,23].

Assessment of Tumor Growth
The effect of cell therapy on LLC growth was assessed by statistical comparison of the tumor node volume in the control and experimental groups at the different observation periods, according to the duration of tumor growth retardation and tumor growth inhibition index (TGII) [24] TGII = ( where Vc and Ve are the average node volume in the control and experimental groups.

Assessment of Tumor Volume
Linear dimensions of tumor nodes were measured in orthogonal planes and their volume was calculated in the elliptical approximation [25]. The tumor was measured with a caliper and the volume of the tumors was calculated by the formula:

Statistical Analysis
Statistical analysis was performed by methods of variational statistics using the SPSS 12.0 software (SPSS Inc., Chicago, IL, USA). The arithmetic mean (M), error of the mean (m), and the probability value (p) were calculated. The difference between the two compared values was significant at p < 0.05.

Study of CCR7 Marker Expression by Reprogrammed CD8 + T-Lymphocytes of C57BL/6 Mice Bone Marrow In Vitro
We studied the expression of chemokine receptor CCR7 by CD8 + T-lymphocytes in vitro to evaluate the effectiveness of the reprogramming ( Figure 2). Reprogrammed CD8 + T-lymphocytes have higher expression levels of chemokine receptor CCR7 than naive CD8 + T-lymphocytes in CD8 + T-lymphocyte culture ( Figure 2). Exhaustion of reprogrammed CD8 + T-lymphocytes was performed to assess the persistence of the changes caused by the MEK inhibitor and nivolumab. Exhaustion did not cause changes in the CCR7 expression by reprogrammed CD8 + T-lymphocytes, which indicates that the changes induced by the MEK inhibitor and nivolumab are stable ( Figure 2).

Study of Apoptosis of Naive and Reprogrammed CD8 + T-Lymphocytes in the Lewis Lung Carcinoma Cell Culture
At the end of the cultivation cycle of naive CD8 + T-lymphocytes, the percentage of dead cells was 2.05% of the total cell number in the standard medium. Reprogrammed

Control of CD8 + T-Lymphocytes
At the end of the cultivation cycle of naive CD8 + T-lymphocytes, the percentage of dead cells was 2.05% of the total cell number in the standard medium. Reprogrammed CD8 + T-lymphocytes are more stable under cultivation: 0.42% of the cells were in apoptosis ( Figure 3).

Cocultivation of CD8 + T-Lymphocytes with Cancer Cells
In a 0.25:1 dilution, we observed that naive CD8 + T-lymphocytes displayed increa apoptosis in LLC cell culture. The level of apoptosis was more than two-fold highe comparison with that of the control lymphocytes.
An increase in the concentration of naive CD8 + T-lymphocytes in the LLC cell ture caused a decrease in apoptosis comparable with that in the control group.
Reprogrammed CD8 + T-lymphocytes of bone marrow were more resistant to cytotoxic effect of cancer cells in comparison with naive CD8 + T-lymphocytes. This is dicated by a decrease in apoptosis of reprogrammed CD8 + T-lymphocytes in 0.25:1; and 2.5:1 dilution (Figure 3).
These results confirm that CD8 + T-lymphocytes, when treated with MEKi a nivolumab in vitro, become T-cells, having high stable CCR7 expression and being sistant to the cytotoxic effect of cancer cells and distinct from naive T-lymphocytes. -for comparison with the group "naive CD8 + T-lymphocytes + tumor cells" by Mann-Whitney test (p < 0.05).

