LFA-1 Knockout Leads to CD4 + and CD8 + T Cells Differentiation Disorder in Thymus Gland and is Related with ERK Signaling Pathway in Mice

LFA-1 Knockout Leads to CD4 + and CD8 + T Cells Differentiation Disorder in Thymus Gland and is Related with ERK Signaling Pathway in Mice | 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 LFA-1 Knockout Leads to CD4 + and CD8 + T Cells Differentiation Disorder in Thymus Gland and is Related with ERK Signaling Pathway in Mice 秀琼 蒙, Yiting Huang, Yunxia Kuang, Hongliang Ma, Zhengyang Li, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4337853/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 T cell precursors from fetal liver bone marrow migrate into the thymus to initiate their development, including double-negative selection, double-positive selection, and single-positive selection. Subsequently, fully matured single-positive CD4 + T cells or CD8 + T cells traverse the bloodstream to the peripheral tissues, executing immune functions. Lymphocyte function-associated antigen-1 (LFA-1) is invovuled with thymic cortical epithelial cells facilitate positive selection. But LFA-1 mediates signaling pathways in thymic keep unknown. Here, Knockout LFA-1 displayed thymic atrophy and aberrant structural alterations in the cortical and medullary of the thymus in mice. And the cells populations of thymocytes during the positive and negative selection process was observed, characterized by CD4 + T cells increased and CD8 + T cells decreased. Furthermore, LFA-1 inhibitor also impact on thymic development. A significant downregulation of pERK1/2 in MAPK signaling pathway. The thymus gland medullary atrophy still was observed in LFA-1 knockout mice with tail vein tumor metastasis, along with CD4 + T lymphocytes increased and a reduced CD8 + T cells. The Genome Databases revealed that mutations in LFA-1 in clinical patients, suggesting that LFA-1 mutation individuals maybe affect the CD8+ T cells function. This study indicated that LFA-1 regulates the differentiation of CD4 + T and CD8 + T cells in the thymus, implying that LFA-1 mutation in health individuals may influence the tumor immunity or therapy when they get tumor. LFA-1 Transgenic mice T cells differentiation CD4+ T cells CD8+ T cells ERK Signaling Pathway Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Introduction The thymus is the central immunological organ responsible for the differentiation and maturation of T cell[ 1 ]. The process of T-cell differentiation and development entails the migration of hematopoietic stem cells from the bone marrow into the thymus, where they undergo a complex developmental process, transitioning from the cortical to the medullary regions, ultimately differentiating into mature T cells that migrate to peripheral immune organs to exert their functions. The cortex and medulla of the thymus serve as critical sites for the maturation and selection of T cells[ 2 ]. During the onset of infections, autoimmune diseases, and malignancies, the thymus frequently exhibits acute atrophy and functional decline[ 3 ]. The differentiation and development of naive T cells are directly impacted by the size of the thymic organ, as well as the structure and functionality of the thymic microenvironment. Disruption of T-cell homeostasis, thymic atrophy, or structural and functional disorders within the thymic microenvironment can result in immunodeficiency or autoimmune diseases[ 4 ]. Lymphocyte function-associated antigen-1 (LFA-1) plays an instrumental role in regulating leukocyte adhesion and T cell activation[ 5 ]. As one of the integrin family, LFA-1 is composed of the alpha chain CD11a and the beta chain CD18 and is expressed on the majority of leukocytes. It is one of the principal leukocyte integrins on T cells and contributes to the orchestration of the immunological synapse (IS), which includes binding with antigen-presenting cells (APCs), as well as mediating T cell interactions, proliferation, and the induction of T cell effector functions. The adhesion of LFA-1 to its ligand, intercellular adhesion molecule-1 (ICAM-1), facilitates firm adhesion to endothelial cells, prolongs contact with APCs, and anchors to target cells, thereby mediating the targets[ 6 ]. Studies suggested that LFA-1 plays a key role in the formation of the immunological synapse and cooperates with the TCR to modulate T cell activation comprehensively. The immunological synapse constitutes a specialized structure formed between T cells and antigen-presenting cells (APCs) or target cells, which functions to enhance T cell activation and signal transduction[ 7 ]. Mice of LFA-1 (CD11a, ITGAL) knockout exhibit impairments in leukocyte adhesion, lymphocyte proliferation, and tumor rejection. Within the thymus, LFA-1 emerges as an indispensable lymphocyte integrin, critically essential for progenitor cell ingress and the generation of common lymphoid progenitors[ 8 ]. Integratedly, integrins play a pivotal role throughout the entire lifespan of T-cells, from their differentiation in the thymus to their migration to the peripheral lymphoid tissues, ultimately exerting a significant function in the immunological synapses within the tissue, while simultaneously furnishing the T-cells with requisite co-stimulatory signal receptors[ 9 ]. Nonetheless, reports are few about concerning the LFA-1 in the stages of T-cell development within the thymus, as well as the underlying mechanisms. Our previous reports indicated that LFA-1 knockout affected Treg cells and inhibitor tumor growth[ 10 ]. Here, our recent data showed that thymic atrophy was present in LFA-1 knockout mice, and within these thymuses, disorder in the structure of the cortical and medullary regions were evident. An increased CD4 + T cells and a decreased CD8 + T cells in LFA-1 knockout mice. Additionally, the downregulation of the MAPK/ERK pathway in LFA-1 knockout mice suggests a potential interaction between LFA-1 and this signaling pathway. Now, some genes have been successfully pinpointed as targets for cancer therapy, with targeted treatments emerging as an efficacious means of substantially ameliorating survival rates in tumor patients[ 11 ]. We think that LFA-1 mutation would cause CD4 + and CD8 + cells function, altering the health individuals tumor immune. In conclusion, our results indicated the role of LFA-1 in T cell development and the involvement of the MAPK/ERK signaling pathway in the thymus. Although the specific mechanisms are not yet fully clear, it has provided valuable clues and directions for future study, particularly in exploring LFA-1 as an immunomodulatory target, and also suggest that some insights in the molecular mechanisms of thymic CD4 + and CD8 + T cell development and LFA-1. 2. Materials and Methods Mice LFA-1 knockout mice (LFA-1 −/− ) were purchased from The Jackson Laboratory (B6.129S7-Itgaltm1Bll/J, number:005257. C57BL/6J mice were obtained from the Guangdong Medical Laboratory Animal Center, Guangzhou, China. The LFA-1 genotype was identified using polymerase chain reaction (PCR) with specific primers (Supplementary Table 1 and S. Figure 1 ). Mice were housed under specific pathogen-free conditions in the Animal Center of Guangdong Pharmaceutical University. The mice were kept at standard room temperature and humidity, alternating light and dark (12 h:12 h). We raised and managed the mice strictly according to the standards of the animal experimental center of Guangdong Pharmaceutical University (license: SCYK(YUE)2017 − 0125). All animals experiment protocols complied with Guangdong Pharmaceutical University and were approved by the Animal Ethics Committee of Guangdong Pharmaceutical University (Animal Ethics Approval Number: gdpulacspf 2021002). Cell Culture B16-F10 melanoma cells (Shanghai Institute of Cell Biology, Chinese Academy of Sciences) were cultured in Dulbecco's Modification of Eagle's Medium (DMEM) containing 10% fetal bovine serum (FBS; GIBCO) and 1% penicillin-streptomycin. The cell was incubated in a humidified chamber containing 5% CO 2 at 37°C, and it was confirmed with no mycoplasma contamination by PCR detection. H&E and IHC Thymus and spleen tissue sections (2–3 µm) were prepared by 4% Formalin-fixed, paraffin-embedded. After deparaffinization, sections were stained with hematoxylin for 3 minutes and eosin for 30 seconds and were then dried and sealed with neutral resin. IHC staining was performed as described on the Abcam website. Antigen retrieval was performed in citrate buffer (pH 6.0) at high temperature and pressure for 15 minutes. Next, the tissue sections were incubated overnight at 4°C with primary antibodies, including anti-CD11a (1:100, Abcam, ab186873, USA), anti-CD3 (1:100, Abcam, ab16669, USA), anti-CD4 (1:100, CST 25229s) and anti-CD8 (1:100, CST 98941T). Then, the sections were treated with secondary antibodies (1:200, ZSGB-BIO, ZB-2301) at 37°C for one hour and color was developed with DAB chromogenic solution. The sections were counterstained with hematoxylin, dehydrated in graded alcohols, cleared in xylene, and sealed with neutral resin. Digital imaging was performed using a microscope (Olympus CX31, New York, USA). qPCR Total RNA was extracted from mouse thymus with TRIzol reagent (Invitrogen, USA) according to the instructions. All reverse transcription reactions were set up using an EVO M-MLV RT Kit (Accurate Biology AG, China). Real-time PCR was performed using the SYBR® Green Premix Pro Taq HS qPCR Kit (AG11701, Accurate Biology AG) on a LightCycler 96 real-time fluorescent quantitative PCR detection system (Roche). Data were normalized to the endogenous control GAPDH, and fold changes were calculated using relative quantification (2^-∆∆Ct). Flow Cytometry Single-cell suspensions of mouse thymus and spleen were prepared, and red blood cells in the spleen were lysed using red blood cell lysis buffer. Each sample was stained with specific antibodies in various channels, including FITC channel anti-mouse CD44 antibody (BioLegend, 103005, USA), APC channel anti-mouse CD25 antibody (BioLegend,101909, USA), PerCP-Cyanine5.5 anti-mouse CD3 antibody (BioLegend,100218, USA), PE channel anti-mouse CD4 antibody (BioLegend, 100408, USA), and Brilliant Violet 421TM anti-mouse CD8a antibody (BioLegend, 100738, USA). Samples were incubated at 4°C for one hour, washed twice with PBS, and then analyzed on an LSRII flow cytometer (BD Biosciences). Western Blotting Thymus tissues were lysed in a lysis buffer containing 1% protease and phosphatase inhibitor on ice and centrifuged, and the supernatant was collected as the protein sample. Protein quantification was performed using the BCA protein assay. Equal amounts of protein were loaded, and after electrophoresis, transfer, and blocking, the membranes were incubated with primary antibodies overnight at 4°C, followed by secondary antibody incubation. The primary antibodies: anti-CD4(1:100, CST 25229s), anti-CD8(1:100, CST 98941T), anti-Erk (CST,4695T), p-Erk (CST,4370T), SAPK/JNK(CST,9252T), p-SAPK/JNK(CST,4668T), p38(CST,8690), P-P38(CST,8203T), GAPDH(CST,5174S). Mice model of LFA-1 Inhibitor BIRT377 BIRT377 (Tocris; Cat# 4776) in anhydrous ethanol (22.156 mg/mL) was stored at 2–8 ℃. The vehicle was sterile water containing ethanol. On the day of injection, the vehicle was heated in bath at 37 ℃, and BIRT377 concentration is 500 ng/20 µL for i.v. injection[ 12 ]. Animals were injected within one hour of BIRT377 dilution. BIRT377 (20 µL per mouse) was injected through the tail vein into mice in the experimental group (n = 6), and mice in the control group (n = 6) were injected with 20 µL of vehicle. Three times a week for four weeks, the mice were euthanized, and thymus tissues were then excised for subsequent analysis. Tail vein metastasis model B16F10 melanoma cells (2×10^6 cells per mouse) were injected subcutaneously into LFA-1 −/− mice (n = 6) and LFA-1 +/+ mice (n = 6) through the tail vein. Mouse body weight was recorded once a day. After 2 weeks, these mice were sacrificed, and their thymus tissues were harvested and preserved for further analysis. RNA sequencing The total RNA of thymus of LFA-1 −/− mice (n = 4) and LFA-1 +/+ mice (n = 4) were extracted, and cDNA libraries were constructed, which was sequenced on the Illumina sequencing platform. The DESeq algorithm was utilized to filter the differentially expressed genes. Statistical analysis Unpaired tests were performed on the statistical data using GraphPad Prism 9 software, p value of less than 0.05 was considered statistically significant. Data are shown as the mean ± standard deviation (SD) values. 