Stability of T Cell and NK Cell Profiles in Peripheral Blood Mononuclear Cells After Multiple Immune Cell Therapy Sessions

Stability of T Cell and NK Cell Profiles in Peripheral Blood Mononuclear Cells After Multiple Immune Cell Therapy Sessions | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Stability of T Cell and NK Cell Profiles in Peripheral Blood Mononuclear Cells After Multiple Immune Cell Therapy Sessions Karina Karina, anabel abiyyah Nugroho, Irsyah Afini, Difky Ernanda, and 17 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6390155/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Immunotherapy represents a significant advancement in oncology, characterized by the ex vivo expansion of innate and adaptive immune cells to combat cancer. Despite its demonstrated efficacy in both preclinical and clinical studies, assessing the immune system's response through peripheral blood mononuclear cells (PBMC) post-therapy remains crucial. This study examined the expression profiles of T cell and NK cell markers in the PBMC of healthy individuals and cancer patients (breast, lung, and colon cancer) following five immune cell therapy sessions using flow cytometry, specifically evaluating cells with T cell (CD3 and CD8) and NK Cell (CD56 and NKG2D) markers. The results indicated no substantial changes in the proportions of CD3 + and CD8 + for T cells, nor NK cells expressing CD56 and NKG2D, across healthy subjects, and patients with breast, lung, and colon cancer after five sessions of immune cell therapy. These findings suggest that immune cell therapy does not induce significant alterations in T cell populations or NK cell activity, thereby preserving immune system homeostasis. Consequently, this research supports the notion that immune cell therapy operates effectively and safely, without disrupting immune equilibrium, and offers valuable insights for the development of enhanced cancer treatment modalities in the future. Immune Cell Therapy T Cell Stability NK Cell Stability Cancer Treatment Immune Homeostasis Figures Figure 1 Figure 2 1. Introduction The prevailing conventional strategy for cancer treatment involves the surgical excision of the tumor, followed by adjuvant radiotherapy utilizing X-rays and/or chemotherapy. Despite chemotherapy's success in decreasing morbidity and mortality rates, it is notable that nearly all chemotherapeutic agents exert cytotoxic effects on healthy cells. 1 . Consequently, global research efforts are focused on identifying alternative and novel treatments for cancer patients. A promising new approach is immune cell therapy, a type of immune cell therapy. This technique involves the ex vivo expansion of innate and adaptive immune cells, derived from autologous blood, which are subsequently reinfused into the patient to target and combat tumors. 2 , 3 Cancer immune cell therapy represents one of the most substantial advancements in oncology in recent years, exhibiting remarkable antitumor activity and enduring clinical benefits across diverse malignancies. Recent studies have demonstrated that the infusion of autologous activated lymphocytes, specifically targeted to tumor antigens, can induce prolonged responses in patients with chemotherapy-resistant luminal metastatic breast cancer. This approach, which utilizes mutant protein-specific tumor-infiltrating lymphocytes (TILs) in conjunction with IL-2 and pembrolizumab, has shown significant efficacy in these patients. 4 In clinical trials, tumor-I nfiltrating lymphocytes (TILs) have demonstrated efficacy in various immunogenic cancers, including melanoma and human papillomavirus-associated cancers. Additionally, TILs exhibit considerable variability in their specificity and avidity for undefined antigens. In melanoma, both TILs and peripheral blood lymphocytes, when stimulated in vitro with the patient's tumor, have been shown to effectively eradicate the tumor. 5 Another study involving patients with metastatic colorectal cancer demonstrated that the infusion of CD8 + cells targeting the mutant KRAS gene mediated effective antitumor immunotherapy against cancers expressing KRAS G12D and HLA-C. 6 Meta-analyses of immunotherapy studies, including those involving activated T lymphocytes, cytokine-induced NK cells, and dendritic cell vaccine therapy, have demonstrated their effectiveness in treating gastric and lung cancer. 7 These successes suggest that cellular immunotherapy will be a pivotal future therapy for cancer patients. According to the Journal of the Indonesian Radiation Oncology Society, the most prevalent cancers among patients in Indonesia are breast cancer (36.1%), cervical cancer (17.3%), nasopharyngeal cancer (8.2%), lung cancer (7.4%), rectal cancer (6.9%), leukemia (6.7%), ovarian cancer (6.3%), lymphoma (5.3%), colon cancer (4.0%), and prostate cancer (2.0%). Therefore, this study aims to analyze the profiles of T cells and NK cells in patients with breast, colon, and lung cancer. The focus on solid cancers for this initial study is based on research by Prihantono et al. (2023), which reported 6,203 cases of solid cancer (79.3%) in Indonesia, resulting in 1,063 deaths (61.7% of all cancer-related deaths), compared to 1,621 cases of non-solid cancer (20.7%), resulting in 678 deaths (38.3%) 8 . Although decades of preclinical and clinical studies conducted globally have demonstrated that cell immunotherapy is safe and effective, only a limited number have evaluated the immune system's response in peripheral blood mononuclear cells, particularly following multiple cell immunotherapy sessions. Therefore, it is essential to adequately verify the safety of cell immunotherapy in both healthy individuals and cancer patients after five sessions. This study aims to analyze the profiles of T cell and NK cell markers in the peripheral blood of healthy individuals and cancer patients post-immunotherapy. To gain insights into the human immune system, the composition of Peripheral Blood Mononuclear Cells (PBMC) will be examined. 9 This PBMC analysis will focus on profiling NK cells and T cells in the patient's body. Natural killer (NK) cells and cytotoxic T cells (CD8+) are critical immune cells that play a pivotal role in defending the body against harmful invaders. 10 NK cells, through their cytotoxic functions, induce apoptosis and participate in the regulation of immune responses. They may also contribute to the pathogenesis of various immune-mediated diseases, including ankylosing spondylitis, Behçet's disease, multiple sclerosis, rheumatoid arthritis, psoriasis, systemic lupus erythematosus, and type-1 diabetes. 11 Additionally, NK cells are positioned between the innate and adaptive immune systems, serving as a "bridge" between the two. They also play a role in inflammation and immune regulation. 12 Moreover, dysfunction in the regulatory functions of NK cells can lead to inadequate control of T cell responses, thereby contributing to the development of certain disorders. 13 T cells are involved in a range of immune responses associated with infections, cancer, autoimmune diseases, and allergic conditions. 14 Therefore, it is crucial to assess the immune system by evaluating NK cell and T cell marker profiles in the blood of both healthy individuals and cancer patients following cell immunotherapy. 2. Materials and Methods Research design This study employed an experimental design, incorporating both control and treatment groups to assess the effects of the intervention. The subjects were divided into two groups: one consisting of healthy individuals and the other consisting of patients diagnosed with cancer, with each group comprising 10 subjects. Blood samples from both healthy individuals and cancer patients, collected on the 14th day of cell immunotherapy, will be submitted to Hayandralab for analysis. Each subject will provide blood samples on five separate occasions 15 . The samples will be isolated and characterized according to the research parameters. Blood Sample Collection from Healthy and Cancer Patients Pre- and Post- Cell Immunotherapy. This experiment, conducted at Hayandra Laboratory, involved healthy individuals and cancer patients, with each group providing 10 blood samples on the 14th day following immune cell therapy. Blood samples were collected and submitted five times, with a 2-week interval or on the 14th day post-therapy. Healthy blood donors were required to be over 18 years old, in good health based on medical history and physical examination, and willing to participate by signing informed consent. Cancer blood donors needed to be 18 years or older, of either gender, with a confirmed diagnosis of breast, lung, or colon cancer at stages 1–4, at least four weeks after the last chemotherapy or radiation therapy, and meet specific laboratory criteria (leukocyte count ≥ 3,000/µl, neutrophil count ≥ 1,500/µl, platelet count ≥ 80,000/µl, hemoglobin ≥ 9.0 g/dl, serum creatinine ≤ 1.5 mg/dl, and serum bilirubin ≤ 2.5 × upper limit of institutional normal range), as well as sign informed consent. Exclusion criteria for both groups included the presence of HIV, hepatitis, psychiatric conditions, drug addiction, pregnancy, autoimmune disorders, or any other condition that could prevent participation or endanger subject safety. 