Cocultivation of CD8 + T-Lymphocytes with Cancer Cells
In a 0.25:1 dilution, we observed that naive CD8 + T-lymphocytes displayed increased apoptosis in LLC cell culture. The level of apoptosis was more than two-fold higher in comparison with that of the control lymphocytes.
An increase in the concentration of naive CD8 + T-lymphocytes in the LLC cell culture caused a decrease in apoptosis comparable with that in the control group.
Reprogrammed CD8 + T-lymphocytes of bone marrow were more resistant to the cytotoxic effect of cancer cells in comparison with naive CD8 + T-lymphocytes. This is indicated by a decrease in apoptosis of reprogrammed CD8 + T-lymphocytes in 0.25:1; 1:1, and 2.5:1 dilution (Figure 3).
These results confirm that CD8 + T-lymphocytes, when treated with MEKi and nivolumab in vitro, become T-cells, having high stable CCR7 expression and being resistant to the cytotoxic effect of cancer cells and distinct from naive T-lymphocytes.

Cytotoxicity of Reprogrammed CD8 + T-Lymphocytes Cocultured with Lewis Lung Carcinoma Cells
The ratios of CD8 + T-lymphocytes to cancer cells, which were observed in the LLC cell culture, were 0:1, 0.25:1, 1:1, 2.5:1, 5:1, and 10:1. Naive and reprogrammed CD8 + T-lymphocytes induced apoptosis of cancer cells at the ratio of 0.25:1. The maximum level of tumor cell apoptosis was observed at a 10:1 cell ratio (CD8 + T-lymphocytes:cancer cells). Cytotoxicity of reprogrammed CD8 + T-lymphocytes was higher in comparison with that of naive CD8 + T-lymphocytes in the same ratios ( Figure 4).

Cytotoxicity of Reprogrammed CD8 + T-Lymphocytes Cocultured with Lewis Lung Carcinoma Cells
The ratios of CD8 + T-lymphocytes to cancer cells, which were observed in the LLC cell culture, were 0:1, 0.25:1, 1:1, 2.5:1, 5:1, and 10:1. Naive and reprogrammed CD8 + T-lymphocytes induced apoptosis of cancer cells at the ratio of 0.25:1. The maximum level of tumor cell apoptosis was observed at a 10:1 cell ratio (CD8 + T-lymphocytes:cancer cells). Cytotoxicity of reprogrammed CD8 + T-lymphocytes was higher in comparison with that of naive CD8 + T-lymphocytes in the same ratios ( Figure 4). Together, these results demonstrate that MEKi not only results in increased CCR7 expression, but also enhances the functionality of reprogrammed CD8 + T-lymphocytes. Together, these results demonstrate that MEKi not only results in increased CCR7 expression, but also enhances the functionality of reprogrammed CD8 + T-lymphocytes.

Evaluation of Migration of Naive and Reprogrammed CD8 + T-Lymphocytes Derived from Donor-Mice Bone Marrow into the Lungs of Recipient Mice with Lewis Lung Carcinoma
Naive and reprogrammed CD8 + T-lymphocytes derived from donor-mice bone marrow were preliminarily stained with vital dye CFSE. The analysis was performed 60 min after intravenous administration of CFSE-labeled CD8 + T-lymphocytes derived from donormice bone marrow to recipient mice with LLC.
Reprogrammed CD8 + T-lymphocytes of bone marrow migrated more actively to the lungs of mice with LLC. Thus, the number of CFSE-labeled reprogrammed CD45 + CD8 + T-lymphocytes in the lungs of recipient mice with LLC was significantly higher than CFSE-labeled naive CD45 + CD8 + T-lymphocytes (8.9 times) ( Figure 5).

Evaluation of Migration of Naive and Reprogrammed CD8 + T-Lymphocytes Derived fro Donor-Mice Bone Marrow into the Lungs of Recipient Mice with Lewis Lung Carcinoma
Naive and reprogrammed CD8 + T-lymphocytes derived from donor-mice marrow were preliminarily stained with vital dye CFSE. The analysis was performe min after intravenous administration of CFSE-labeled CD8 + T-lymphocytes derived donor-mice bone marrow to recipient mice with LLC.
Reprogrammed CD8 + T-lymphocytes of bone marrow migrated more actively t lungs of mice with LLC. Thus, the number of CFSE-labeled reprogrammed CD45 + C T-lymphocytes in the lungs of recipient mice with LLC was significantly higher CFSE-labeled naive CD45 + CD8 + T-lymphocytes (8.9 times) ( Figure 5). We found that reprogrammed with MEKi and nivolumab CD8 + T-lymphocytes present at higher frequencies in the lung of mice with LLC after cell administra compared to naive CD8 + T-lymphocytes.