3. Results 3.1 LFA-1 highly expression in cortex but lowly expression in thymic medulla. The LFA-1 composed of an alpha subunit CD11a and a beta subunit CD18, it across the membranes of T lymphocytes, B lymphocytes, monocytes, macrophages, and neutrophils. The analysis of Human Protein Atlas (HPA) database revealed that mRNA expression of LFA-1 is in body organs such as the thymus, lymph nodes, and spleen, with the thymus exhibiting more expression than other organ (Fig. 1 A). Further NCBI database indicate that within 8-week-old C57BL/6J mice, LFA-1 is most abundantly posited in the thymus (Fig. 1 B). Subsequent immunohistochemical of CD11a expression in the thymic of 8-week-old C57 mice suggest that sparse expression in the medulla, while the cortex predominated in highly expression. Within the cortical region, cellular expressions varied, some cells with high, some cells intermediate, and some cells low levels, and yet the precise cellular type have not been determined (Figs. 1 C-D). The immunohistochemical results showed that the high expression of LFA-1 in thymic tissues. The thymus as an immune organ responsible for self-limiting, self-tolerant T cells, and the disruption of T cell homeostasis, thymic atrophy, and structural or functional derangement of the thymic can cause immunodeficiencies or autoimmune diseases. Most LFA-1 positive cells maintain in cortex. 3.2 LFA-1 knockout causes thymic atrophy and structural disruption in mice To investigate if LFA-1 knockout altered the thymic structure or impacts on the differentiation of thymic T cell subsets in mice., we dissected the thymus and spleen of LFA-1 −/− mice and LFA-1 +/+ mice at 8, 18, and 50 weeks of age. The results showed that the thymus of both LFA-1 −/− and LFA-1 +/+ mice both exhibited atrophy with increasing age, and the degree of atrophy intensified with age. Compared to the control group, the extent of thymic atrophy in LFA-1 −/− mice were more (Fig. 2 A). At different age stages, the size, weight, and thymus index (thymus index mg/g = thymus mass mg/body weight g) of LFA-1 −/− mice were significantly lower than those of LFA-1 +/+ mice, and the thymus index decreased significantly with age in LFA-1 −/− mice than that in LFA-1 +/+ mice (Fig. 2 B, *P < 0.05, **P < 0.01, ***P < 0.001). Subsequently, using hematoxylin and eosin (H&E) staining to observe the thymus of mice at different ages under a microscope. Compared to the thymus of LFA-1 +/+ mice, the thymus structure of LFA-1 −/− mice underwent significant changes, with a blurry boundary between the cortex and medulla, irregular size and shape of the medulla, and fusion phenomena (Fig. 2 C). The ratio of medulla to cortex in LFA-1 −/− mice was significantly reduced, as quantified using Image J software (Fig. 2 D, ***P < 0.001). With increasing age, the degree of thymic atrophy and the severity of cortex-medulla fusion in LFA-1 −/− mice became more significant and obvious. 3.3 LFA-1 knockout unbalance the percentage of DN1 ~ 4 Stage cells in thymus Above results indicate that LFA-1 KO significantly affects the structure of the mouse thymus, leading to thymic medulla fusion and a decrease in medulla/cortex ratio. As the thymus is the initial site for the development of T-cell immune function and the site for T cells differentiation. The specific differentiation of T-cells in the thymus is as follows: Initially, lymphoid progenitor cells from the bone marrow or embryo enter the thymus, referred to as early thymic progenitor cells, namely ETP cells. T-cell differentiation in the thymus is divided into three stages. In the first stage, thymic cells in the cortex neither express CD4 nor CD8 on their surface, hence called double-negative (DN) cells[ 13 ]. DN cells are further classified into DN1 ~ 4 stages based on grouping by CD44 and CD25. In the DN3 stage, T-cell receptor (TCR) undergoes β-selection, doubling the number of double-positive (DP) cells expressing both CD4 and CD8. In the second stage, thymic cells undergo positive selection in the cortex, allowing the survival of DP cells[ 14 ]. Subsequently, DP cells migrate from the cortex to the medulla, where they undergo negative selection to differentiate into CD4 single-positive or CD8 single-positive T-cells. Finally, these cells exit through blood vessels to exert immune functions in the periphery (Fig. 3 A)[ 15 ]. Western blotting results suggest that CD4 expression increase and a reduction in CD8 expression in the thymus of LFA-1 −/− mice (Fig. 3 B). Flow cytometry was performed on thymic cells from LFA-1 +/+ mice and LFA-1 −/− mice, representing the development of T-cells in the thymus based on subgroups defined by CD25 and CD44. The results show that the percentage of thymic cells in DN1, DN2, and DN4 stages is reduced in LFA-1 −/− mice, but increased in the DN3 stage (Fig. 3 C, * P < 0.05, ***P < 0.001). This indicates that LFA-1 KO affects the double-negative phase of T-cell development in the thymus, suggesting that LFA-1 knockout impact T-cell development through the double-negative phase. Thymocytes, following the double-negative cells in the cortex, enter the medulla through blood vessels at the cortico-medullary junction, commencing positive selection. Then, we further employed antibodies against CD4 and CD8 to stain thymocytes and differentiated the following subgroups: DP (CD3 + CD4 + CD8+). The results revealed a significant decrease in the percentage of LFA-1 −/− thymic T cells within the DP and CD8 subgroups, while a significant increase was observed in the CD4 subgroup (Fig. 3 D, *** P < 0.001). Collectively, these experimental find that the development of thymic T cells in LFA-1 −/− mice is hindered. Given that initial T cells differentiate into CD4 + and CD8 + T cells in the thymus before traversing the blood vessels at the cortico-medullary junction to reach peripheral immune organs to perform immune functions, and as spleen being a principal peripheral immune organ, we also conducted flow cytometric analysis on the spleen. The results exhibited a significant increase in the proportion of CD4 + T cells and a decrease in CD8 + T cells in the spleens of LFA-1 −/− mice (Fig. 3 E, ***P < 0.001). Thus, the alteration of T cells in the spleens of LFA-1 −/− mice parallels that in the thymus, suggesting that the thymic development defects in LFA-1 −/− mice consequently affect the proportion and quantity of T cells in peripheral immune organs. To further explore whether the LFA-1 knockout affects the distribution of CD4 + and CD8 + T cell expression in the thymus, IHC was used to detect the expression of CD4 + and CD8 + T cells in the thymus. Our data demonstrated an increase in CD3 + T cell expression within the medullary regions of LFA-1 −/− mice’s thymus, an increment in CD4 + T cells, and a decremented expression of CD8 + T cells in the medulla (Fig. 3 F). 3.4 The thymic T cells is abnormal in tail vein tumor metastasis model To observe changes in thymic T cells of LFA-1 −/− mice within the tumor immune environment, we constructed a tumor lung metastasis model through injecting B16F10 melanoma cells into LFA-1 −/− and LFA-1 +/+ tail mice tail. The results indicated that mice with LFA-1 deficiency have an increased number of tumors in lung tissue (Figs. 4 A&B, **P < 0.01). Furthermore, the result show that post-tumor-burden LFA-1 −/− mice presented a conspicuous weight decline and significant atrophy of the thymus and a decrease in thymic weight (Figs. 4 C-E, ***P < 0.001). Further H&E staining of the mice thymic revealed that post-tumor LFA-1 −/− mice had pronounced medullary atrophy and a significant reduction in the medulla/cortex ratio (Fig. 6 F, ***P < 0.001), implying that LFA-1 affects the development and differentiation of thymic T cells in mice of the tumor bearing. Subsequent flow cytometric analysis of thymic T cells from post-tumor mice indicated a significant increase in the proportion of CD4 + T cells and a significant decrease in CD8 + T cells in the thymus of LFA-1 −/− mice (Fig. 6 G, * P < 0.05, **P < 0.01, ***P < 0.001). This variation aligns with the results prior to tumor bearing mice; therefore, we conjecture that post-tumor alterations in CD4 + and CD8 + T cells in the mouse thymus may also be affected the tumor microenvironment. 3.5 LFA-1 Inhibitor also induced the development of thymic T cells BIRT 377 is an inhibitor impeding the interaction between intercellular adhesion molecule-1 (ICAM-1) and lymphocyte function-associated antigen-1 (LFA-1)[ 16 , 17 ]. It has been validated that BIRT 377 can reduce the aggregation of CD3 + T cells and also inhibit the production of IL-2 in vivo. Here, we observed the developmental state of mouse thymic T cells using LFA-1 inhibitor model. Four-week-old C57 mice were divided into a BIRT377 injection group and control group, administered via intravenous injection thrice weekly for four weeks, with tissue collection post-treatment at week eight (Fig. 4 A). During this time, mouse activity and weight changes were observed and recorded. The results showed no significant difference in body weight between the two groups (Fig. 4 B). The spleen size, weight, and spleen index of the BIRT 377-treated group were comparable to the control group (Fig. 4 C&D). However, there was a noticeable change in thymus size in the BIRT 377 group, with a significant decrease in thymus weight and index (Fig. 4 C&D, * P < 0.05). Subsequently, H&E staining of thymus and spleen tissues from both groups revealed that the boundary between the cortex and medulla in the thymus became unclear in the BIRT 377-treated group, showing a trend of medullary atrophy (Fig. 5 E). Flow cytometry analysis of thymic cells from both groups indicated no difference in the percentage of DN1 ~ 4 stage cells and T cells in the BIRT 377-treated group (Fig. 5 G&I). Similarly, H&E staining and flow cytometry analysis of spleen tissues showed atrophy of the white pulp, with a blurred boundary between white and red pulp in the BIRT 377-treated group (Fig. 5 F). Flow cytometry analysis revealed no difference in the percentage of CD4 + and CD8 + T cells in the BIRT 377 group (Fig. 5 H). In summary, the results suggest that postnatally blocking LFA-1 with BIRT 377 also affects thymic T cell development. 3.6 LFA-1 knockout downregulated the ERK1/2 signaling pathway Above results suggest that the LFA-1 knockout cause the differentiation abnormal of thymic T cells. To further explore the underlying mechanisms. mRNA transcriptome sequencing of thymus tissue was carried out with eight-week-old LFA-1 −/− and LFA-1 +/+ mice (Figs. 4 A-C). Because of antigen-presenting cells (APCs) being activated, naive CD4 + T cells differentiate into functionally distinct T-cell lineages, including Th1, Th2, Th17, and regulatory T (Treg) cell. Therefore, the corresponding factors or genes of T-cell subsets were analyzed, and the sequencing data showed that the transcription factor Foxp3 in the thymus of the LFA-1 −/− mice increased significantly (S. Figure 2 A-E). and cell surface molecules and effectors corresponding to CD8 + T-cell subsets were analyze, the chemokines CCL4 was increased significantly and NK cell surface chemokine CCR6 was significantly reduced (S. Figure 2 F-G). Selecting three upregulated and three downregulated differentially expressed genes (DEGs) within the thymus tissue, qRT-PCR technology was utilized to validation. The outcomes affirmed (Fig. 4 D) that the expression trends of the chosen differential genes were consistent with the sequencing data. Through KEGG functional enrichment analysis, it found that after LFA-1 knockout, most genes in mouse thymus were mainly enriched in KEGG pathways such as the Rap1 signaling pathway, MAPK signaling pathway and VEGF signaling pathway. As Rap1 and MAPK were the most significantly signaling pathways, we analyzed the expression of relevant differentially expressed genes in these two pathways. The results suggested that the LFA-1 could regulate the Rap1 and MAPK signaling pathways (Fig. 4 E). Here, we focused on the MAPK pathway. The MAPK pathway in various cellular processes, inclusive of cell proliferation, differentiation, and migration. In mice, at least c-Jun N-terminal kinases (SAPK/JNK), p38 proteins, and the extracellular signal-regulated kinases (p42/44 MAPK) are expressed[ 18 ]. Among them, JNK and p38 have same functions, all concerned with inflammation, apoptosis, and growth; while p42/44 MAPK are tasked with controlling cellular growth and differentiation[ 19 – 21 ]. Next, the MAPK pathway in the thymus tissue of LFA-1 −/− and LFA-1 +/+ mice, as well as BIRT377 model, was determined through WB. The results showed that an uptrend in SAPK/JNK and p38 in the thymus following LFA-1 knockout, with a prominent downtrend in p42/44(Fig. 4 F&G). The LFA-1 knockout is correlates with MAPK pathway-associated proteins, particularly the extracellular signal-regulated kinases (p42/44). Simultaneously, the CD8a and CD8b promotor in the database have many motifs binding TFAP2B and a significant downregulation of the TFAP2B transcription factor (Fig. 4 H and 4 I), suggesting that LFA-1 knockout affects the CD8a and CD8b during thymic cell differentiation maybe is by influencing the CD8 promotor motif. 