16 , 17 Peripheral Blood Mononuclear Cell Isolation Peripheral blood mononuclear cell (PBMC) isolation was performed in a Biosafety Cabinet (BSC). Blood, collected in a 1-liter heparin tube (approximately 3 ml), was mixed well, aspirated, and transferred into a T75 flask, with the blood volume recorded. HBSS was added to the flask in a 1:1 ratio and mixed thoroughly. Ficoll was added in a 1:2 ratio, creating a 10 ml tube mixture (3 ml Ficoll and 6 ml diluted blood). The diluted blood was carefully layered over the Ficoll and then centrifuged at 2,500 rpm for 10 minutes at room temperature (acceleration 7, deceleration 1) using a Thermo Scientific Centrifuge SL40. The plasma layer was discarded, and the turbid interphase layer was collected into two 10 ml conical tubes. HBSS was added to each tube to a total volume of 10 ml and mixed thoroughly by pipetting up and down with a new 10 ml serological pipette. The tubes were then centrifuged at 300g for 10 minutes at room temperature (speed 7, deceleration 7). The supernatant was discarded, and the pellet was resuspended in 10 ml of HBSS. The suspension was centrifuged again at 200g for 10 minutes at room temperature (speed 7, deceleration 7), and the supernatant was discarded completely. The final pellet was resuspended in 1 ml of growth medium. Characterization of Peripheral Blood Mononuclear Cells via Flow Cytometry A suspension of peripheral blood mononuclear cells (PBMCs) at a concentration of at least 1 × 10 5 cells/ml was transferred into a 5 ml round-bottom tube. The suspension was then centrifuged at 1,200 rpm for 5 minutes. After centrifugation, 300 µl of PBS was added to the tube (labeled with the sample code "unstained") and the mixture was vortexed. Subsequently, 100 µl of this suspension was transferred to a new tube (labeled with the specific sample code). To the new tube, 5 µl of the following antibodies were added: (1) Anti-Human CD3 FITC clone Hit3a (Tonbo Bioscience 35-0039-T100), (2) Anti-Human CD19 PerCP-Cyanine 5.5 clone HIB19 (Tonbo Bioscience 65-0199-T100), (3) Anti-Human CD56 (N CAM) PE clone MY31 (Tonbo Bioscience 50-0564-T100), (4) Anti-Human CD8 APC clone SK1 (Tonbo Bioscience 20-0087-T100), and (5) PE-CyTM7 Mouse Anti-Human CD314 (NKG2D) clone 1D11 (BD PharmingenTM 562365). The sample was then incubated in the dark for 15 minutes. Following incubation, 1 ml of PBS was added, vortexed, and the mixture was centrifuged again at 1,200 rpm for 5 minutes. Afterward, 300 µl of PBS was added to the pellet, and the sample was analyzed using a flow cytometer. Statistical Data Analysis Normal data are presented as mean ± standard deviation (SD). Statistical comparisons between different treatments were conducted using one-way analysis of variance (ANOVA). The analysis was performed with a significance level of α = 0.05 and a confidence level of 95%. 3. Results a. Hematology Data of Research Blood Samples The demographic data analysis revealed that the average age in the healthy group was 58.3 years. Breast cancer patients had a slightly younger average age of 54.7 years compared to the healthy group. Colon cancer patients had an average age of 56.4 years, which was similar to that of the healthy group. In contrast, lung cancer patients had an average age of 65.9 years, which was notably older than the other groups. Table 1 Hematology Data of Research Blood Samples Characteristic Healthy People Breast Cancer Colon Cancer Lung Cancer p Average ± SD Age (Years) 58,3 ± 16,91 54,7 ± 14,05 56,4 ± 14,05 65,9 6 ± 9,86 0,319 Leukocyte Count \(\:7\times\:\) 10 3 µl ± 2,4 6,5 \(\:\times\:\) 10 3 µl ± 2,2 \(\:\text{9,8}\times\:\) 10 3 µl ± 6,2* \(\:\text{6,4}\:\times\:\) 10 3 µl ± 2,4 0,037 Neutrophil Count \(\:\text{5,2}\times\:\) 10 3 µl ± 2,2 \(\:\text{4,1}\times\:\) 10 3 µl ± 1,3 \(\:\text{7,3}\times\:\) 10 3 µl ± 5,7 \(\:\text{4,3}\times\:\) 10 3 µl ± 1,7 0,288 Platelet Count \(\:285\times\:\) 10 3 µl ± 77,4 \(\:243\times\:\) 10 3 µl ± 109,9 \(\:\text{395,5}\times\:\) 10 3 µl ± 182,7* \(\:\text{262,3}\:\times\:\) 10 3 µl ± 103,6 0,37 Hemoglobin 13,7 g/dl ± 1,7* 11,6 g/dl ± 2,9 11 g/dl ± 2,7 11,8 g/dl ± 1,6 0,001 * It is the result of Duncan's analysis which indicates a statistically significant difference in the same parameters. Regarding hematological parameters in Table 1 ., the average leukocyte count in healthy individuals was 7,000 µl. In comparison, breast cancer and lung cancer patients had slightly lower leukocyte counts of 6.5 × 10³ µl and 6.4 × 10³ µl, respectively. Conversely, colon cancer patients exhibited a higher average leukocyte count of 9.8 × 10³ µl. The average neutrophil count in healthy individuals was 5,200 µl, which was elevated compared to breast cancer and lung cancer patients, who had average neutrophil counts of 4.1 × 10³ µl and 4.3 × 10³ µl, respectively. Among the groups, colon cancer patients had the highest average neutrophil count, at 7.3 × 10³ µl. The average platelet count in healthy individuals is 285,000 µl. Breast cancer patients exhibit a lower platelet count of 243 × 10³ µl, whereas colon cancer patients have the highest platelet count, at 395.5 × 10³ µl. Lung cancer patients have a platelet count of 262.3 × 10³ µl, which is slightly lower than that of healthy individuals. Regarding hemoglobin levels, healthy individuals have an average of 13.7 g/dl, which is higher than the levels observed in cancer patients. Breast cancer and colon cancer patients have hemoglobin levels of 11.6 g/dl and 11 g/dl, respectively, while lung cancer patients have a hemoglobin level of 11.8 g/dl, which is slightly lower than that of healthy individuals. Overall, cancer patients exhibited distinct values for leukocytes, neutrophils, platelets, and hemoglobin compared to healthy individuals. These variations highlight differences in health conditions and physiological responses to disease, with notable discrepancies among different cancer types. Statistically, significant differences were observed in leukocyte counts (p = 0.037) and hemoglobin levels (p = 0.001) between the groups. No significant differences were found in age (p = 0.319), neutrophil counts (p = 0.288), or platelet counts (p = 0.37). b. Percentage of T cells in healthy and cancer patients Analysis of the T cell population in peripheral blood mononuclear cells (PBMCs) from blood samples obtained after five rounds of immune cell therapy revealed no significant changes in the percentage of CD3 + and CD8 + T cells across healthy individuals, breast cancer, colon cancer, and lung cancer patients (Fig. 1 ). Statistical analysis yielded non-significant p-values for each group: healthy individuals (0.938), breast cancer patients (0.848), colon cancer patients (0.916), and lung cancer patients (0.701). The proportion of CD3 + T cells, representing the total T cell population, showed no significant increase or decrease, indicating no abnormal alterations in the T cell population in the bloodstream following five rounds of immune cell therapy. Additionally, the percentage of CD8 + T cells, a subset of CD3 + T cells with cytotoxic activity against infected and cancerous cells, also showed no significant differences following five sessions of immune cell therapy. Statistical analysis revealed non-significant p-values for each group: healthy individuals (0.640), breast cancer patients (0.522), colon cancer patients (0.633), and lung cancer patients (0.832). These results suggest that CD8 + T cells are effectively targeting infected or cancerous cells without causing a significant increase in their overall blood concentration. This indicates that despite repeated immune cell therapy, CD8 + T cells maintain stable levels in the bloodstream, reflecting a focused immune response on the targeted areas rather than an increase in CD8 + T cells in the general circulation. Thus, the immune cell therapy appears to effectively direct the immune response to specific sites without inducing an unnecessary elevation of CD3 + and CD8 + T cells in peripheral blood. c. NK cell percentage in healthy and cancer patients Analysis of the percentage of NK cells with NKG2D and CD56 markers after five rounds of immune cell therapy revealed no significant changes (Fig. 2 ). NKG2D, an activation receptor on NK cells critical for identifying and destroying infected or cancerous cells, did not show a significant increase. Statistical analysis yielded non-significant p-values for each group: healthy individuals (0.999), breast cancer patients (0.522), colon cancer patients (0.909), and lung cancer patients (0.560). These results suggest that immune cell therapy does not induce abnormal alterations in the ability of NK cells to recognize and attack target cells, and the immune surveillance function of NK cells remains within the normal range. Additionally, the percentage of NK cells expressing CD56, a crucial marker of NK cell maturity and activity, remained stable with no significant changes. Statistical analysis showed non-significant p-values for each group: healthy individuals (0.202), breast cancer patients (0.848), colon cancer patients (0.542), and lung cancer patients (0.623). This stability indicates that immune cell therapy does not lead to an excessive increase or decrease in NK cell activity, thus preserving a normal balance in the blood circulation. Overall, the lack of significant changes in NK cells with NKG2D and CD56 markers after five sessions of immune cell therapy suggests that the treatment does not provoke excessive immune reactions or disrupt immune system homeostasis. These findings support the notion that immune cell therapy can be administered safely without causing unwanted alterations in the NK cell population in the bloodstream, thereby maintaining a balanced and healthy immune function. Consequently, these results reinforce the understanding that immune cell therapy is effective and specific, without inducing excessive or misdirected immune responses. 