Histological Examination
On the 7th day of the orthotopic model of LLC, well-vascularized large tumor n composed of atypical cells were detected in the lungs of C57BL/6 mice. This group of was characterized by cellular and nuclear polymorphism. Multinucleated giant cells We found that reprogrammed with MEKi and nivolumab CD8 + T-lymphocytes were present at higher frequencies in the lung of mice with LLC after cell administration, compared to naive CD8 + T-lymphocytes.

Tumor Growth
Therapy with reprogrammed CD8 + T-lymphocytes of bone marrow caused an increase in TGII. The value of TGII after cell therapy with reprogrammed CD8 T-lymphocytes was 57%. Moreover, we observed an increase in tumor volume and the average number of metastases (Table 1).

Effect of Reprogrammed CD8 + T-Lymphocytes on Cancer Cells and Cancer Stem Cells in the Lungs and Blood of Mice with Lewis Lung Carcinoma
The LLC model caused a significant increase in the number of CSCs with different phenotypes in the lungs of C57BL/6 mice on the 7th day of the experiment: Axl + Axl + CD117 + , EGF + CD44 + Sox2 + , EGF + Sox2 + , CD44 + Sox2 + , CD117 + Sox2 +

Tumor Growth
Therapy with reprogrammed CD8 + T-lymphocytes of bone marrow caused an increase in TGII. The value of TGII after cell therapy with reprogrammed CD8 + T-lymphocytes was 57%. Moreover, we observed an increase in tumor volume and the average number of metastases (Table 1).
The injection of reprogrammed CD8 + T-lymphocytes derived from the bone marrow significantly reduced the number of cancer cells and CSCs population in the blood and lungs of recipient mice with LLC on the d7 (Figure 7). However, populations of cells Axl + and Axl + CD117 + in the blood was increased after reprogrammed CD8 + T-lymphocyte administration.
Injection of reprogrammed CD8 + T-lymphocytes caused an increase in the T-lymphocyte population in the blood of recipient mice with LLC compared to mice with LLC without treatment on the 7th day of the experiment ( Figure 8). The exception was highly differentiated memory T-lymphocytes (CD8 + CD62L − CD44 + ), whose number was lower compared to the intact control.
After T-lymphocyte therapy, the T-lymphocyte population in the lungs of recipient mice with LLC was lower in comparison with the animals of LLC group without treatment (Figure 8). At the same time, the number of CD45RA + CD197 hi CD62L + CD95 + CD8 + , CD3 + CD8 + , CD3 + CD8 + PD-1 + and CD3 + CD8 + PD-L1 + cells were comparable to that in intact animals.  Injection of reprogrammed CD8 + T-lymphocytes caused an increase in the T-lymphocyte population in the blood of recipient mice with LLC compared to mice with LLC without treatment on the 7th day of the experiment ( Figure 8). The exception was highly differentiated memory T-lymphocytes (CD8 + CD62L -CD44 + ), whose number was lower compared to the intact control.