3.7 LFA-1 change is correlation with the prognosis of tumor patients and exhibits genetic mutations in individuals and Above results indicate that the knockout of LFA-1 affects the differentiation of thymic CD4 + and CD8 + T cells in mice. Next, using TIMER database, we found that the expression of LFA-1 is significantly higher in various tumor tissues than in adjacent tissues (Fig. 7 A). and it is positively correlated between LFA-1 (ITGAL) and CD4, CD8, and immune cell infiltration in cutaneous melanoma (SKCM), and we reported in our previous published paper that LFA-1 is related with CD4 and CD8 in other type cancer of patients. The expression level of LFA-1 is positively correlated with tumor cell purity and positively correlated with CD4 + T cell and CD8 + T cell infiltration (Fig. 7 B). From the analysis of the cBioPortal for Cancer Genomics databases, it found that multiple mutation sites in the ITGAL gene in populations (Fig. 7 C), the database shows a significant difference in survival prognosis between the LFA-1 mutation group and the normal group in the patients (Fig. 7 D, E & F). The data also showed that significant differences between the CD4 and CD8 groups group in the LFA-1 changed population (Fig. 7 G). These results suggests that LFA-1 affect CD4 + and CD8 + cells immunity or treatment in tumor microenvironment. It provides us the hypothesis that the individuals immune is various with individual genetic mutations of T cells, which maybe cause good or bad prognosis in tumor patients. 4. Discussion Our previously published data indicated that the knockout of LFA-1 can inhibit the proliferation of subcutaneous tumor, and a decrease in Treg cells[ 10 ]. However, the mechanisms underlying this remain unclear. In this study, our experimental results revealed thymic atrophy in LFA-1 knockout mice, alongside a constriction of the medullary area. The experiments showed that LFA-1 knockout affected on the differentiation and maturation of thymic T cells. The presence of CD4 + T cells increased and a decrease of CD8 + T cells in both the thymus and peripheral immune organs. The thymus serves as a crucial site for T cell development, with key processes such as differentiation, maturation, and output in the thymic microenvironment provided by thymic epithelial cells (TECs), including cortical TECs (cTECs) and medullary TECs (mTECs). These cells regulate positive and negative selection of thymic T cells, and the interaction between immature thymic cells and TEC subpopulations is vital for the development and maturation of T cells[ 22 ]. Cell migration of thymic cells within the thymic microenvironment occurs throughout the developmental process, with cells moving from the cortical to medullary regions, and eventually exiting to the periphery[ 23 ]. Beginning at the corticomedullary junction, lymphoid progenitor cells from the bone marrow first become early thymic precursors (ETPs) through the reception of notch ligands (DLL4) and interleukin-7 (IL-7) from cTECs[ 24 ]. ETPs, also referred to as "double-negative" (DN) thymic cells due to the lack of CD4 and CD8 co-receptor expression, progress through DN1-4 stages with coordinated expression of CD44 and CD25 on their cell surfaces. DN1/ETPs migrate to the subcapsular cortical region, triggering development into DN2 and DN3 stages, where TCRβ chain expression occurs at DN3[ 25 ], leading to maturation into "double-positive" thymic cells expressing both CD4 and CD8 co-receptors[ 26 ]. During positive selection, thymic cells differentiate into single-positive CD4 + or CD8 + T cells, which then migrate to the thymic medulla. Subsequent negative selection mediated by mTECs leads to the elimination of autoreactive T cells through apoptosis, while positively selected single-positive T cells become functionally distinct CD4 helper T cells or CD8 cytotoxic T cells, ready for output to the periphery[ 26 ]. Our flow cytometry data indicated that, compared to LFA-1 +/+ mice, LFA-1 −/− mice exhibit varying degrees of reduction in cell proportions at the DN1, DN2, and DN4 stages, with an increase in DN3 cells. This suggests that the developmental defects of T cells in the thymus due to LFA-1 knockout begin as early as the DN stage, and the percentage of cells at the DP stage is significantly reduced, indicating a deficiency in positive selection of thymic cells due to the absence of LFA-1. This developmental ensures the peripheral accumulation of naive CD4 + and CD8 + T cells, as well as CD4 + regulatory T cells (Tregs), within the blood and lymphoid tissues[ 27 ]. At the DN stage, thymocytes must undergo TCRβ chain rearrangement, pairing with the pre-TCRα chain to form a TCR complex, a process termed “β-selection”[ 28 ]. The pre-TCR signals, active from the DN3a stage through to the CD4 + CD8 + DP proliferative burst stage, see the invariable pre-Tα chain expression disappear, to be replaced by TCRα upon successful V-J gene rearrangement[ 29 ]. Several studies have suggested that optimal pre-TCR signal transduction—potentially ligand-dependent or independent—may play a critical role in establishing the mature TCRβ repertoire in the mature mammalian T cells[ 30 , 31 ]. During β-selection, pre-TCR-mediated signaling curtails further rearrangement of TCRβ alleles through a process called allelic exclusion[ 32 ], concurrent with the activation of Notch signaling and cellular proliferation, ultimately propelling the αβ-T cell lineage to mature into the DP stage[ 33 , 34 ]. BIRT377 is an inhibitor of LFA-1 that has been confirmed to block the binding between LFA-1 and ICAM-1, consequently suppressing the interaction between T cells and antigen presentation[ 35 ]. In our study, BIRT377 impacts the differentiation and development of thymic T cells. And it also revealed a reduction in ERK kinase in the thymus of LFA-1 knockout mice, ultimately leading to a decrease in the number of CD8 + T cells that undergo positive selection in the thymus. Contrary to previous findings, we speculate that LFA-1, during the differentiation and development of thymic T cells, exerts negative regulation on T cell positive selection through the MAPK signaling pathway[ 36 ]. However, the precise mechanisms underlying this action need do more in LFA-1 KO mice. Typically, active MAPK1 is crucial for the success of positive selection. Elevated levels of MAPK1 activity promote the lineage commitment to CD4 + single positive (SP) T cells, whereas reduced MAPK1 activity leads to an increased count of CD8 + SP T cells—the duration of MAPK1 activation appears pivotal in the developmental process of CD4 + versus CD8 + T cells[ 37 ]. Our studies also reveal reduced ERK kinase in the thymus of LFA-1 knockout mice, resulting in diminished numbers of CD8 + T cells completing their positive selection. Hence, we hypothesize that LFA-1, during the differentiation and maturation of thymic T cells, negatively regulates positive selection via the MAPK signaling pathway, although the exact mechanisms remain to be elucidated and are currently under investigation. Lastly, in tumor patients, the LFA-1 mutations has been correlated with variations in the quantity or functionality of CD8 + T cells, indicating that LFA-1 may be a critical factor influencing tumor immune responses and patient prognosis. This discovery provides a basis for potentially enhancing understand for why the same TNM stage of patients show different response for tumor growth, especially some patients live more life spans than other patients although we did not clear it if these patients have normal LFA-1 and other patients with LFA-1 mutation. Since the mechanisms underlying the role of LFA-1 are not fully understood, more should be investigated the intracellular signaling pathways responsible for abnormal thymocyte differentiation in our body, particularly focusing on the dynamic regulation involving the MAPK/ERK pathway. Next investigations should employ single-cell sequencing techniques, such as single-cell RNA sequencing (scRNA-seq), to intricately explore the transcriptional changes induced by LFA-1 knockout in thymic cells. Additionally, high-resolution cellular imaging techniques can be utilized to observe the movement and subset distribution of T cell precursors in the thymus of LFA-1 knockout mice, providing clues on how T cells position themselves and interact with thymic epithelial cells (TECs). In conclusion, this study offers researcher for understanding the molecular mechanisms of thymic T cell development and has provided valuable clues for future studies, particularly in exploring LFA-1 mutation in heathen individuals and our body immune ability had exists when one born. Abbreviations LFA-1 （ ITGAL ） : Lymphocyte function-associated antigen-1 Treg: Regulatory T cells ICAM-1: Intercellular adhesion molecule 1 H&E: Hematoxylin-Eosin staining IHC: Immunohistochemistry DAB: Diaminobenzidine PBS: Phosphate-buffered saline GAPDH: Glyceraldehyde 3-phosphate dehydrogenase qRT-PCR: Quantitative reverse transcription-polymerase chain reaction SKCM: skin cutaneous melanoma Declarations Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Acknowledgments The authors thank Dr. Zhang Qianqian, Qi Cuiling and Zheng Lingyun for advice in our manuscript and data analysis. Funding This work was supported by grants from National Natural Science Foundation of China (Grant ID: 81773118 to Jiangchao Li), and Science and Technology Planning Project of Guangdong Province, China 2023A0505050153. Ethics declarations Ethics approval and consent to participate All mice were housed in the specific pathogen-free barrier facility of Animal Center of Guangdong Pharmaceutical University with license number SCYK (Guangdong) 2017-0125. The Animal Ethics Review Committee approves all Guangdong Pharmaceutical University animal experiments; the animal ethics approval number: gdpulacspf 2021002. Consent for publication The consents for publication from all authors were obtained. Conflict of Interest The authors declare that they have no conflicts of interest. Authors' contributions The authors contributed equally: Xiuqiong Meng, Yiting Huang. Conception idea and scheme design: Xiuqiong Meng, Yiting Huang, Jiangchao Li. Acquisition of experiments data: Xiuqiong Meng, Yiting Huang Hongliang Ma, Ruyu Zeng, Yunxia Kuang, Zhengyang Li. Data statistics and analysis: Xiuqiong Meng, Yiting Huang, Zhengyang Li. Writing and revising manuscripts: Xiuqiong Meng, Jiangchao Li. Manage transgenic mice: Xiuqiong Meng, Yiting Huang, Hongliang Ma. Supervision and Funding: Jiangchao Li. Jugao Chen All authors read and approved the final manuscript. References Pearse G: Normal structure, function and histology of the thymus . Toxicol Pathol 2006, 34 (5):504-514. Bhandoola A, von Boehmer H, Petrie HT, Zúñiga-Pflücker JC: Commitment and developmental potential of extrathymic and intrathymic T cell precursors: plenty to choose from . Immunity 2007, 26 (6):678-689. Mandal D, Lahiry L, Bhattacharyya A, Bhattacharyya S, Sa G, Das T: Tumor-induced thymic involution via inhibition of IL-7R alpha and its JAK-STAT signaling pathway: protection by black tea . Int Immunopharmacol 2006, 6 (3):433-444. Chung B, Montel-Hagen A, Ge S, Blumberg G, Kim K, Klein S, Zhu Y, Parekh C, Balamurugan A, Yang OO, Crooks GM: Engineering the human thymic microenvironment to support thymopoiesis in vivo . Stem Cells 2014, 32 (9):2386-2396. Gulla S, Reddy MC, Reddy VC, Chitta S, Bhanoori M, Lomada D: Role of thymus in health and disease . Int Rev Immunol 2023, 42 (5):347-363. Reina M, Espel E: Role of LFA-1 and ICAM-1 in Cancer . Cancers (Basel) 2017, 9 (11). Walling BL, Kim M: LFA-1 in T Cell Migration and Differentiation . Front Immunol 2018, 9 :952. Hogg N, Patzak I, Willenbrock F: The insider's guide to leukocyte integrin signalling and function . Nat Rev Immunol 2011, 11 (6):416-426. Bivona TG, Doebele RC: A framework for understanding and targeting residual disease in oncogene-driven solid cancers . Nat Med 2016, 22 (5):472-478. Niu T, Li Z, Huang Y, Ye Y, Liu Y, Ye Z, Jiang L, He X, Wang L, Li J: LFA-1 knockout inhibited the tumor growth and is correlated with treg cells . Cell Commun Signal 2023, 21 (1):233. Thakur V, Kutty