4. Discussion Demographic data analysis of healthy individuals and cancer patients revealed significant differences in several hematological parameters, reflecting varying physiological conditions and immune responses to cancer. Notably, colon cancer patients exhibited higher mean leukocyte counts, suggesting a more pronounced inflammatory response. Additionally, the elevated platelet counts observed in colon cancer patients may indicate reactive thrombocytosis, commonly associated with chronic inflammation or neoplastic conditions. 18 . The lower hemoglobin levels observed in all cancer patient groups compared to healthy individuals may be attributed to several factors, including bleeding, hemolysis, impaired red blood cell production, or anemia, which affects approximately 40% of cancer patients. 19 This variation is expected in the context of cancer, as the disease frequently induces significant alterations in hematological parameters. Cancer can impact the production, distribution, and destruction of blood cells, contributing to these changes. The analysis of blood samples collected after five rounds of immune cell therapy revealed no significant changes in the percentages of CD3 + and CD8 + T cells, as well as NK cells expressing CD56 and NKG2D, across various groups including healthy individuals and patients with breast cancer, colon cancer, and lung cancer. The high p-values associated with these parameters indicate that immune cell therapy did not induce significant fluctuations in the populations of these T cells and NK cells. CD3 + T cells, representing the total T cell population 20 , 21 , showed no significant increase or decrease after five rounds of immune cell therapy, indicating that the therapy did not induce excessive T cell proliferation or a reduction that could suggest an impaired immune response in the patient's PBMCs. The stability in the proportion of CD3 + T cells suggests that the therapy operates within safe limits, preserving immune system homeostasis. Similarly, CD8 + T cells, which are responsible for cytotoxic activity against infected and cancerous cells through antigen recognition in the context of MHC class I molecules 22 , 23 , also displayed no significant changes in their percentage. This indicates that CD8 + T cells remain focused on their targets without causing unnecessary increases in overall blood circulation, and the immune response remains specifically targeted, avoiding excessive systemic reactions. The stability of both CD3 + and CD8 + T cell proportions reflects that T cell tolerance is preserved, which is crucial for preventing overreaction to the body's own antigens and avoiding autoimmune damage 24 . Maintaining T cell tolerance ensures that the immune response induced by the therapy remains effective while protecting healthy tissues from damage. These findings contrast with those observed in allogeneic CAR T cell therapy. In such studies, patients frequently exhibit abnormal T cell subsets, as evidenced by flow cytometry showing CD8 expression without corresponding CD3 expression 25 . This discrepancy is likely attributable to the differing origins of the cells used in the therapies. Autologous immunotherapy utilizes T cells derived from the patient’s own body, which are generally more compatible and better at maintaining immune homeostasis. Conversely, allogeneic CAR T cell therapy employs T cells from external donors, which can induce unwanted immune responses, including the development of abnormal T cell subsets and disruption of immune balance 26 . Additionally, the standard procedure for allogeneic CAR T cell production often includes the depletion of TCR + cells to achieve specific therapeutic goals. 27 However, previous research has demonstrated that TCR signaling is essential for maintaining T cell homeostasis and long-term survival in vivo 28 , potentially explaining why allogeneic CAR T cell therapy might cause greater immune disruption compared to autologous immune cell therapy. Maintaining the normal balance of CD3 + and CD8 + T cells in the blood is crucial for optimal immune system function. T cells are primary regulators of the immune response, modulating the activity of other immune cells and influencing the behavior of endothelial and parenchymal cells. They are essential for protecting against viral and bacterial infections 29 , 30 . Deviations in CD3 + or CD8 + T cell populations can trigger or exacerbate autoimmune diseases 31 and may also result in diminished immune function. The analysis of NK cells marked by NKG2D and CD56 after five rounds of immune cell therapy revealed no significant changes. NKG2D, an activation receptor crucial for NK cell recognition and destruction of infected or cancerous cells 32 , did not show a significant increase. Similarly, the percentage of NK cells expressing CD56, a marker indicative of NK cell maturity and activity, remained stable without significant fluctuations. This stability suggests that immune cell therapy does not induce excessive increases or decreases in NK cell activity, thereby preserving the normal balance of NK cells in the bloodstream. It also implies that the NK cells' ability to detect and target abnormal cells remains intact, ensuring effective immune surveillance. As essential components of innate immunity, NK cells respond swiftly to bodily abnormalities 33 , and immune cell therapy does not impair this critical function. Overall, the lack of significant changes in NK cell percentages with NKG2D and CD56 markers indicates that immune cell therapy does not provoke undue immune responses or disrupt immune system homeostasis. NK cells and T cells interact through cytokine production and response, engagement with dendritic cells, and mechanisms of target cell destruction 34 . These interactions play a critical role in modulating the local immune environment and enhancing the immune response to infections and malignancies. 34 – 36 . Their collaborative actions ensure a rapid and targeted defense against diverse threats. The absence of abnormalities in both T and NK cells suggests that the immune cell therapy does not induce excessive immune reactions or adverse effects in the circulating T and NK cell populations. This supports the notion that immune cell therapy functions as a precise intervention, targeting areas requiring immune action while maintaining overall immune system homeostasis. Consequently, despite the stability of T and NK cell percentages, these findings underscore the safety and specificity of immune cell therapy in managing various disease conditions, affirming its potential as a focused therapeutic approach. 5. Conclusion The analysis of blood samples after five rounds of immune cell therapy revealed no significant changes in the percentages of CD3 + and CD8 + T cells among healthy individuals and patients with breast, colon, and lung cancers. The high P values suggest that immune cell therapy does not induce substantial fluctuations in these T cell populations, thereby preserving immune system homeostasis. Similarly, the percentages of NK cells marked by NKG2D and CD56 also remained stable, indicating that immune cell therapy does not lead to excessive NK cell activity and maintains normal blood circulation balance. Overall, the stability in both T cell and NK cell populations signifies that immune cell therapy is effective and safe, without disrupting immune system equilibrium. These results reinforce the notion that immune cell therapy can operate efficiently without inducing excessive or adverse immune responses. Declarations Ethical Approval All authors confirm that all experiments involving the use of human samples (blood) were performed in accordance with relevant guidelines and regulations. The experimental protocols were approved by the Ethics Committee at BRIN (Badan Riset dan Inovasi Nasional), Indonesia. Consent to Participate A formal informed consent form was signed by each participant before they were included in the study. Consent to Publish Not applicable Funding The study was funded by Yayasan Hayandra in Indonesia. Author Contribution K. Karina was the primary supervising doctor for this study, conceptualised and planned the experiments, offered constructive criticism and edits, supplied reagents, supplies, and analytic tools, and approved the final paper. The experiments were also planned and executed by A. Nugroho, who also prepared the text, collected and put together the data, analysed and evaluated the results. I. Rosadi and I. Afini draughted the text and carried out the experiments. T.P. Sibuea, A. Maryam, J.A. Biben, N. Nadia, N.R. Marina, K.A. Pamungkas, and L.D.K. Lay were the supervising doctors and provided the reagents, materials, and analysis tools for this study. Performers of the trials were D. Ernanda, H. Hafizh, A. Azhari, C. B. Sepanya, Y.A. Widyanti, N. Aini, S. Sobariah, T. Widyastuti, A. Zakiyah, and P. Pannindya. Acknowledgment We would like to express our heartfelt appreciation to Hayandralab for providing the essential facilities and resources that made this research possible. Special thanks to the entire Hayandralab team for their unwavering support and dedication throughout the study. We also extend our gratitude to our peers and reviewers for their insightful feedback and constructive guidance, which significantly improved the quality of this manuscript. 