Discussion
Designing optimal (neo)adjuvant therapy is a crucial aspect of the treatment of lung cancer. Standard methods of chemotherapy, radiotherapy, and immunotherapy represent current standard strategies for treatment. However, in some cases with high metastatic activity and high levels of circulating tumor cells and CSC, the efficacy of standard treatment methods is insufficient and results in treatment failure, relapse, and ultimately reduced patient survival. Currently, much attention is paid to the search for targeted therapy of lung cancer. Targeted therapy for non-small-cell lung cancer (NSCLC) involves drugs that inhibit angiogenesis, and inhibitors of kinases EGFR, ROS1, MET, and RET. Targeted therapies for small-cell lung cancer (SCLC) are still limited compared with other forms of lung cancer. In some cases, PD-1/PDL-1 checkpoint inhibitors in combination with other therapies are used as targets for the treatment of SCLC [26]. Cancer vaccines and gene therapy for lung cancer are undergoing clinical trials [27]. Early cancer diagnosis, monitoring of the tumor growth progression and correct determination of histological subtype and tumor mutation are making treatment of lung cancer cumbersome and costly. Additionally, tumor heterogeneity, specific tumor microenvironment, and complex intercellular interaction between tumor and normal cells lead to the escape of the tumor from drugs and reduce the overall effectiveness of antitumor therapy.
Actively proliferating tumor cells are generally targets for drugs. Meanwhile, progression, metastasis, and recurrence of lung cancer, as well as resistance to therapy, are related to CSCs [15,28]. CSCs with a long resting state (G0 arrest) and high activity of the efflux transport system are insensitive to anticancer drugs [29]. Thus, treatment of lung cancer would largely benefit from early detection of CSCs and targeted CSCs elimination.
Currently, the search for therapies is aimed at enhancing antitumor immunity modifying immune cells. Development of CAR T-lymphocytes is one approach for the treatment of malignant tumors. CAR T-lymphocytes recognize specific tumor antigens and induce the eradication of tumor cells. CAR T-lymphocyte therapy has been approved for the treatment of blood cancers (leukemia and lymphoma). Clinical evaluation of efficacy and safety of TCR T-lymphocyte therapy in solid cancers (NCT04044859, NCT03132792, NCT03967223, NCT03907852), including NSCLC (NCT02592577, NCT02408016) is still ongoing [30]. The source of cells for modification is the patient's lymphocytes. Persistent tumor antigen exposure generally causes an exhaustion of T-lymphocytes. Exhausted T-lymphocytes lose effector functions and persistently express inhibitory molecules such as PD-1, Tim-3, and CD160 [31]. Interestingly, modified CAR T-lymphocytes can be exhausted similarly to endogenous T-lymphocytes [32]. This prompts us to search for new approaches for cancer treatment.
MEK inhibition forms a pharmacologically feasible method that can be used to enhance different immune therapeutic approaches, including combination with priming agents, immune modulators or enhancing the efficacy of cellular therapies. Importantly, an MEK inhibitor is approved by the US Federal Drug Administration and is in use for anticancer treatment (NCT02407405, NCT00888134, NCT03109301) [10]. However, a lack of specificity and frequent side effects after systemic administration of a MEK inhibitor have been observed [7]. We propose an approach to reprogramming that involves combined use of MEK and PD-1 inhibitors. Bone marrow CD8 + T-lymphocytes were targets for modification. Inhibition of MEK1/2 leads to alteration of T-lymphocyte metabolism by modulating the ERK1/2 metabolic pathway. As a result, population of T memory stem cells (Tscm) has been generated [33]. MEKi-induced Tscm cells showed strong cellular activation, high antigen-specific responses, and long-term survival. Verma V et al. showed that MEK inhibition improved mitochondrial function of T-lymphocytes, inducing the formation of Tscm with CD45RA + CD197 hi CD62L + CD95 + CD8 + phenotype [10]. In order to protect T-lymphocytes from the immunosuppressive effect of tumors, we considered it appropriate to use an immune checkpoint inhibitor to block the PD/PDL-1 signaling pathway. Another requirement for successful reprogramming was the "training" of CD8 + T-lymphocytes with the entire spectrum of tumor antigens. The full set of antigens contained in the tumor cell lysate during training provided access for immune cells to a whole spectrum of epitopes for the formation of a tumor-specific CD8 + T-lymphocyte population. Thus, the risk of tumor escape from the immune response was reduced [34].