RV: Recent advances in nanotheranostics for triple negative breast cancer treatment . J Exp Clin Cancer Res 2019, 38 (1):430. Sanchez JJ, Sanchez JE, Noor S, Ruffaner-Hanson CD, Davies S, Wagner CR, Jantzie LL, Mellios N, Savage DD, Milligan ED: Targeting the β2-integrin LFA-1, reduces adverse neuroimmune actions in neuropathic susceptibility caused by prenatal alcohol exposure . Acta Neuropathol Commun 2019, 7 (1):54. Barnett JB: Consequences of Blocking the Choreography of Double Negative Thymocyte Maturation . In: Signaling Mechanisms Regulating T Cell Diversity and Function. edn. Edited by Soboloff J, Kappes DJ. Boca Raton (FL): CRC Press/Taylor & Francis© 2017 Taylor & Francis Group, LLC.; 2018: 1-16. Takaba H, Takayanagi H: The Mechanisms of T Cell Selection in the Thymus . Trends Immunol 2017, 38 (11):805-816. Starr TK, Jameson SC, Hogquist KA: Positive and negative selection of T cells . Annu Rev Immunol 2003, 21 :139-176. Evans R, Patzak I, Svensson L, De Filippo K, Jones K, McDowall A, Hogg N: Integrins in immunity . J Cell Sci 2009, 122 (Pt 2):215-225. Woska JR, Jr., Shih D, Taqueti VR, Hogg N, Kelly TA, Kishimoto TK: A small-molecule antagonist of LFA-1 blocks a conformational change important for LFA-1 function . J Leukoc Biol 2001, 70 (2):329-334. Cargnello M, Roux PP: Activation and function of the MAPKs and their substrates, the MAPK-activated protein kinases . Microbiol Mol Biol Rev 2011, 75 (1):50-83. Keshet Y, Seger R: The MAP kinase signaling cascades: a system of hundreds of components regulates a diverse array of physiological functions . Methods Mol Biol 2010, 661 :3-38. Sabio G, Davis RJ: TNF and MAP kinase signalling pathways . Semin Immunol 2014, 26 (3):237-245. Plotnikov A, Zehorai E, Procaccia S, Seger R: The MAPK cascades: signaling components, nuclear roles and mechanisms of nuclear translocation . Biochim Biophys Acta 2011, 1813 (9):1619-1633. Wang HX, Pan W, Zheng L, Zhong XP, Tan L, Liang Z, He J, Feng P, Zhao Y, Qiu YR: Thymic Epithelial Cells Contribute to Thymopoiesis and T Cell Development . Front Immunol 2019, 10 :3099. Takahama Y: Journey through the thymus: stromal guides for T-cell development and selection . Nat Rev Immunol 2006, 6 (2):127-135. Peschon JJ, Morrissey PJ, Grabstein KH, Ramsdell FJ, Maraskovsky E, Gliniak BC, Park LS, Ziegler SF, Williams DE, Ware CB et al : Early lymphocyte expansion is severely impaired in interleukin 7 receptor-deficient mice . J Exp Med 1994, 180 (5):1955-1960. Gascoigne NR, Rybakin V, Acuto O, Brzostek J: TCR Signal Strength and T Cell Development . Annu Rev Cell Dev Biol 2016, 32 :327-348. Fehling HJ, Krotkova A, Saint-Ruf C, von Boehmer H: Crucial role of the pre-T-cell receptor alpha gene in development of alpha beta but not gamma delta T cells . Nature 1995, 375 (6534):795-798. Palmer E: Negative selection--clearing out the bad apples from the T-cell repertoire . Nat Rev Immunol 2003, 3 (5):383-391. Ziegler SF: FOXP3: of mice and men . Annu Rev Immunol 2006, 24 :209-226. Dutta A, Zhao B, Love PE: New insights into TCR β-selection . Trends Immunol 2021, 42 (8):735-750. Michie AM, Zúñiga-Pflücker JC: Regulation of thymocyte differentiation: pre-TCR signals and beta-selection . Semin Immunol 2002, 14 (5):311-323. Mallis RJ, Bai K, Arthanari H, Hussey RE, Handley M, Li Z, Chingozha L, Duke-Cohan JS, Lu H, Wang JH et al : Pre-TCR ligand binding impacts thymocyte development before αβTCR expression . Proc Natl Acad Sci U S A 2015, 112 (27):8373-8378. Mallis RJ, Arthanari H, Lang MJ, Reinherz EL, Wagner G: NMR-directed design of pre-TCRβ and pMHC molecules implies a distinct geometry for pre-TCR relative to αβTCR recognition of pMHC . J Biol Chem 2018, 293 (3):754-766. Das DK, Mallis RJ, Duke-Cohan JS, Hussey RE, Tetteh PW, Hilton M, Wagner G, Lang MJ, Reinherz EL: Pre-T Cell Receptors (Pre-TCRs) Leverage Vβ Complementarity Determining Regions (CDRs) and Hydrophobic Patch in Mechanosensing Thymic Self-ligands . J Biol Chem 2016, 291 (49):25292-25305. Brady BL, Steinel NC, Bassing CH: Antigen receptor allelic exclusion: an update and reappraisal . J Immunol 2010, 185 (7):3801-3808. Woska JR, Jr., Last-Barney K, Rothlein R, Kroe RR, Reilly PL, Jeanfavre DD, Mainolfi EA, Kelly TA, Caviness GO, Fogal SE et al : Small molecule LFA-1 antagonists compete with an anti-LFA-1 monoclonal antibody for binding to the CD11a I domain: development of a flow-cytometry-based receptor occupancy assay . J Immunol Methods 2003, 277 (1-2):101-115. Li D, Molldrem JJ, Ma Q: LFA-1 regulates CD8+ T cell activation via T cell receptor-mediated and LFA-1-mediated Erk1/2 signal pathways . J Biol Chem 2009, 284 (31):21001-21010. Germain RN: T-cell development and the CD4-CD8 lineage decision . Nat Rev Immunol 2002, 2 (5):309-322. Additional Declarations No competing interests reported. Supplementary Files SupplementaryData.docx 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-4337853","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":299875485,"identity":"76a9ae40-1c4f-4e8e-8f8f-aff90ee77791","order_by":0,"name":"秀琼 蒙","email":"","orcid":"","institution":"Guangdong Pharmaceutical University","correspondingAuthor":false,"prefix":"","firstName":"秀琼","middleName":"","lastName":"蒙","suffix":""},{"id":299875486,"identity":"4dc82561-8123-4566-9ad0-60592e364ff1","order_by":1,"name":"Yiting Huang","email":"","orcid":"","institution":"Guangdong Pharmaceutical University","correspondingAuthor":false,"prefix":"","firstName":"Yiting","middleName":"","lastName":"Huang","suffix":""},{"id":299875487,"identity":"35f7b555-b9b8-41b8-990a-986dc4207a0d","order_by":2,"name":"Yunxia Kuang","email":"","orcid":"","institution":"Guangdong Pharmaceutical University","correspondingAuthor":false,"prefix":"","firstName":"Yunxia","middleName":"","lastName":"Kuang","suffix":""},{"id":299875488,"identity":"0275ddc7-f7c5-45c0-9556-35492df6d85e","order_by":3,"name":"Hongliang Ma","email":"","orcid":"","institution":"Guangdong Pharmaceutical University","correspondingAuthor":false,"prefix":"","firstName":"Hongliang","middleName":"","lastName":"Ma","suffix":""},{"id":299875489,"identity":"85627350-5703-46f5-82bf-ea7f68a8ab51","order_by":4,"name":"Zhengyang Li","email":"","orcid":"","institution":"Guangdong Pharmaceutical University","correspondingAuthor":false,"prefix":"","firstName":"Zhengyang","middleName":"","lastName":"Li","suffix":""},{"id":299875490,"identity":"d1fabc2e-87ee-42eb-9e70-a2dd8c89ebc3","order_by":5,"name":"Ruyu Zeng","email":"","orcid":"","institution":"Guangdong Pharmaceutical University","correspondingAuthor":false,"prefix":"","firstName":"Ruyu","middleName":"","lastName":"Zeng","suffix":""},{"id":299875491,"identity":"222d1c8a-b0e3-4713-8973-957cdefed9a0","order_by":6,"name":"Jugao Chen","email":"","orcid":"","institution":"Shenzhen People’s Hospital, Second Clinical Medical College of Jinan University, First Affiliated Hospital of Southern University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Jugao","middleName":"","lastName":"Chen","suffix":""},{"id":299875492,"identity":"b0d5e570-58e9-47a8-9ef6-e9a012837745","order_by":7,"name":"Jiangchao Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+0lEQVRIiWNgGAWjYBACPmaGhAMghgEDA+MDBjYGKBsPYEPSwmxAnBYYA6iMTYI4LewMDw/83FErb85++Fjll7I7iQ3szdskGGru4HXYwd4zxw139qSl3ZY59yyxgedYmQTDsWf4/cLbdizB4ECO2W3JtsOJDRI5ZhKMDYfx2/IXpOX8G7NisBb5N4S1HOZtq0kwuJFjxvgRbAsPEVpk2w4YbrjxLFma4dwz4zaetGKLhGO4tfDzn0n++LatTt7gfPLBjz/K7sj2sx/eeONDDW4tDAw8CUACooCZh+EAJGoS8GhgYGA/ACTqwEzGHwwH8KodBaNgFIyCkQkAA7RaXIRxnSEAAAAASUVORK5CYII=","orcid":"","institution":"Guangdong Pharmaceutical University","correspondingAuthor":true,"prefix":"","firstName":"Jiangchao","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2024-04-28 12:10:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4337853/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4337853/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":56279717,"identity":"209c1fff-c870-4afb-8ab7-2192e6a9e3f0","added_by":"auto","created_at":"2024-05-10 20:46:14","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":11950749,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLFA-1 highly expresses in cortex but lowly in thymic medulla\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eA.LFA-1 exhibits the highly expression level in human thymic tissue. (\u003ca href=\"https://www.proteinatlas.org/\"\u003ehttps://www.proteinatlas.org/\u003c/a\u003e) B. The analysis of data indicated that LFA-1 is highly expression in human thymic tissue. (\u003ca href=\"https://www.ncbi.nlm.nih.gov/\"\u003ehttps://www.ncbi.nlm.nih.gov/\u003c/a\u003e) \u0026nbsp;C-D. The expression of LFA-1(CD11a or named ITGAL) in mouse thymic tissue which were stained with CD11a antibody.\u003c/p\u003e","description":"","filename":"Fig.1Mengetal.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4337853/v1/4117e73a3f23a5478ff45470.jpg"},{"id":56279718,"identity":"87ddf4c8-3a43-40fa-b457-a43f1e6df9fd","added_by":"auto","created_at":"2024-05-10 20:46:14","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":8738091,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLFA-1 knockout causes thymic atrophy and structural disruption in mice\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eA. The size of the thymus in LFA-1 knockout mice and WT mice were changed at different age stages（n = 4 /group）. B. The thymus index of mice at different age stages (Thymus index = thymus weight/body weight × 10) (n = 4 /group; mean ± SE; *p \u0026lt; 0.05, **p \u0026lt; 0.01). C. To observe the structural features of the thymus at different age stages, thymic tissues were stained with H\u0026amp;E staining. D. The ratio of thymic medullary area to cortical area in mice at different age stages (n = 3/group; mean ± SE; ***P \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"Fig.2Mengetal.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4337853/v1/e9c8335a9ca6df2e4e685088.jpg"},{"id":56279716,"identity":"e4b78f7d-14b8-481c-b07c-a24fe9405cc8","added_by":"auto","created_at":"2024-05-10 20:46:14","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":37636764,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLFA-1 knockout disorder the percentage of DN1~4 Stage cells in thymus.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. A schematic diagram of thymic T-cell differentiation and development process. B. Expression of CD4 and CD8 protein in thymus of LFA-1\u003csup\u003e+/+\u003c/sup\u003e mice and LFA-1\u003csup\u003e-/-\u003c/sup\u003e mice by Western Blot. C. The percentage of DN1~DN4 cells in thymus of LFA-1\u003csup\u003e+/+\u003c/sup\u003e mice and LFA-1\u003csup\u003e-/-\u003c/sup\u003e mice by flow cytometry (n = 4 each group; mean ± SE; *p \u0026lt; 0.05, ***p \u0026lt; 0.001). D. The percentage of CD4+T cells and CD8 +T cells in thymus of LFA-1\u003csup\u003e+/+\u003c/sup\u003e mice and LFA-1\u003csup\u003e-/- \u003c/sup\u003emice by flow cytometry (n = 4 /group; mean ± SE; ***p \u0026lt; 0.001). E. In spleen, the percentage of CD4+T cells and CD8 +T cells of LFA-1\u003csup\u003e+/+\u003c/sup\u003e mice and LFA-1\u003csup\u003e-/- \u003c/sup\u003emice by flow cytometry (n = 4 / group; mean ± SE; ***p \u0026lt; 0.001). F. IHC staining of CD4 and CD8 in thymus of LFA-1\u003csup\u003e+/+\u003c/sup\u003e mice and LFA-1\u003csup\u003e-/-\u003c/sup\u003e mice.\u003c/p\u003e","description":"","filename":"Fig.3Mengetal.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4337853/v1/70587e448757ea5763c9f4de.jpg"},{"id":56279713,"identity":"9403eeb4-0783-41bf-aa5c-9482f8b6e6a0","added_by":"auto","created_at":"2024-05-10 20:46:10","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":10601738,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe abnormal development of thymic T cells also presents in tail vein tumor metastasis model.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA\u0026amp;B. The growth of tumors in lung tissues in a model of pulmonary metastasis (n=9, ** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01). C-E. The size changes of thymus, body weight changes and thymic index in LFA-1\u003csup\u003e+/+\u003c/sup\u003e and LFA-1\u003csup\u003e-/- \u003c/sup\u003egroups of mice with tail vein tumors metastasis model (B16 cell lines) (n=3, * P \u0026lt; 0.05). F. The thymic medullary cortical area changes in LFA-1\u003csup\u003e-/-\u003c/sup\u003e + B16 mice compared to LFA-1\u003csup\u003e+/+\u003c/sup\u003e+ B16 mice (n=3, *P\u0026lt;0.05). G. The distribution of CD4+ T and CD8+T cells in the spleen of LFA-1\u003csup\u003e-/-\u003c/sup\u003e + B16 mice compare with LFA-1\u003csup\u003e+/+\u003c/sup\u003e+ B16 mice (n=4, *P\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"Fig.4Mengetal.