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Curr Opin Virol . 2021;51:127-133. doi:10.1016/j.coviro.2021.09.017 Suárez-Fueyo A, Crispín JC, Tsokos GC. T Cells. In: Dubois’ Lupus Erythematosus and Related Syndromes . Elsevier; 2019:116-124. doi:10.1016/B978-0-323-47927-1.00010-4 Sun L, Su Y, Jiao A, Wang X, Zhang B. T cells in health and disease. Signal Transduct Target Ther . 2023;8(1):235. doi:10.1038/s41392-023-01471-y Siemaszko J, Marzec-Przyszlak A, Bogunia-Kubik K. NKG2D Natural Killer Cell Receptor—A Short Description and Potential Clinical Applications. Cells . 2021;10(6):1420. doi:10.3390/cells10061420 Jiang H, Jiang J. Balancing act: the complex role of NK cells in immune regulation. Front Immunol . 2023;14. doi:10.3389/fimmu.2023.1275028 Paul S, Lal G. The Molecular Mechanism of Natural Killer Cells Function and Its Importance in Cancer Immunotherapy. Front Immunol . 2017;8. doi:10.3389/fimmu.2017.01124 Bozward AG, Warricker F, Oo YH, Khakoo SI. Natural Killer Cells and Regulatory T Cells Cross Talk in Hepatocellular Carcinoma: Exploring Therapeutic Options for the Next Decade. Front Immunol . 2021;12. doi:10.3389/fimmu.2021.643310 Yang Y, Yang F, Huang Z, et al. T cells, NK cells, and tumor-associated macrophages in cancer immunotherapy and the current state of the art of drug delivery systems. Front Immunol . 2023;14. doi:10.3389/fimmu.2023.1199173 Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6390155","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":456329249,"identity":"c8501aba-db89-4d45-89f8-f24cd908cd0e","order_by":0,"name":"Karina Karina","email":"","orcid":"","institution":"Universitas Pembangunan Nasional Veteran Jakarta Fakultas Kedokteran","correspondingAuthor":false,"prefix":"","firstName":"Karina","middleName":"","lastName":"Karina","suffix":""},{"id":456329250,"identity":"065fdbc6-c8e3-4d83-a9c4-3eb8528d32d0","order_by":1,"name":"anabel abiyyah Nugroho","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABAElEQVRIiWNgGAWjYHACNgaGAoYEIMnAzFABIpkbiNBiANNyBkQyEqmFAay4DUQR0MI/u/nZgw8Gdnl8EjmGnwvn1UbztwO1/KjYhlOLxJ1j5oYzDJKL2SRyjKVnbjueO+MwYwNjz5nbuK25kWAmzWPAnNgmkWMgzbvtWG4DUAvQhbi1yN9I/yb9x6AepMX4N++cY7nzCWkxuJFjJs1gcBikxUyat6EmdwMhLYY3csoNewyOF7PxPCuz5jl2IHcjUMtBfH6Ru5G+7cGPiuo8+fbkzbd5aupy550/fBAogsf7cCCQYQAkD4PZB4hQDwT8xx8AyTriFI+CUTAKRsGIAgBcxlpeN48abQAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0009-0005-9522-8634","institution":"University of Indonesia: Universitas Indonesia","correspondingAuthor":true,"prefix":"","firstName":"anabel","middleName":"abiyyah","lastName":"Nugroho","suffix":""},{"id":456329251,"identity":"7af1af06-5e7c-4165-bcd6-ff5f14411a27","order_by":2,"name":"Irsyah Afini","email":"","orcid":"","institution":"Universitas 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Sepanya","email":"","orcid":"","institution":"HayandraLab","correspondingAuthor":false,"prefix":"","firstName":"Cris","middleName":"Gaby","lastName":"Sepanya","suffix":""},{"id":456329258,"identity":"bc17d46c-3296-4ebd-a145-7492a315064d","order_by":9,"name":"Yasinta Ayu Widyawati","email":"","orcid":"","institution":"HayandraLab","correspondingAuthor":false,"prefix":"","firstName":"Yasinta","middleName":"Ayu","lastName":"Widyawati","suffix":""},{"id":456329259,"identity":"4a7e0a93-15f1-4b4b-a734-cc585a526d44","order_by":10,"name":"Noor Aini","email":"","orcid":"","institution":"HayandraLab","correspondingAuthor":false,"prefix":"","firstName":"Noor","middleName":"","lastName":"Aini","suffix":""},{"id":456329260,"identity":"b4117308-929d-458d-bc9f-e4fe78823272","order_by":11,"name":"Siti Sobariah","email":"","orcid":"","institution":"HayandraLab","correspondingAuthor":false,"prefix":"","firstName":"Siti","middleName":"","lastName":"Sobariah","suffix":""},{"id":456329261,"identity":"4bffce9d-7ca8-4437-a8fc-a08d5047539a","order_by":12,"name":"Tias Widyastuti","email":"","orcid":"","institution":"HayandraLab","correspondingAuthor":false,"prefix":"","firstName":"Tias","middleName":"","lastName":"Widyastuti","suffix":""},{"id":456329262,"identity":"cbe5ea73-19cc-4d95-9b81-7af4d22ffdb5","order_by":13,"name":"Alfida Zakiyah","email":"","orcid":"","institution":"HayandraLab","correspondingAuthor":false,"prefix":"","firstName":"Alfida","middleName":"","lastName":"Zakiyah","suffix":""},{"id":456329263,"identity":"1246a145-3e1a-4ac9-a10e-3bc374538ad4","order_by":14,"name":"Prayoga Pannindya","email":"","orcid":"","institution":"HayandraLab","correspondingAuthor":false,"prefix":"","firstName":"Prayoga","middleName":"","lastName":"Pannindya","suffix":""},{"id":456329264,"identity":"af4e4803-875e-43bd-a5d2-1a7a72108ee1","order_by":15,"name":"Azza Maryam","email":"","orcid":"","institution":"HayandraLab","correspondingAuthor":false,"prefix":"","firstName":"Azza","middleName":"","lastName":"Maryam","suffix":""},{"id":456329265,"identity":"1319e0ec-6746-43d9-a2fd-c54fc844f812","order_by":16,"name":"Johannes Albert Biben","email":"","orcid":"","institution":"Klinik Hayandra","correspondingAuthor":false,"prefix":"","firstName":"Johannes","middleName":"Albert","lastName":"Biben","suffix":""},{"id":456329266,"identity":"1bc381ec-9dd7-453e-945d-681cb27512b3","order_by":17,"name":"Nadia Nadia","email":"","orcid":"","institution":"Klinik Hayandra","correspondingAuthor":false,"prefix":"","firstName":"Nadia","middleName":"","lastName":"Nadia","suffix":""},{"id":456329267,"identity":"c78ce612-f67d-4a20-a74f-b31749672487","order_by":18,"name":"Nungki Ratna Martina","email":"","orcid":"","institution":"Klinik Hayandra","correspondingAuthor":false,"prefix":"","firstName":"Nungki","middleName":"Ratna","lastName":"Martina","suffix":""},{"id":456329268,"identity":"389a04f4-b698-4f9d-b09c-b867e18170c2","order_by":19,"name":"Kuswan Ambar Pamungkas","email":"","orcid":"","institution":"Klinik Hayandra","correspondingAuthor":false,"prefix":"","firstName":"Kuswan","middleName":"Ambar","lastName":"Pamungkas","suffix":""},{"id":456329269,"identity":"2cb5d015-99cb-4581-acff-f4b666ccdd4c","order_by":20,"name":"Lydia Dewi Kemala Lay","email":"","orcid":"","institution":"Klinik Hayandra","correspondingAuthor":false,"prefix":"","firstName":"Lydia","middleName":"Dewi Kemala","lastName":"Lay","suffix":""}],"badges":[],"createdAt":"2025-04-07 04:55:53","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-6390155/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6390155/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":82897243,"identity":"6b60e442-40da-4ea6-b190-74d988cf3fd9","added_by":"auto","created_at":"2025-05-16 13:03:00","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":70328,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePercentage of T cell in healthy people, breast cancer, colon cancer and lung cancer. \u003c/strong\u003e(a) CD 3+ percentage; (b) CD 8+ percentage\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6390155/v1/1d62787fe52d04f0f1515410.jpg"},{"id":82897241,"identity":"b6588f42-9ba9-44b1-8bd6-dc05763b82b4","added_by":"auto","created_at":"2025-05-16 13:02:59","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":67950,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePercentage of NK cells in healthy people, breast cancer, colon cancer and lung cancer. \u003c/strong\u003e(a) Percentage of CD NKG2D+; (b) Percentage of CD 56+\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6390155/v1/b8d85e305775c897b2fd17c4.jpg"},{"id":82901213,"identity":"2216f8a8-b701-4b42-898a-57122d30124b","added_by":"auto","created_at":"2025-05-16 13:27:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":955235,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6390155/v1/42d26df3-18d3-4ab4-a046-363e7c02211a.pdf"}],"financialInterests":"","formattedTitle":"Stability of T Cell and NK Cell Profiles in Peripheral Blood Mononuclear Cells After Multiple Immune Cell Therapy Sessions","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThe prevailing conventional strategy for cancer treatment involves the surgical excision of the tumor, followed by adjuvant radiotherapy utilizing X-rays and/or chemotherapy. Despite chemotherapy's success in decreasing morbidity and mortality rates, it is notable that nearly all chemotherapeutic agents exert cytotoxic effects on healthy cells. \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Consequently, global research efforts are focused on identifying alternative and novel treatments for cancer patients. A promising new approach is immune cell therapy, a type of immune cell therapy. This technique involves the ex vivo expansion of innate and adaptive immune cells, derived from autologous blood, which are subsequently reinfused into the patient to target and combat tumors.