In our study, we showed that reprogramming combined with preliminary training increased CCR7 expression by CD8 + T-lymphocytes ( Figure 2). CCR7 is known to be characteristic of T memory stem cells [34,35]. Interestingly, CD8 + T-lymphocytes showed high expression of CCR7 even in exhaustion. The stability of new properties of reprogrammed cells is important for increasing the effectiveness in cell therapy, since transplanted T-lymphocytes, including modified ones, are affected by various damaging (exhausting) factors (tumor microenvironment, cytokines) [32].
Based on the results of our in vitro experiments, we conclude that reprogrammed T-lymphocytes in LLC cell culture were more resistant to apoptosis in comparison with naive T-lymphocytes (Figure 3). On the other hand, inhibitors of PD-1 and MEK in combination with the training of T-lymphocytes allowed for the increase in cytotoxic activity of T-lymphocytes ( Figure 4).
During cell therapy, transplanted T-lymphocytes should migrate to the tumor for effective implementation of cytotoxic function. We observed an increase in CD8 + T-lymphocyte numbers in the lungs of recipient mice with LLC after intravenous administration of CFSElabeled reprogrammed CD8 + T-lymphocytes of donor mice. As shown on Figure 5, CD8 + T-lymphocyte numbers were more than 8.9 times higher than in cell therapy with naive T-lymphocytes.
The antitumor effect of reprogrammed T-lymphocytes was confirmed by results of our in vivo experiments. In the LLC orthotopic model, well-vascularized large tumor nodes consisted of atypical cells were found in the lungs of mice (day 7). These cells were characterized by cellular and nuclear polymorphism. Multinucleated giant cells were found in the general population of atypical cells. Many cells were in mitosis. Multiple small foci of necrosis were observed in the tumor tissue ( Figure 6). Intravenous administration of reprogrammed CD8 + T-lymphocytes reduced the number of tumor emboli in vessels, perivascular, and peribronchial metastases in the lungs of recipient mice in LLC orthotopic model. The number of lung metastases after the treatment with reprogrammed T-lymphocytes decreased (Table 1). In the present study, we aimed to model lung cancer, since in our opinion, cell therapy with reprogrammed CD8+ T-lymphocytes can be more effective to CSCs, while radiation therapy or chemotherapy are not effective on CSCs.
We hypothesized that CSCs, as well as tumor cells, are potential targets for reprogrammed CD8 + T-lymphocytes. Indeed, all studied populations of CSCs (Axl + , Axl + CD117 + , EGF + CD44 + Sox2 + , EGF + Sox2 + , CD44 + Sox2 + , CD117 + Sox2 + , CD117 + EGF + CD44 + Sox2 + ) in the lungs of recipient mice significantly decreased after cell therapy (day 7) (Figure 7). The number of CSCs in the blood had declining trends. It is shown that these cells can thus act as potential contributors to tumor progression and formation of metastases. Since these cell types are present in the blood, we hypothesize that they are potential markers of the disease prognosis and targets of the treatment.
Thus, results of our study indicate the effectiveness of reprogramming to enhance the antitumor activity of T-lymphocytes. However, we paid attention to an increase in the content of T-lymphocytes with a high proliferative potential (CD8 + CD62L + CD197 + CD45RA + ) and memory cells (CD8 + CD62L − CD44 + ) in the lungs and blood in addition to the data indicating the antitumor activity of reprogrammed CD8 + T-lymphocytes in vitro and in vivo ( Figure 8). These data indirectly confirm CD8 + T-lymphocytes high proliferative activity which is important for cell therapy of lung cancer.
Our work is limited by the capacity of animal models to mimic the human lung cancer. Thus, further evaluation in more mouse experimental systems and randomized trials is needed.

Conclusions
In our study, we demonstrate that reprogramming by inhibiting the MAPK/ERK pathway through MEK1/2i and blockade of the PD/PDL-1 signaling pathway by a human monoclonal antibody, nivolumab, enhances antitumor activity of CD8 + T-lymphocytes in the LLC orthotopic model. At the same time, various populations of cancer stem cells are potential targets for reprogrammed CD8 + T-lymphocytes.