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4337853/v1/fb325bc04eacccd6bc5213a0.jpg"},{"id":56279714,"identity":"6c139057-5a3a-4236-84bf-18252a5fdbd1","added_by":"auto","created_at":"2024-05-10 20:46:11","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1499565,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLFA-1 inhibitor disrupts the development of thymic T cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA．A schematic diagram of the LFA-1 inhibitor model construction. The schematic diagram made on this site (\u003ca href=\"https://app.biorender.com/\"\u003ehttps://app.biorender.com/\u003c/a\u003e). \u0026nbsp;B. Body weight changes in the mouse model treated with this LFA-1 inhibitor. C. The general map of the spleen and thymus in control group and BIRT377 group (n = 4 / group). D. Spleen index (spleen weight/body weight) and thymus index (thymus index = thymus weight/body weight × 10) in control group and BIRT377 group (n = 4 / group, mean ± SE; *p \u0026lt; 0.05). E\u0026amp;F. The structural features of the thymus and spleen in control group and BIRT377 group, thymus and spleen tissues were stained with H\u0026amp;E staining. G. The percentage of DN1~DN4 cells in thymus of two groups by flow cytometry (n = 4 / group; mean ± SE, P \u0026gt; 0.05). H. The CD4+T cells and CD8 +T cells in thymus of control group and BIRT377 group detected by flow cytometry (n = 4 / group; mean ± SE, P \u0026gt; 0.05). I. Expression of CD4+T cells and CD8 +T cells in thymus of control group and BIRT377 group by flow cytometry (n = 4/group; mean ± SE, P \u0026gt; 0.05).\u003c/p\u003e","description":"","filename":"Fig.5Mengetal.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4337853/v1/dd3cf614b7c7feb5b7006625.jpg"},{"id":56279721,"identity":"923dfa87-e795-42fa-8f60-477ea993a3b2","added_by":"auto","created_at":"2024-05-10 20:46:16","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":11143151,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLFA-1 knockout downregulated the ERK1/2 signaling pathway\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. The mRNA sequencing of thymus showed that LFA-1 knockout up-regulated 3301 genes and down-regulated 339 genes. B. The Gene Ontology (GO) functional enrichment analysis of differential genes. C. The differential gene KEGG functional enrichment analysis indicate that the LFA-1 knockout mice primarily regulate signaling pathways such as Rap1 and MAPK. D. The relative expression levels of upregulated and downregulated genes in the thymus transcriptome sequencing results (**p \u0026lt; 0.01, ***p \u0026lt; 0.001). E. The FPKM ratios of genes associated with the Rap1 and MAPK pathways in thymic sequencing data analysis (*p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001). F. Western blot data indicated the expression of proteins related to the MAPK signaling pathway in the thymus of LFA-1\u003csup\u003e+/+\u003c/sup\u003e mice and LFA-1\u003csup\u003e-/-\u003c/sup\u003e mice. G. Western blot analysis of the proteins level related to the MAPK signaling pathway in the thymus of control group and BIRT377 group. H\u0026amp;I. The mRNA sequencing of LFA-1 KO thymus showed transcription factor changes, which binding to CD8a or CD8b promotor, TFAP2B is dramatically downregulated. And The CD8a and CD8b promotor include lots of putative sites to being bind by TFAP2B.\u003c/p\u003e","description":"","filename":"Fig.6Mengetal.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4337853/v1/a289f65f43241c5434ea4e46.jpg"},{"id":56279720,"identity":"aa04c4c2-6fc6-43c6-a599-5141ee9c064a","added_by":"auto","created_at":"2024-05-10 20:46:15","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":9813359,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eITGAL changes is correlation with prognosis of tumor patients and ITGAL mutations exist in the health individuals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. In this study, LFA-1, also maned ITGAL. The expression of ITGAL is significantly higher in various tumor tissues than in adjacent tissues. B. The expression level of ITGAL is positively correlated correlated with CD4\u003csup\u003e+ \u003c/sup\u003eT cell and CD8\u003csup\u003e+\u003c/sup\u003e T cell infiltration in tumor. C. The analysis of the cBioPortal for Cancer Genomics databases, multiple mutation sites in the ITGAL gene in populations. D-F. The analysis of database shows a significant difference in survival prognosis between the ITGAL mutation or other changes group and the normal group in the patients. G\u0026amp;H. There is a significant difference between the CD4 and CD8 groups group in the LFA-1 changed population, suggesting mutation sites exits in ITAGL of health individual or tumor patients.\u003c/p\u003e","description":"","filename":"Fig.7Mengetal.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4337853/v1/55fe5170ec96b3e0986abdb3.jpg"},{"id":56279712,"identity":"78622189-c2f0-4eda-86f4-0fe906b7f92d","added_by":"auto","created_at":"2024-05-10 20:46:07","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":7964957,"visible":true,"origin":"","legend":"\u003cp\u003eLegend not included with this version\u003c/p\u003e","description":"","filename":"Fig.8Mengetal.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4337853/v1/75d91cc18d129562ee724773.jpg"},{"id":56280840,"identity":"6a7517a3-acc8-4d07-8d3a-8d7c57e6044e","added_by":"auto","created_at":"2024-05-10 21:03:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":100790131,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4337853/v1/f2ae74b0-dd48-4654-81e2-9d66db0708a2.pdf"},{"id":56279719,"identity":"b522720a-4066-4d2b-b2de-bd5c02cf59ce","added_by":"auto","created_at":"2024-05-10 20:46:15","extension":"docx","order_by":12,"title":"","display":"","copyAsset":false,"role":"supplement","size":562731,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryData.docx","url":"https://assets-eu.researchsquare.com/files/rs-4337853/v1/1a52e4096e67bfce0d17db16.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"LFA-1 Knockout Leads to CD4 + and CD8 + T Cells Differentiation Disorder in Thymus Gland and is Related with ERK Signaling Pathway in Mice","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe thymus is the central immunological organ responsible for the differentiation and maturation of T cell[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The process of T-cell differentiation and development entails the migration of hematopoietic stem cells from the bone marrow into the thymus, where they undergo a complex developmental process, transitioning from the cortical to the medullary regions, ultimately differentiating into mature T cells that migrate to peripheral immune organs to exert their functions.\u003c/p\u003e \u003cp\u003eThe cortex and medulla of the thymus serve as critical sites for the maturation and selection of T cells[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. During the onset of infections, autoimmune diseases, and malignancies, the thymus frequently exhibits acute atrophy and functional decline[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The differentiation and development of naive T cells are directly impacted by the size of the thymic organ, as well as the structure and functionality of the thymic microenvironment. Disruption of T-cell homeostasis, thymic atrophy, or structural and functional disorders within the thymic microenvironment can result in immunodeficiency or autoimmune diseases[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eLymphocyte function-associated antigen-1 (LFA-1) plays an instrumental role in regulating leukocyte adhesion and T cell activation[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. As one of the integrin family, LFA-1 is composed of the alpha chain CD11a and the beta chain CD18 and is expressed on the majority of leukocytes. It is one of the principal leukocyte integrins on T cells and contributes to the orchestration of the immunological synapse (IS), which includes binding with antigen-presenting cells (APCs), as well as mediating T cell interactions, proliferation, and the induction of T cell effector functions. The adhesion of LFA-1 to its ligand, intercellular adhesion molecule-1 (ICAM-1), facilitates firm adhesion to endothelial cells, prolongs contact with APCs, and anchors to target cells, thereby mediating the targets[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Studies suggested that LFA-1 plays a key role in the formation of the immunological synapse and cooperates with the TCR to modulate T cell activation comprehensively. The immunological synapse constitutes a specialized structure formed between T cells and antigen-presenting cells (APCs) or target cells, which functions to enhance T cell activation and signal transduction[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMice of LFA-1 (CD11a, ITGAL) knockout exhibit impairments in leukocyte adhesion, lymphocyte proliferation, and tumor rejection. Within the thymus, LFA-1 emerges as an indispensable lymphocyte integrin, critically essential for progenitor cell ingress and the generation of common lymphoid progenitors[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Integratedly, integrins play a pivotal role throughout the entire lifespan of T-cells, from their differentiation in the thymus to their migration to the peripheral lymphoid tissues, ultimately exerting a significant function in the immunological synapses within the tissue, while simultaneously furnishing the T-cells with requisite co-stimulatory signal receptors[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Nonetheless, reports are few about concerning the LFA-1 in the stages of T-cell development within the thymus, as well as the underlying mechanisms.\u003c/p\u003e \u003cp\u003eOur previous reports indicated that LFA-1 knockout affected Treg cells and inhibitor tumor growth[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Here, our recent data showed that thymic atrophy was present in LFA-1 knockout mice, and within these thymuses, disorder in the structure of the cortical and medullary regions were evident. An increased CD4\u003csup\u003e+\u003c/sup\u003e T cells and a decreased CD8\u003csup\u003e+\u003c/sup\u003e T cells in LFA-1 knockout mice. Additionally, the downregulation of the MAPK/ERK pathway in LFA-1 knockout mice suggests a potential interaction between LFA-1 and this signaling pathway. Now, some genes have been successfully pinpointed as targets for cancer therapy, with targeted treatments emerging as an efficacious means of substantially ameliorating survival rates in tumor patients[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. We think that LFA-1 mutation would cause CD4\u0026thinsp;+\u0026thinsp;and CD8\u0026thinsp;+\u0026thinsp;cells function, altering the health individuals tumor immune.\u003c/p\u003e \u003cp\u003eIn conclusion, our results indicated the role of LFA-1 in T cell development and the involvement of the MAPK/ERK signaling pathway in the thymus. Although the specific mechanisms are not yet fully clear, it has provided valuable clues and directions for future study, particularly in exploring LFA-1 as an immunomodulatory target, and also suggest that some insights in the molecular mechanisms of thymic CD4\u0026thinsp;+\u0026thinsp;and CD8\u0026thinsp;+\u0026thinsp;T cell development and LFA-1.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cp\u003eMice\u003c/p\u003e \u003cp\u003eLFA-1 knockout mice (LFA-1\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e) were purchased from The Jackson Laboratory (B6.129S7-Itgaltm1Bll/J, number:005257. C57BL/6J mice were obtained from the Guangdong Medical Laboratory Animal Center, Guangzhou, China. The LFA-1 genotype was identified using polymerase chain reaction (PCR) with specific primers (Supplementary Table\u0026nbsp;1 and S. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Mice were housed under specific pathogen-free conditions in the Animal Center of Guangdong Pharmaceutical University. The mice were kept at standard room temperature and humidity, alternating light and dark (12 h:12 h). We raised and managed the mice strictly according to the standards of the animal experimental center of Guangdong Pharmaceutical University (license: SCYK(YUE)2017\u0026thinsp;\u0026minus;\u0026thinsp;0125). All animals experiment protocols complied with Guangdong Pharmaceutical University and were approved by the Animal Ethics Committee of Guangdong Pharmaceutical University (Animal Ethics Approval Number: gdpulacspf 2021002).