\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e Cancer immune cell therapy represents one of the most substantial advancements in oncology in recent years, exhibiting remarkable antitumor activity and enduring clinical benefits across diverse malignancies. Recent studies have demonstrated that the infusion of autologous activated lymphocytes, specifically targeted to tumor antigens, can induce prolonged responses in patients with chemotherapy-resistant luminal metastatic breast cancer. This approach, which utilizes mutant protein-specific tumor-infiltrating lymphocytes (TILs) in conjunction with IL-2 and pembrolizumab, has shown significant efficacy in these patients.\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e In clinical trials, tumor-I nfiltrating lymphocytes (TILs) have demonstrated efficacy in various immunogenic cancers, including melanoma and human papillomavirus-associated cancers. Additionally, TILs exhibit considerable variability in their specificity and avidity for undefined antigens. In melanoma, both TILs and peripheral blood lymphocytes, when stimulated in vitro with the patient's tumor, have been shown to effectively eradicate the tumor.\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e Another study involving patients with metastatic colorectal cancer demonstrated that the infusion of CD8\u0026thinsp;+\u0026thinsp;cells targeting the mutant KRAS gene mediated effective antitumor immunotherapy against cancers expressing KRAS G12D and HLA-C.\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e Meta-analyses of immunotherapy studies, including those involving activated T lymphocytes, cytokine-induced NK cells, and dendritic cell vaccine therapy, have demonstrated their effectiveness in treating gastric and lung cancer.\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e These successes suggest that cellular immunotherapy will be a pivotal future therapy for cancer patients.\u003c/p\u003e\u003cp\u003e According to the Journal of the Indonesian Radiation Oncology Society, the most prevalent cancers among patients in Indonesia are breast cancer (36.1%), cervical cancer (17.3%), nasopharyngeal cancer (8.2%), lung cancer (7.4%), rectal cancer (6.9%), leukemia (6.7%), ovarian cancer (6.3%), lymphoma (5.3%), colon cancer (4.0%), and prostate cancer (2.0%). Therefore, this study aims to analyze the profiles of T cells and NK cells in patients with breast, colon, and lung cancer. The focus on solid cancers for this initial study is based on research by Prihantono et al. (2023), which reported 6,203 cases of solid cancer (79.3%) in Indonesia, resulting in 1,063 deaths (61.7% of all cancer-related deaths), compared to 1,621 cases of non-solid cancer (20.7%), resulting in 678 deaths (38.3%) \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eAlthough decades of preclinical and clinical studies conducted globally have demonstrated that cell immunotherapy is safe and effective, only a limited number have evaluated the immune system's response in peripheral blood mononuclear cells, particularly following multiple cell immunotherapy sessions. Therefore, it is essential to adequately verify the safety of cell immunotherapy in both healthy individuals and cancer patients after five sessions. This study aims to analyze the profiles of T cell and NK cell markers in the peripheral blood of healthy individuals and cancer patients post-immunotherapy. To gain insights into the human immune system, the composition of Peripheral Blood Mononuclear Cells (PBMC) will be examined.\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e This PBMC analysis will focus on profiling NK cells and T cells in the patient's body. Natural killer (NK) cells and cytotoxic T cells (CD8+) are critical immune cells that play a pivotal role in defending the body against harmful invaders.\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e NK cells, through their cytotoxic functions, induce apoptosis and participate in the regulation of immune responses. They may also contribute to the pathogenesis of various immune-mediated diseases, including ankylosing spondylitis, Beh\u0026ccedil;et's disease, multiple sclerosis, rheumatoid arthritis, psoriasis, systemic lupus erythematosus, and type-1 diabetes.\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e Additionally, NK cells are positioned between the innate and adaptive immune systems, serving as a \"bridge\" between the two. They also play a role in inflammation and immune regulation.\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e Moreover, dysfunction in the regulatory functions of NK cells can lead to inadequate control of T cell responses, thereby contributing to the development of certain disorders.\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e T cells are involved in a range of immune responses associated with infections, cancer, autoimmune diseases, and allergic conditions.\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e Therefore, it is crucial to assess the immune system by evaluating NK cell and T cell marker profiles in the blood of both healthy individuals and cancer patients following cell immunotherapy.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cp\u003e \u003cb\u003eResearch design\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThis study employed an experimental design, incorporating both control and treatment groups to assess the effects of the intervention. The subjects were divided into two groups: one consisting of healthy individuals and the other consisting of patients diagnosed with cancer, with each group comprising 10 subjects. Blood samples from both healthy individuals and cancer patients, collected on the 14th day of cell immunotherapy, will be submitted to Hayandralab for analysis. Each subject will provide blood samples on five separate occasions\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. The samples will be isolated and characterized according to the research parameters.\u003c/p\u003e \u003cp\u003e \u003cb\u003eBlood Sample Collection from Healthy and Cancer Patients Pre- and Post- Cell Immunotherapy.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThis experiment, conducted at Hayandra Laboratory, involved healthy individuals and cancer patients, with each group providing 10 blood samples on the 14th day following immune cell therapy. Blood samples were collected and submitted five times, with a 2-week interval or on the 14th day post-therapy. Healthy blood donors were required to be over 18 years old, in good health based on medical history and physical examination, and willing to participate by signing informed consent. Cancer blood donors needed to be 18 years or older, of either gender, with a confirmed diagnosis of breast, lung, or colon cancer at stages 1\u0026ndash;4, at least four weeks after the last chemotherapy or radiation therapy, and meet specific laboratory criteria (leukocyte count\u0026thinsp;\u0026ge;\u0026thinsp;3,000/\u0026micro;l, neutrophil count\u0026thinsp;\u0026ge;\u0026thinsp;1,500/\u0026micro;l, platelet count\u0026thinsp;\u0026ge;\u0026thinsp;80,000/\u0026micro;l, hemoglobin\u0026thinsp;\u0026ge;\u0026thinsp;9.0 g/dl, serum creatinine\u0026thinsp;\u0026le;\u0026thinsp;1.5 mg/dl, and serum bilirubin\u0026thinsp;\u0026le;\u0026thinsp;2.5 \u0026times; upper limit of institutional normal range), as well as sign informed consent. Exclusion criteria for both groups included the presence of HIV, hepatitis, psychiatric conditions, drug addiction, pregnancy, autoimmune disorders, or any other condition that could prevent participation or endanger subject safety.\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e \u003cb\u003ePeripheral Blood Mononuclear Cell Isolation\u003c/b\u003e \u003c/p\u003e \u003cp\u003ePeripheral blood mononuclear cell (PBMC) isolation was performed in a Biosafety Cabinet (BSC). Blood, collected in a 1-liter heparin tube (approximately 3 ml), was mixed well, aspirated, and transferred into a T75 flask, with the blood volume recorded. HBSS was added to the flask in a 1:1 ratio and mixed thoroughly. Ficoll was added in a 1:2 ratio, creating a 10 ml tube mixture (3 ml Ficoll and 6 ml diluted blood). The diluted blood was carefully layered over the Ficoll and then centrifuged at 2,500 rpm for 10 minutes at room temperature (acceleration 7, deceleration 1) using a Thermo Scientific Centrifuge SL40. The plasma layer was discarded, and the turbid interphase layer was collected into two 10 ml conical tubes. HBSS was added to each tube to a total volume of 10 ml and mixed thoroughly by pipetting up and down with a new 10 ml serological pipette. The tubes were then centrifuged at 300g for 10 minutes at room temperature (speed 7, deceleration 7). The supernatant was discarded, and the pellet was resuspended in 10 ml of HBSS. The suspension was centrifuged again at 200g for 10 minutes at room temperature (speed 7, deceleration 7), and the supernatant was discarded completely. The final pellet was resuspended in 1 ml of growth medium.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCharacterization of Peripheral Blood Mononuclear Cells via Flow Cytometry\u003c/b\u003e \u003c/p\u003e \u003cp\u003eA suspension of peripheral blood mononuclear cells (PBMCs) at a concentration of at least 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/ml was transferred into a 5 ml round-bottom tube. The suspension was then centrifuged at 1,200 rpm for 5 minutes. After centrifugation, 300 \u0026micro;l of PBS was added to the tube (labeled with the sample code \"unstained\") and the mixture was vortexed. Subsequently, 100 \u0026micro;l of this suspension was transferred to a new tube (labeled with the specific sample code). To the new tube, 5 \u0026micro;l of the following antibodies were added: (1) Anti-Human CD3 FITC clone Hit3a (Tonbo Bioscience 35-0039-T100), (2) Anti-Human CD19 PerCP-Cyanine 5.5 clone HIB19 (Tonbo Bioscience 65-0199-T100), (3) Anti-Human CD56 (N CAM) PE clone MY31 (Tonbo Bioscience 50-0564-T100), (4) Anti-Human CD8 APC clone SK1 (Tonbo Bioscience 20-0087-T100), and (5) PE-CyTM7 Mouse Anti-Human CD314 (NKG2D) clone 1D11 (BD PharmingenTM 562365). The sample was then incubated in the dark for 15 minutes. Following incubation, 1 ml of PBS was added, vortexed, and the mixture was centrifuged again at 1,200 rpm for 5 minutes. Afterward, 300 \u0026micro;l of PBS was added to the pellet, and the sample was analyzed using a flow cytometer.\u003c/p\u003e \u003cp\u003e \u003cb\u003eStatistical Data Analysis\u003c/b\u003e \u003c/p\u003e \u003cp\u003eNormal data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Statistical comparisons between different treatments were conducted using one-way analysis of variance (ANOVA). The analysis was performed with a significance level of α\u0026thinsp;=\u0026thinsp;0.05 and a confidence level of 95%.\u003c/p\u003e"},{"header":"3. Results","content":"\u003cp\u003e \u003cb\u003ea. Hematology Data of Research Blood Samples\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe demographic data analysis revealed that the average age in the healthy group was 58.3 years. Breast cancer patients had a slightly younger average age of 54.7 years compared to the healthy group. Colon cancer patients had an average age of 56.4 years, which was similar to that of the healthy group. In contrast, lung cancer patients had an average age of 65.9 years, which was notably older than the other groups.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eHematology Data of Research Blood Samples\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eCharacteristic\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eHealthy People\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBreast Cancer\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eColon Cancer\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eLung Cancer\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003ep\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"5\" nameend=\"c6\" namest=\"c2\"\u003e \u003cp\u003eAverage\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge (Years)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e58,3\u0026thinsp;\u0026plusmn;\u0026thinsp;16,91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e54,7\u0026thinsp;\u0026plusmn;\u0026thinsp;14,05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e56,4 \u0026plusmn; 14,05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e65,9 6 \u0026plusmn; 9,86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0,319\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLeukocyte Count\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:7\\times\\:\\)\u003c/span\u003e\u003c/span\u003e10\u003csup\u003e3\u003c/sup\u003e \u0026micro;l\u0026thinsp;\u0026plusmn;\u0026thinsp;2,4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e6,5\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\times\\:\\)\u003c/span\u003e\u003c/span\u003e10\u003csup\u003e3\u003c/sup\u003e \u0026micro;l\u0026thinsp;\u0026plusmn;\u0026thinsp;2,2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\text{9,8}\\times\\:\\)\u003c/span\u003e\u003c/span\u003e10\u003csup\u003e3\u003c/sup\u003e \u0026micro;l\u0026thinsp;\u0026plusmn;\u0026thinsp;6,2*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\text{6,4}\\:\\times\\:\\)\u003c/span\u003e\u003c/span\u003e10\u003csup\u003e3\u003c/sup\u003e \u0026micro;l\u0026thinsp;\u0026plusmn;\u0026thinsp;2,4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0,037\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNeutrophil Count\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\text{5,2}\\times\\:\\)\u003c/span\u003e\u003c/span\u003e10\u003csup\u003e3\u003c/sup\u003e \u0026micro;l\u0026thinsp;\u0026plusmn;\u0026thinsp;2,2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\text{4,1}\\times\\:\\)\u003c/span\u003e\u003c/span\u003e10\u003csup\u003e3\u003c/sup\u003e \u0026micro;l\u0026thinsp;\u0026plusmn;\u0026thinsp;1,3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\text{7,3}\\times\\:\\)\u003c/span\u003e\u003c/span\u003e10\u003csup\u003e3\u003c/sup\u003e \u0026micro;l\u0026thinsp;\u0026plusmn;\u0026thinsp;5,7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\text{4,3}\\times\\:\\)\u003c/span\u003e\u003c/span\u003e10\u003csup\u003e3\u003c/sup\u003e \u0026micro;l\u0026thinsp;\u0026plusmn;\u0026thinsp;1,7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0,288\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePlatelet Count\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:285\\times\\:\\)\u003c/span\u003e\u003c/span\u003e10\u003csup\u003e3\u003c/sup\u003e \u0026micro;l\u0026thinsp;\u0026plusmn;\u0026thinsp;77,4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:243\\times\\:\\)\u003c/span\u003e\u003c/span\u003e10\u003csup\u003e3\u003c/sup\u003e \u0026micro;l\u0026thinsp;\u0026plusmn;\u0026thinsp;109,9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\text{395,5}\\times\\:\\)\u003c/span\u003e\u003c/span\u003e10\u003csup\u003e3\u003c/sup\u003e \u0026micro;l\u0026thinsp;\u0026plusmn;\u0026thinsp;182,7*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\text{262,3}\\:\\times\\:\\)\u003c/span\u003e\u003c/span\u003e10\u003csup\u003e3\u003c/sup\u003e \u0026micro;l\u0026thinsp;\u0026plusmn;\u0026thinsp;103,6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0,37\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHemoglobin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e13,7 g/dl\u0026thinsp;\u0026plusmn;\u0026thinsp;1,7*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e11,6 g/dl\u0026thinsp;\u0026plusmn;\u0026thinsp;2,9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e11 g/dl\u0026thinsp;\u0026plusmn;\u0026thinsp;2,7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e11,8 g/dl\u0026thinsp;\u0026plusmn;\u0026thinsp;1,6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0,001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e* It is the result of Duncan's analysis which indicates a statistically significant difference in the same parameters.\u003c/p\u003e \u003cp\u003eRegarding hematological parameters in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e., the average leukocyte count in healthy individuals was 7,000 \u0026micro;l. In comparison, breast cancer and lung cancer patients had slightly lower leukocyte counts of 6.5 \u0026times; 10\u0026sup3; \u0026micro;l and 6.4 \u0026times; 10\u0026sup3; \u0026micro;l, respectively. Conversely, colon cancer patients exhibited a higher average leukocyte count of 9.8 \u0026times; 10\u0026sup3; \u0026micro;l. The average neutrophil count in healthy individuals was 5,200 \u0026micro;l, which was elevated compared to breast cancer and lung cancer patients, who had average neutrophil counts of 4.1 \u0026times; 10\u0026sup3; \u0026micro;l and 4.3 \u0026times; 10\u0026sup3; \u0026micro;l, respectively. Among the groups, colon cancer patients had the highest average neutrophil count, at 7.3 \u0026times; 10\u0026sup3; \u0026micro;l.\u003c/p\u003e \u003cp\u003eThe average platelet count in healthy individuals is 285,000 \u0026micro;l. Breast cancer patients exhibit a lower platelet count of 243 \u0026times; 10\u0026sup3; \u0026micro;l, whereas colon cancer patients have the highest platelet count, at 395.5 \u0026times; 10\u0026sup3; \u0026micro;l. Lung cancer patients have a platelet count of 262.3 \u0026times; 10\u0026sup3; \u0026micro;l, which is slightly lower than that of healthy individuals. Regarding hemoglobin levels, healthy individuals have an average of 13.7 g/dl, which is higher than the levels observed in cancer patients. Breast cancer and colon cancer patients have hemoglobin levels of 11.6 g/dl and 11 g/dl, respectively, while lung cancer patients have a hemoglobin level of 11.8 g/dl, which is slightly lower than that of healthy individuals.\u003c/p\u003e \u003cp\u003eOverall, cancer patients exhibited distinct values for leukocytes, neutrophils, platelets, and hemoglobin compared to healthy individuals. These variations highlight differences in health conditions and physiological responses to disease, with notable discrepancies among different cancer types. Statistically, significant differences were observed in leukocyte counts (p\u0026thinsp;=\u0026thinsp;0.037) and hemoglobin levels (p\u0026thinsp;=\u0026thinsp;0.001) between the groups. No significant differences were found in age (p\u0026thinsp;=\u0026thinsp;0.319), neutrophil counts (p\u0026thinsp;=\u0026thinsp;0.288), or platelet counts (p\u0026thinsp;=\u0026thinsp;0.37).