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCell Culture\u003c/p\u003e \u003cp\u003eB16-F10 melanoma cells (Shanghai Institute of Cell Biology, Chinese Academy of Sciences) were cultured in Dulbecco's Modification of Eagle's Medium (DMEM) containing 10% fetal bovine serum (FBS; GIBCO) and 1% penicillin-streptomycin. The cell was incubated in a humidified chamber containing 5% CO\u003csub\u003e2\u003c/sub\u003e at 37\u0026deg;C, and it was confirmed with no mycoplasma contamination by PCR detection.\u003c/p\u003e \u003cp\u003eH\u0026amp;E and IHC\u003c/p\u003e \u003cp\u003eThymus and spleen tissue sections (2\u0026ndash;3 \u0026micro;m) were prepared by 4% Formalin-fixed, paraffin-embedded. After deparaffinization, sections were stained with hematoxylin for 3 minutes and eosin for 30 seconds and were then dried and sealed with neutral resin. IHC staining was performed as described on the Abcam website. Antigen retrieval was performed in citrate buffer (pH 6.0) at high temperature and pressure for 15 minutes. Next, the tissue sections were incubated overnight at 4\u0026deg;C with primary antibodies, including anti-CD11a (1:100, Abcam, ab186873, USA), anti-CD3 (1:100, Abcam, ab16669, USA), anti-CD4 (1:100, CST 25229s) and anti-CD8 (1:100, CST 98941T). Then, the sections were treated with secondary antibodies (1:200, ZSGB-BIO, ZB-2301) at 37\u0026deg;C for one hour and color was developed with DAB chromogenic solution. The sections were counterstained with hematoxylin, dehydrated in graded alcohols, cleared in xylene, and sealed with neutral resin. Digital imaging was performed using a microscope (Olympus CX31, New York, USA).\u003c/p\u003e \u003cp\u003eqPCR\u003c/p\u003e \u003cp\u003eTotal RNA was extracted from mouse thymus with TRIzol reagent (Invitrogen, USA) according to the instructions. All reverse transcription reactions were set up using an EVO M-MLV RT Kit (Accurate Biology AG, China). Real-time PCR was performed using the SYBR\u0026reg; Green Premix Pro Taq HS qPCR Kit (AG11701, Accurate Biology AG) on a LightCycler 96 real-time fluorescent quantitative PCR detection system (Roche). Data were normalized to the endogenous control GAPDH, and fold changes were calculated using relative quantification (2^-∆∆Ct).\u003c/p\u003e \u003cp\u003eFlow Cytometry\u003c/p\u003e \u003cp\u003eSingle-cell suspensions of mouse thymus and spleen were prepared, and red blood cells in the spleen were lysed using red blood cell lysis buffer. Each sample was stained with specific antibodies in various channels, including FITC channel anti-mouse CD44 antibody (BioLegend, 103005, USA), APC channel anti-mouse CD25 antibody (BioLegend,101909, USA), PerCP-Cyanine5.5 anti-mouse CD3 antibody (BioLegend,100218, USA), PE channel anti-mouse CD4 antibody (BioLegend, 100408, USA), and Brilliant Violet 421TM anti-mouse CD8a antibody (BioLegend, 100738, USA). Samples were incubated at 4\u0026deg;C for one hour, washed twice with PBS, and then analyzed on an LSRII flow cytometer (BD Biosciences).\u003c/p\u003e \u003cp\u003eWestern Blotting\u003c/p\u003e \u003cp\u003eThymus tissues were lysed in a lysis buffer containing 1% protease and phosphatase inhibitor on ice and centrifuged, and the supernatant was collected as the protein sample. Protein quantification was performed using the BCA protein assay. Equal amounts of protein were loaded, and after electrophoresis, transfer, and blocking, the membranes were incubated with primary antibodies overnight at 4\u0026deg;C, followed by secondary antibody incubation. The primary antibodies: anti-CD4(1:100, CST 25229s), anti-CD8(1:100, CST 98941T), anti-Erk (CST,4695T), p-Erk (CST,4370T), SAPK/JNK(CST,9252T), p-SAPK/JNK(CST,4668T), p38(CST,8690), P-P38(CST,8203T), GAPDH(CST,5174S).\u003c/p\u003e \u003cp\u003eMice model of LFA-1 Inhibitor BIRT377\u003c/p\u003e \u003cp\u003eBIRT377 (Tocris; Cat# 4776) in anhydrous ethanol (22.156 mg/mL) was stored at 2\u0026ndash;8 ℃. The vehicle was sterile water containing ethanol. On the day of injection, the vehicle was heated in bath at 37 ℃, and BIRT377 concentration is 500 ng/20 \u0026micro;L for i.v. injection[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Animals were injected within one hour of BIRT377 dilution. BIRT377 (20 \u0026micro;L per mouse) was injected through the tail vein into mice in the experimental group (n\u0026thinsp;=\u0026thinsp;6), and mice in the control group (n\u0026thinsp;=\u0026thinsp;6) were injected with 20 \u0026micro;L of vehicle. Three times a week for four weeks, the mice were euthanized, and thymus tissues were then excised for subsequent analysis.\u003c/p\u003e \u003cp\u003eTail vein metastasis model\u003c/p\u003e \u003cp\u003eB16F10 melanoma cells (2\u0026times;10^6 cells per mouse) were injected subcutaneously into LFA-1\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice (n\u0026thinsp;=\u0026thinsp;6) and LFA-1\u003csup\u003e+/+\u003c/sup\u003e mice (n\u0026thinsp;=\u0026thinsp;6) through the tail vein. Mouse body weight was recorded once a day. After 2 weeks, these mice were sacrificed, and their thymus tissues were harvested and preserved for further analysis.\u003c/p\u003e \u003cp\u003eRNA sequencing\u003c/p\u003e \u003cp\u003eThe total RNA of thymus of LFA-1\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice (n\u0026thinsp;=\u0026thinsp;4) and LFA-1\u003csup\u003e+/+\u003c/sup\u003e mice (n\u0026thinsp;=\u0026thinsp;4) were extracted, and cDNA libraries were constructed, which was sequenced on the Illumina sequencing platform. The DESeq algorithm was utilized to filter the differentially expressed genes.\u003c/p\u003e \u003cp\u003eStatistical analysis\u003c/p\u003e \u003cp\u003eUnpaired tests were performed on the statistical data using GraphPad Prism 9 software, p value of less than 0.05 was considered statistically significant. Data are shown as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) values.\u003c/p\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e3.1 LFA-1 highly expression in cortex but lowly expression in thymic medulla.\u003c/h2\u003e \u003cp\u003eThe LFA-1 composed of an alpha subunit CD11a and a beta subunit CD18, it across the membranes of T lymphocytes, B lymphocytes, monocytes, macrophages, and neutrophils. The analysis of Human Protein Atlas (HPA) database revealed that mRNA expression of LFA-1 is in body organs such as the thymus, lymph nodes, and spleen, with the thymus exhibiting more expression than other organ (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Further NCBI database indicate that within 8-week-old C57BL/6J mice, LFA-1 is most abundantly posited in the thymus (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Subsequent immunohistochemical of CD11a expression in the thymic of 8-week-old C57 mice suggest that sparse expression in the medulla, while the cortex predominated in highly expression. Within the cortical region, cellular expressions varied, some cells with high, some cells intermediate, and some cells low levels, and yet the precise cellular type have not been determined (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC-D). The immunohistochemical results showed that the high expression of LFA-1 in thymic tissues. The thymus as an immune organ responsible for self-limiting, self-tolerant T cells, and the disruption of T cell homeostasis, thymic atrophy, and structural or functional derangement of the thymic can cause immunodeficiencies or autoimmune diseases. Most LFA-1 positive cells maintain in cortex.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e3.2 LFA-1 knockout causes thymic atrophy and structural disruption in mice\u003c/h2\u003e \u003cp\u003eTo investigate if LFA-1 knockout altered the thymic structure or impacts on the differentiation of thymic T cell subsets in mice., we dissected the thymus and spleen of LFA-1\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice and LFA-1\u003csup\u003e+/+\u003c/sup\u003e mice at 8, 18, and 50 weeks of age. The results showed that the thymus of both LFA-1\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e and LFA-1\u003csup\u003e+/+\u003c/sup\u003e mice both exhibited atrophy with increasing age, and the degree of atrophy intensified with age. Compared to the control group, the extent of thymic atrophy in LFA-1\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice were more (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). At different age stages, the size, weight, and thymus index (thymus index mg/g\u0026thinsp;=\u0026thinsp;thymus mass mg/body weight g) of LFA-1\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice were significantly lower than those of LFA-1\u003csup\u003e+/+\u003c/sup\u003e mice, and the thymus index decreased significantly with age in LFA-1\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice than that in LFA-1\u003csup\u003e+/+\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, *P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **P\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Subsequently, using hematoxylin and eosin (H\u0026amp;E) staining to observe the thymus of mice at different ages under a microscope. Compared to the thymus of LFA-1\u003csup\u003e+/+\u003c/sup\u003e mice, the thymus structure of LFA-1\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice underwent significant changes, with a blurry boundary between the cortex and medulla, irregular size and shape of the medulla, and fusion phenomena (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). The ratio of medulla to cortex in LFA-1\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice was significantly reduced, as quantified using Image J software (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, ***P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). With increasing age, the degree of thymic atrophy and the severity of cortex-medulla fusion in LFA-1\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice became more significant and obvious.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e3.3 LFA-1 knockout unbalance the percentage of DN1\u0026thinsp;~\u0026thinsp;4 Stage cells in thymus\u003c/h2\u003e \u003cp\u003eAbove results indicate that LFA-1 KO significantly affects the structure of the mouse thymus, leading to thymic medulla fusion and a decrease in medulla/cortex ratio. As the thymus is the initial site for the development of T-cell immune function and the site for T cells differentiation. The specific differentiation of T-cells in the thymus is as follows: Initially, lymphoid progenitor cells from the bone marrow or embryo enter the thymus, referred to as early thymic progenitor cells, namely ETP cells. T-cell differentiation in the thymus is divided into three stages. In the first stage, thymic cells in the cortex neither express CD4 nor CD8 on their surface, hence called double-negative (DN) cells[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. DN cells are further classified into DN1\u0026thinsp;~\u0026thinsp;4 stages based on grouping by CD44 and CD25. In the DN3 stage, T-cell receptor (TCR) undergoes β-selection, doubling the number of double-positive (DP) cells expressing both CD4 and CD8. In the second stage, thymic cells undergo positive selection in the cortex, allowing the survival of DP cells[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Subsequently, DP cells migrate from the cortex to the medulla, where they undergo negative selection to differentiate into CD4 single-positive or CD8 single-positive T-cells. Finally, these cells exit through blood vessels to exert immune functions in the periphery (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA)[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Western blotting results suggest that CD4 expression increase and a reduction in CD8 expression in the thymus of LFA-1\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Flow cytometry was performed on thymic cells from LFA-1\u003csup\u003e+/+\u003c/sup\u003e mice and LFA-1\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice, representing the development of T-cells in the thymus based on subgroups defined by CD25 and CD44. The results show that the percentage of thymic cells in DN1, DN2, and DN4 stages is reduced in LFA-1\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice, but increased in the DN3 stage (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, * P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, ***P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). This indicates that LFA-1 KO affects the double-negative phase of T-cell development in the thymus, suggesting that LFA-1 knockout impact T-cell development through the double-negative phase. Thymocytes, following the double-negative cells in the cortex, enter