\u003c/p\u003e\u003cp\u003e \u003cb\u003eb. Percentage of T cells in healthy and cancer patients\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAnalysis of the T cell population in peripheral blood mononuclear cells (PBMCs) from blood samples obtained after five rounds of immune cell therapy revealed no significant changes in the percentage of CD3\u0026thinsp;+\u0026thinsp;and CD8\u0026thinsp;+\u0026thinsp;T cells across healthy individuals, breast cancer, colon cancer, and lung cancer patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Statistical analysis yielded non-significant p-values for each group: healthy individuals (0.938), breast cancer patients (0.848), colon cancer patients (0.916), and lung cancer patients (0.701). The proportion of CD3\u0026thinsp;+\u0026thinsp;T cells, representing the total T cell population, showed no significant increase or decrease, indicating no abnormal alterations in the T cell population in the bloodstream following five rounds of immune cell therapy.\u003c/p\u003e \u003cp\u003eAdditionally, the percentage of CD8\u0026thinsp;+\u0026thinsp;T cells, a subset of CD3\u0026thinsp;+\u0026thinsp;T cells with cytotoxic activity against infected and cancerous cells, also showed no significant differences following five sessions of immune cell therapy. Statistical analysis revealed non-significant p-values for each group: healthy individuals (0.640), breast cancer patients (0.522), colon cancer patients (0.633), and lung cancer patients (0.832). These results suggest that CD8\u0026thinsp;+\u0026thinsp;T cells are effectively targeting infected or cancerous cells without causing a significant increase in their overall blood concentration. This indicates that despite repeated immune cell therapy, CD8\u0026thinsp;+\u0026thinsp;T cells maintain stable levels in the bloodstream, reflecting a focused immune response on the targeted areas rather than an increase in CD8\u0026thinsp;+\u0026thinsp;T cells in the general circulation. Thus, the immune cell therapy appears to effectively direct the immune response to specific sites without inducing an unnecessary elevation of CD3\u0026thinsp;+\u0026thinsp;and CD8\u0026thinsp;+\u0026thinsp;T cells in peripheral blood.\u003c/p\u003e\u003cp\u003e \u003cb\u003ec. NK cell percentage in healthy and cancer patients\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAnalysis of the percentage of NK cells with NKG2D and CD56 markers after five rounds of immune cell therapy revealed no significant changes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). NKG2D, an activation receptor on NK cells critical for identifying and destroying infected or cancerous cells, did not show a significant increase. Statistical analysis yielded non-significant p-values for each group: healthy individuals (0.999), breast cancer patients (0.522), colon cancer patients (0.909), and lung cancer patients (0.560). These results suggest that immune cell therapy does not induce abnormal alterations in the ability of NK cells to recognize and attack target cells, and the immune surveillance function of NK cells remains within the normal range.\u003c/p\u003e\u003cp\u003eAdditionally, the percentage of NK cells expressing CD56, a crucial marker of NK cell maturity and activity, remained stable with no significant changes. Statistical analysis showed non-significant p-values for each group: healthy individuals (0.202), breast cancer patients (0.848), colon cancer patients (0.542), and lung cancer patients (0.623). This stability indicates that immune cell therapy does not lead to an excessive increase or decrease in NK cell activity, thus preserving a normal balance in the blood circulation. Overall, the lack of significant changes in NK cells with NKG2D and CD56 markers after five sessions of immune cell therapy suggests that the treatment does not provoke excessive immune reactions or disrupt immune system homeostasis. These findings support the notion that immune cell therapy can be administered safely without causing unwanted alterations in the NK cell population in the bloodstream, thereby maintaining a balanced and healthy immune function. Consequently, these results reinforce the understanding that immune cell therapy is effective and specific, without inducing excessive or misdirected immune responses.\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eDemographic data analysis of healthy individuals and cancer patients revealed significant differences in several hematological parameters, reflecting varying physiological conditions and immune responses to cancer. Notably, colon cancer patients exhibited higher mean leukocyte counts, suggesting a more pronounced inflammatory response. Additionally, the elevated platelet counts observed in colon cancer patients may indicate reactive thrombocytosis, commonly associated with chronic inflammation or neoplastic conditions.\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. The lower hemoglobin levels observed in all cancer patient groups compared to healthy individuals may be attributed to several factors, including bleeding, hemolysis, impaired red blood cell production, or anemia, which affects approximately 40% of cancer patients.\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e This variation is expected in the context of cancer, as the disease frequently induces significant alterations in hematological parameters. Cancer can impact the production, distribution, and destruction of blood cells, contributing to these changes.\u003c/p\u003e \u003cp\u003eThe analysis of blood samples collected after five rounds of immune cell therapy revealed no significant changes in the percentages of CD3\u0026thinsp;+\u0026thinsp;and CD8\u0026thinsp;+\u0026thinsp;T cells, as well as NK cells expressing CD56 and NKG2D, across various groups including healthy individuals and patients with breast cancer, colon cancer, and lung cancer. The high p-values associated with these parameters indicate that immune cell therapy did not induce significant fluctuations in the populations of these T cells and NK cells.\u003c/p\u003e \u003cp\u003eCD3\u0026thinsp;+\u0026thinsp;T cells, representing the total T cell population\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e, showed no significant increase or decrease after five rounds of immune cell therapy, indicating that the therapy did not induce excessive T cell proliferation or a reduction that could suggest an impaired immune response in the patient's PBMCs. The stability in the proportion of CD3\u0026thinsp;+\u0026thinsp;T cells suggests that the therapy operates within safe limits, preserving immune system homeostasis. Similarly, CD8\u0026thinsp;+\u0026thinsp;T cells, which are responsible for cytotoxic activity against infected and cancerous cells through antigen recognition in the context of MHC class I molecules\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, also displayed no significant changes in their percentage. This indicates that CD8\u0026thinsp;+\u0026thinsp;T cells remain focused on their targets without causing unnecessary increases in overall blood circulation, and the immune response remains specifically targeted, avoiding excessive systemic reactions. The stability of both CD3\u0026thinsp;+\u0026thinsp;and CD8\u0026thinsp;+\u0026thinsp;T cell proportions reflects that T cell tolerance is preserved, which is crucial for preventing overreaction to the body's own antigens and avoiding autoimmune damage\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Maintaining T cell tolerance ensures that the immune response induced by the therapy remains effective while protecting healthy tissues from damage.\u003c/p\u003e \u003cp\u003eThese findings contrast with those observed in allogeneic CAR T cell therapy. In such studies, patients frequently exhibit abnormal T cell subsets, as evidenced by flow cytometry showing CD8 expression without corresponding CD3 expression\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. This discrepancy is likely attributable to the differing origins of the cells used in the therapies. Autologous immunotherapy utilizes T cells derived from the patient\u0026rsquo;s own body, which are generally more compatible and better at maintaining immune homeostasis. Conversely, allogeneic CAR T cell therapy employs T cells from external donors, which can induce unwanted immune responses, including the development of abnormal T cell subsets and disruption of immune balance\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Additionally, the standard procedure for allogeneic CAR T cell production often includes the depletion of TCR\u0026thinsp;+\u0026thinsp;cells to achieve specific therapeutic goals. \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e However, previous research has demonstrated that TCR signaling is essential for maintaining T cell homeostasis and long-term survival in vivo\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e, potentially explaining why allogeneic CAR T cell therapy might cause greater immune disruption compared to autologous immune cell therapy.