the medulla through blood vessels at the cortico-medullary junction, commencing positive selection. Then, we further employed antibodies against CD4 and CD8 to stain thymocytes and differentiated the following subgroups: DP (CD3\u0026thinsp;+\u0026thinsp;CD4\u0026thinsp;+\u0026thinsp;CD8+). The results revealed a significant decrease in the percentage of LFA-1\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e thymic T cells within the DP and CD8 subgroups, while a significant increase was observed in the CD4 subgroup (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, *** P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Collectively, these experimental find that the development of thymic T cells in LFA-1\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice is hindered. Given that initial T cells differentiate into CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e T cells in the thymus before traversing the blood vessels at the cortico-medullary junction to reach peripheral immune organs to perform immune functions, and as spleen being a principal peripheral immune organ, we also conducted flow cytometric analysis on the spleen. The results exhibited a significant increase in the proportion of CD4\u003csup\u003e+\u003c/sup\u003e T cells and a decrease in CD8\u003csup\u003e+\u003c/sup\u003e T cells in the spleens of LFA-1\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, ***P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Thus, the alteration of T cells in the spleens of LFA-1\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice parallels that in the thymus, suggesting that the thymic development defects in LFA-1\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice consequently affect the proportion and quantity of T cells in peripheral immune organs. To further explore whether the LFA-1 knockout affects the distribution of CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e T cell expression in the thymus, IHC was used to detect the expression of CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e T cells in the thymus. Our data demonstrated an increase in CD3\u003csup\u003e+\u003c/sup\u003e T cell expression within the medullary regions of LFA-1\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice\u0026rsquo;s thymus, an increment in CD4\u003csup\u003e+\u003c/sup\u003e T cells, and a decremented expression of CD8\u003csup\u003e+\u003c/sup\u003e T cells in the medulla (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e3.4 The thymic T cells is abnormal in tail vein tumor metastasis model\u003c/h2\u003e \u003cp\u003eTo observe changes in thymic T cells of LFA-1\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice within the tumor immune environment, we constructed a tumor lung metastasis model through injecting B16F10 melanoma cells into LFA-1\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e and LFA-1\u003csup\u003e+/+\u003c/sup\u003e tail mice tail. The results indicated that mice with LFA-1 deficiency have an increased number of tumors in lung tissue (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA\u0026amp;B, **P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Furthermore, the result show that post-tumor-burden LFA-1\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice presented a conspicuous weight decline and significant atrophy of the thymus and a decrease in thymic weight (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC-E, ***P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Further H\u0026amp;E staining of the mice thymic revealed that post-tumor LFA-1\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice had pronounced medullary atrophy and a significant reduction in the medulla/cortex ratio (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003eF, ***P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), implying that LFA-1 affects the development and differentiation of thymic T cells in mice of the tumor bearing. Subsequent flow cytometric analysis of thymic T cells from post-tumor mice indicated a significant increase in the proportion of CD4\u003csup\u003e+\u003c/sup\u003e T cells and a significant decrease in CD8\u003csup\u003e+\u003c/sup\u003e T cells in the thymus of LFA-1\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003eG, * P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **P\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). This variation aligns with the results prior to tumor bearing mice; therefore, we conjecture that post-tumor alterations in CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e T cells in the mouse thymus may also be affected the tumor microenvironment.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e3.5 LFA-1 Inhibitor also induced the development of thymic T cells\u003c/h2\u003e \u003cp\u003eBIRT 377 is an inhibitor impeding the interaction between intercellular adhesion molecule-1 (ICAM-1) and lymphocyte function-associated antigen-1 (LFA-1)[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. It has been validated that BIRT 377 can reduce the aggregation of CD3\u003csup\u003e+\u003c/sup\u003e T cells and also inhibit the production of IL-2 in vivo. Here, we observed the developmental state of mouse thymic T cells using LFA-1 inhibitor model. Four-week-old C57 mice were divided into a BIRT377 injection group and control group, administered via intravenous injection thrice weekly for four weeks, with tissue collection post-treatment at week eight (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). During this time, mouse activity and weight changes were observed and recorded. The results showed no significant difference in body weight between the two groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). The spleen size, weight, and spleen index of the BIRT 377-treated group were comparable to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC\u0026amp;D). However, there was a noticeable change in thymus size in the BIRT 377 group, with a significant decrease in thymus weight and index (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC\u0026amp;D, * P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Subsequently, H\u0026amp;E staining of thymus and spleen tissues from both groups revealed that the boundary between the cortex and medulla in the thymus became unclear in the BIRT 377-treated group, showing a trend of medullary atrophy (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). Flow cytometry analysis of thymic cells from both groups indicated no difference in the percentage of DN1\u0026thinsp;~\u0026thinsp;4 stage cells and T cells in the BIRT 377-treated group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eG\u0026amp;I). Similarly, H\u0026amp;E staining and flow cytometry analysis of spleen tissues showed atrophy of the white pulp, with a blurred boundary between white and red pulp in the BIRT 377-treated group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). Flow cytometry analysis revealed no difference in the percentage of CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e T cells in the BIRT 377 group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eH). In summary, the results suggest that postnatally blocking LFA-1 with BIRT 377 also affects thymic T cell development.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.6 LFA-1 knockout downregulated the ERK1/2 signaling pathway\u003c/h2\u003e \u003cp\u003eAbove results suggest that the LFA-1 knockout cause the differentiation abnormal of thymic T cells. To further explore the underlying mechanisms. mRNA transcriptome sequencing of thymus tissue was carried out with eight-week-old LFA-1\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e and LFA-1\u003csup\u003e+/+\u003c/sup\u003e mice (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-C). Because of antigen-presenting cells (APCs) being activated, naive CD4\u0026thinsp;+\u0026thinsp;T cells differentiate into functionally distinct T-cell lineages, including Th1, Th2, Th17, and regulatory T (Treg) cell. Therefore, the corresponding factors or genes of T-cell subsets were analyzed, and the sequencing data showed that the transcription factor Foxp3 in the thymus of the LFA-1\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice increased significantly (S. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-E). and cell surface molecules and effectors corresponding to CD8\u0026thinsp;+\u0026thinsp;T-cell subsets were analyze, the chemokines CCL4 was increased significantly and NK cell surface chemokine CCR6 was significantly reduced (S. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF-G). Selecting three upregulated and three downregulated differentially expressed genes (DEGs) within the thymus tissue, qRT-PCR technology was utilized to validation. The outcomes affirmed (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD) that the expression trends of the chosen differential genes were consistent with the sequencing data.\u003c/p\u003e \u003cp\u003eThrough KEGG functional enrichment analysis, it found that after LFA-1 knockout, most genes in mouse thymus were mainly enriched in KEGG pathways such as the Rap1 signaling pathway, MAPK signaling pathway and VEGF signaling pathway. As Rap1 and MAPK were the most significantly signaling pathways, we analyzed the expression of relevant differentially expressed genes in these two pathways. The results suggested that the LFA-1 could regulate the Rap1 and MAPK signaling pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). Here, we focused on the MAPK pathway. The MAPK pathway in various cellular processes, inclusive of cell proliferation, differentiation, and migration. In mice, at least c-Jun N-terminal kinases (SAPK/JNK), p38 proteins, and the extracellular signal-regulated kinases (p42/44 MAPK) are expressed[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Among them, JNK and p38 have same functions, all concerned with inflammation, apoptosis, and growth; while p42/44 MAPK are tasked with controlling cellular growth and differentiation[\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Next, the MAPK pathway in the thymus tissue of LFA-1\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e and LFA-1\u003csup\u003e+/+\u003c/sup\u003e mice, as well as BIRT377 model, was determined through WB. The results showed that an uptrend in SAPK/JNK and p38 in the thymus following LFA-1 knockout, with a prominent downtrend in p42/44(Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF\u0026amp;G). The LFA-1 knockout is correlates with MAPK pathway-associated proteins, particularly the extracellular signal-regulated kinases (p42/44). Simultaneously, the CD8a and CD8b promotor in the database have many motifs binding TFAP2B and a significant downregulation of the TFAP2B transcription factor (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI), suggesting that LFA-1 knockout affects the CD8a and CD8b during thymic cell differentiation maybe is by influencing the CD8 promotor motif.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e3.7 LFA-1 change is correlation with the prognosis of tumor patients and exhibits genetic mutations in individuals and\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAbove results indicate that the knockout of LFA-1 affects the differentiation of thymic CD4\u0026thinsp;+\u0026thinsp;and CD8\u0026thinsp;+\u0026thinsp;T cells in mice. Next, using TIMER database, we found that the expression of LFA-1 is significantly higher in various tumor tissues than in adjacent tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). and it is positively correlated between LFA-1 (ITGAL) and CD4, CD8, and immune cell infiltration in cutaneous melanoma (SKCM), and we reported in our previous published paper that LFA-1 is related with CD4 and CD8 in other type cancer of patients. The expression level of LFA-1 is positively correlated with tumor cell purity and positively correlated with CD4\u003csup\u003e+\u003c/sup\u003e T cell and CD8\u003csup\u003e+\u003c/sup\u003e T cell infiltration (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). From the analysis of the cBioPortal for Cancer Genomics databases, it found that multiple mutation sites in the ITGAL gene in populations (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC), the database shows a significant difference in survival prognosis between the LFA-1 mutation group and the normal group in the patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD, E \u0026amp; F). The data also showed that significant differences between the CD4 and CD8 groups group in the LFA-1 changed population (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eG). These results suggests that LFA-1 affect CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e cells immunity or treatment in tumor microenvironment. It provides us the hypothesis that the individuals immune is various with individual genetic mutations of T cells, which maybe cause good or bad prognosis in tumor patients.