\u003c/p\u003e \u003cp\u003eMaintaining the normal balance of CD3\u0026thinsp;+\u0026thinsp;and CD8\u0026thinsp;+\u0026thinsp;T cells in the blood is crucial for optimal immune system function. T cells are primary regulators of the immune response, modulating the activity of other immune cells and influencing the behavior of endothelial and parenchymal cells. They are essential for protecting against viral and bacterial infections\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Deviations in CD3\u0026thinsp;+\u0026thinsp;or CD8\u0026thinsp;+\u0026thinsp;T cell populations can trigger or exacerbate autoimmune diseases\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e and may also result in diminished immune function.\u003c/p\u003e \u003cp\u003eThe analysis of NK cells marked by NKG2D and CD56 after five rounds of immune cell therapy revealed no significant changes. NKG2D, an activation receptor crucial for NK cell recognition and destruction of infected or cancerous cells\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e, did not show a significant increase. Similarly, the percentage of NK cells expressing CD56, a marker indicative of NK cell maturity and activity, remained stable without significant fluctuations. This stability suggests that immune cell therapy does not induce excessive increases or decreases in NK cell activity, thereby preserving the normal balance of NK cells in the bloodstream. It also implies that the NK cells' ability to detect and target abnormal cells remains intact, ensuring effective immune surveillance. As essential components of innate immunity, NK cells respond swiftly to bodily abnormalities\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e, and immune cell therapy does not impair this critical function. Overall, the lack of significant changes in NK cell percentages with NKG2D and CD56 markers indicates that immune cell therapy does not provoke undue immune responses or disrupt immune system homeostasis.\u003c/p\u003e \u003cp\u003eNK cells and T cells interact through cytokine production and response, engagement with dendritic cells, and mechanisms of target cell destruction\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. These interactions play a critical role in modulating the local immune environment and enhancing the immune response to infections and malignancies.\u003csup\u003e\u003cspan additionalcitationids=\"CR35\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Their collaborative actions ensure a rapid and targeted defense against diverse threats. The absence of abnormalities in both T and NK cells suggests that the immune cell therapy does not induce excessive immune reactions or adverse effects in the circulating T and NK cell populations. This supports the notion that immune cell therapy functions as a precise intervention, targeting areas requiring immune action while maintaining overall immune system homeostasis. Consequently, despite the stability of T and NK cell percentages, these findings underscore the safety and specificity of immune cell therapy in managing various disease conditions, affirming its potential as a focused therapeutic approach.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThe analysis of blood samples after five rounds of immune cell therapy revealed no significant changes in the percentages of CD3\u0026thinsp;+\u0026thinsp;and CD8\u0026thinsp;+\u0026thinsp;T cells among healthy individuals and patients with breast, colon, and lung cancers. The high P values suggest that immune cell therapy does not induce substantial fluctuations in these T cell populations, thereby preserving immune system homeostasis. Similarly, the percentages of NK cells marked by NKG2D and CD56 also remained stable, indicating that immune cell therapy does not lead to excessive NK cell activity and maintains normal blood circulation balance. Overall, the stability in both T cell and NK cell populations signifies that immune cell therapy is effective and safe, without disrupting immune system equilibrium. These results reinforce the notion that immune cell therapy can operate efficiently without inducing excessive or adverse immune responses.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eEthical Approval\u003c/h2\u003e \u003cp\u003e All authors confirm that all experiments involving the use of human samples (blood) were performed in accordance with relevant guidelines and regulations. The experimental protocols were approved by the Ethics Committee at BRIN (Badan Riset dan Inovasi Nasional), Indonesia.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent to Participate\u003c/strong\u003e \u003cp\u003e A formal informed consent form was signed by each participant before they were included in the study.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent to Publish\u003c/strong\u003e \u003cp\u003eNot applicable\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThe study was funded by Yayasan Hayandra in Indonesia.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e \u003cp\u003eK. Karina was the primary supervising doctor for this study, conceptualised and planned the experiments, offered constructive criticism and edits, supplied reagents, supplies, and analytic tools, and approved the final paper. The experiments were also planned and executed by A. Nugroho, who also prepared the text, collected and put together the data, analysed and evaluated the results. I. Rosadi and I. Afini draughted the text and carried out the experiments. T.P. Sibuea, A. Maryam, J.A. Biben, N. Nadia, N.R. Marina, K.A. Pamungkas, and L.D.K. Lay were the supervising doctors and provided the reagents, materials, and analysis tools for this study. Performers of the trials were D. Ernanda, H. Hafizh, A. Azhari, C. B. Sepanya, Y.A. Widyanti, N. Aini, S. Sobariah, T. Widyastuti, A. Zakiyah, and P. Pannindya.\u003c/p\u003e\u003ch2\u003eAcknowledgment\u003c/h2\u003e \u003cp\u003eWe would like to express our heartfelt appreciation to Hayandralab for providing the essential facilities and resources that made this research possible. Special thanks to the entire Hayandralab team for their unwavering support and dedication throughout the study. We also extend our gratitude to our peers and reviewers for their insightful feedback and constructive guidance, which significantly improved the quality of this manuscript.\u003c/p\u003e\u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eThe datasets generated and/or analysed during the current study are not publicly available due to institutional restrictions on sharing data outside the research team, but are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eDebela DT, Muzazu SG, Heraro KD, et al. New approaches and procedures for cancer treatment: Current perspectives. \u003cem\u003eSAGE Open Med\u003c/em\u003e. 2021;9:205031212110343. doi:10.1177/20503121211034366\u003c/li\u003e\n\u003cli\u003eJiang T, Zhou C. 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T cells, NK cells, and tumor-associated macrophages in cancer immunotherapy and the current state of the art of drug delivery systems. \u003cem\u003eFront Immunol\u003c/em\u003e. 2023;14. doi:10.3389/fimmu.2023.1199173\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Immune Cell Therapy, T Cell Stability, NK Cell Stability, Cancer Treatment, Immune Homeostasis","lastPublishedDoi":"10.21203/rs.3.rs-6390155/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6390155/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eImmunotherapy represents a significant advancement in oncology, characterized by the ex vivo expansion of innate and adaptive immune cells to combat cancer. Despite its demonstrated efficacy in both preclinical and clinical studies, assessing the immune system's response through peripheral blood mononuclear cells (PBMC) post-therapy remains crucial. This study examined the expression profiles of T cell and NK cell markers in the PBMC of healthy individuals and cancer patients (breast, lung, and colon cancer) following five immune cell therapy sessions using flow cytometry, specifically evaluating cells with T cell (CD3 and CD8) and NK Cell (CD56 and NKG2D) markers. The results indicated no substantial changes in the proportions of CD3\u0026thinsp;+\u0026thinsp;and CD8\u0026thinsp;+\u0026thinsp;for T cells, nor NK cells expressing CD56 and NKG2D, across healthy subjects, and patients with breast, lung, and colon cancer after five sessions of immune cell therapy. These findings suggest that immune cell therapy does not induce significant alterations in T cell populations or NK cell activity, thereby preserving immune system homeostasis. Consequently, this research supports the notion that immune cell therapy operates effectively and safely, without disrupting immune equilibrium, and offers valuable insights for the development of enhanced cancer treatment modalities in the future.\u003c/p\u003e","manuscriptTitle":"Stability of T Cell and NK Cell Profiles in Peripheral Blood Mononuclear Cells After Multiple Immune Cell Therapy Sessions","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-16 13:02:53","doi":"10.21203/rs.3.rs-6390155/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d4fc94e2-722f-4534-b089-422cf5bd166a","owner":[],"postedDate":"May 16th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-05-16T13:02:56+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-16 13:02:53","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6390155","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6390155","identity":"rs-6390155","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}