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eOur previously published data indicated that the knockout of LFA-1 can inhibit the proliferation of subcutaneous tumor, and a decrease in Treg cells[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. However, the mechanisms underlying this remain unclear. In this study, our experimental results revealed thymic atrophy in LFA-1 knockout mice, alongside a constriction of the medullary area. The experiments showed that LFA-1 knockout affected on the differentiation and maturation of thymic T cells. The presence of CD4\u003csup\u003e+\u003c/sup\u003e T cells increased and a decrease of CD8\u003csup\u003e+\u003c/sup\u003e T cells in both the thymus and peripheral immune organs.\u003c/p\u003e \u003cp\u003eThe thymus serves as a crucial site for T cell development, with key processes such as differentiation, maturation, and output in the thymic microenvironment provided by thymic epithelial cells (TECs), including cortical TECs (cTECs) and medullary TECs (mTECs). These cells regulate positive and negative selection of thymic T cells, and the interaction between immature thymic cells and TEC subpopulations is vital for the development and maturation of T cells[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Cell migration of thymic cells within the thymic microenvironment occurs throughout the developmental process, with cells moving from the cortical to medullary regions, and eventually exiting to the periphery[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Beginning at the corticomedullary junction, lymphoid progenitor cells from the bone marrow first become early thymic precursors (ETPs) through the reception of notch ligands (DLL4) and interleukin-7 (IL-7) from cTECs[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. ETPs, also referred to as \"double-negative\" (DN) thymic cells due to the lack of CD4 and CD8 co-receptor expression, progress through DN1-4 stages with coordinated expression of CD44 and CD25 on their cell surfaces. DN1/ETPs migrate to the subcapsular cortical region, triggering development into DN2 and DN3 stages, where TCRβ chain expression occurs at DN3[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], leading to maturation into \"double-positive\" thymic cells expressing both CD4 and CD8 co-receptors[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. During positive selection, thymic cells differentiate into single-positive CD4\u003csup\u003e+\u003c/sup\u003e or CD8\u003csup\u003e+\u003c/sup\u003e T cells, which then migrate to the thymic medulla. Subsequent negative selection mediated by mTECs leads to the elimination of autoreactive T cells through apoptosis, while positively selected single-positive T cells become functionally distinct CD4 helper T cells or CD8 cytotoxic T cells, ready for output to the periphery[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Our flow cytometry data indicated that, compared to LFA-1\u003csup\u003e+/+\u003c/sup\u003e mice, LFA-1\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice exhibit varying degrees of reduction in cell proportions at the DN1, DN2, and DN4 stages, with an increase in DN3 cells. This suggests that the developmental defects of T cells in the thymus due to LFA-1 knockout begin as early as the DN stage, and the percentage of cells at the DP stage is significantly reduced, indicating a deficiency in positive selection of thymic cells due to the absence of LFA-1.\u003c/p\u003e \u003cp\u003eThis developmental ensures the peripheral accumulation of naive CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e T cells, as well as CD4\u003csup\u003e+\u003c/sup\u003e regulatory T cells (Tregs), within the blood and lymphoid tissues[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. At the DN stage, thymocytes must undergo TCRβ chain rearrangement, pairing with the pre-TCRα chain to form a TCR complex, a process termed \u0026ldquo;β-selection\u0026rdquo;[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The pre-TCR signals, active from the DN3a stage through to the CD4\u003csup\u003e+\u003c/sup\u003e CD8\u003csup\u003e+\u003c/sup\u003e DP proliferative burst stage, see the invariable pre-Tα chain expression disappear, to be replaced by TCRα upon successful V-J gene rearrangement[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Several studies have suggested that optimal pre-TCR signal transduction\u0026mdash;potentially ligand-dependent or independent\u0026mdash;may play a critical role in establishing the mature TCRβ repertoire in the mature mammalian T cells[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. During β-selection, pre-TCR-mediated signaling curtails further rearrangement of TCRβ alleles through a process called allelic exclusion[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], concurrent with the activation of Notch signaling and cellular proliferation, ultimately propelling the αβ-T cell lineage to mature into the DP stage[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBIRT377 is an inhibitor of LFA-1 that has been confirmed to block the binding between LFA-1 and ICAM-1, consequently suppressing the interaction between T cells and antigen presentation[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. In our study, BIRT377 impacts the differentiation and development of thymic T cells. And it also revealed a reduction in ERK kinase in the thymus of LFA-1 knockout mice, ultimately leading to a decrease in the number of CD8\u003csup\u003e+\u003c/sup\u003e T cells that undergo positive selection in the thymus. Contrary to previous findings, we speculate that LFA-1, during the differentiation and development of thymic T cells, exerts negative regulation on T cell positive selection through the MAPK signaling pathway[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. However, the precise mechanisms underlying this action need do more in LFA-1 KO mice. Typically, active MAPK1 is crucial for the success of positive selection. Elevated levels of MAPK1 activity promote the lineage commitment to CD4\u003csup\u003e+\u003c/sup\u003e single positive (SP) T cells, whereas reduced MAPK1 activity leads to an increased count of CD8\u003csup\u003e+\u003c/sup\u003e SP T cells\u0026mdash;the duration of MAPK1 activation appears pivotal in the developmental process of CD4\u003csup\u003e+\u003c/sup\u003e versus CD8\u003csup\u003e+\u003c/sup\u003e T cells[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOur studies also reveal reduced ERK kinase in the thymus of LFA-1 knockout mice, resulting in diminished numbers of CD8\u003csup\u003e+\u003c/sup\u003e T cells completing their positive selection. Hence, we hypothesize that LFA-1, during the differentiation and maturation of thymic T cells, negatively regulates positive selection via the MAPK signaling pathway, although the exact mechanisms remain to be elucidated and are currently under investigation.\u003c/p\u003e \u003cp\u003eLastly, in tumor patients, the LFA-1 mutations has been correlated with variations in the quantity or functionality of CD8\u003csup\u003e+\u003c/sup\u003e T cells, indicating that LFA-1 may be a critical factor influencing tumor immune responses and patient prognosis. This discovery provides a basis for potentially enhancing understand for why the same TNM stage of patients show different response for tumor growth, especially some patients live more life spans than other patients although we did not clear it if these patients have normal LFA-1 and other patients with LFA-1 mutation. Since the mechanisms underlying the role of LFA-1 are not fully understood, more should be investigated the intracellular signaling pathways responsible for abnormal thymocyte differentiation in our body, particularly focusing on the dynamic regulation involving the MAPK/ERK pathway. Next investigations should employ single-cell sequencing techniques, such as single-cell RNA sequencing (scRNA-seq), to intricately explore the transcriptional changes induced by LFA-1 knockout in thymic cells. Additionally, high-resolution cellular imaging techniques can be utilized to observe the movement and subset distribution of T cell precursors in the thymus of LFA-1 knockout mice, providing clues on how T cells position themselves and interact with thymic epithelial cells (TECs).\u003c/p\u003e \u003cp\u003eIn conclusion, this study offers researcher for understanding the molecular mechanisms of thymic T cell development and has provided valuable clues for future studies, particularly in exploring LFA-1 mutation in heathen individuals and our body immune ability had exists when one born.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e\u003cstrong\u003eLFA-1\u003c/strong\u003e\u003cstrong\u003e（\u003c/strong\u003e\u003cstrong\u003eITGAL\u003c/strong\u003e\u003cstrong\u003e）\u003c/strong\u003e\u003cstrong\u003e: \u0026nbsp;\u003c/strong\u003eLymphocyte function-associated antigen-1\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTreg:\u0026nbsp;\u003c/strong\u003eRegulatory T cells\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eICAM-1:\u0026nbsp;\u003c/strong\u003eIntercellular adhesion molecule 1\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eH\u0026amp;E:\u0026nbsp;\u003c/strong\u003eHematoxylin-Eosin staining\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIHC:\u0026nbsp;\u003c/strong\u003eImmunohistochemistry\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDAB:\u003c/strong\u003e Diaminobenzidine\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePBS:\u003c/strong\u003e Phosphate-buffered saline\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGAPDH:\u0026nbsp;\u003c/strong\u003eGlyceraldehyde 3-phosphate dehydrogenase\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eqRT-PCR:\u0026nbsp;\u003c/strong\u003eQuantitative reverse transcription-polymerase chain reaction\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSKCM:\u003c/strong\u003e\u0026nbsp; skin cutaneous melanoma\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank Dr. Zhang Qianqian, Qi Cuiling and Zheng Lingyun for advice in our manuscript and data analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by grants from National Natural Science Foundation of China (Grant ID: 81773118 to Jiangchao Li), and Science and Technology Planning Project of Guangdong Province, China 2023A0505050153.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEthics approval and consent to participate\u003c/p\u003e\n\u003cp\u003eAll mice were housed in the specific pathogen-free barrier facility of Animal Center of Guangdong Pharmaceutical University with license number SCYK (Guangdong) 2017-0125. The Animal Ethics Review Committee approves all Guangdong Pharmaceutical University animal experiments; the animal ethics approval number: gdpulacspf 2021002.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe consents for publication from all authors were obtained.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors contributed equally: Xiuqiong Meng, Yiting Huang.\u003c/p\u003e\n\u003cp\u003eConception idea and scheme design: Xiuqiong Meng, Yiting Huang, Jiangchao Li.\u003c/p\u003e\n\u003cp\u003eAcquisition of experiments data: Xiuqiong Meng, Yiting Huang Hongliang Ma, Ruyu Zeng, Yunxia Kuang, Zhengyang Li.\u003c/p\u003e\n\u003cp\u003eData statistics and analysis: Xiuqiong Meng, Yiting Huang, Zhengyang Li.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWriting and revising manuscripts: Xiuqiong Meng, Jiangchao Li.\u003c/p\u003e\n\u003cp\u003eManage transgenic mice: Xiuqiong Meng, Yiting Huang, Hongliang Ma.\u003c/p\u003e\n\u003cp\u003eSupervision and Funding: Jiangchao Li.\u0026nbsp;Jugao Chen\u003c/p\u003e\n\u003cp\u003eAll authors read and approved the final manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003ePearse G: \u003cstrong\u003eNormal structure, function and histology of the thymus\u003c/strong\u003e. \u003cem\u003eToxicol Pathol \u003c/em\u003e2006, \u003cstrong\u003e34\u003c/strong\u003e(5):504-514.\u003c/li\u003e\n\u003cli\u003eBhandoola A, von Boehmer H, Petrie HT, Z\u0026uacute;\u0026ntilde;iga-Pfl\u0026uuml;cker JC: \u003cstrong\u003eCommitment and developmental potential of extrathymic and intrathymic T cell precursors: plenty to choose from\u003c/strong\u003e. \u003cem\u003eImmunity \u003c/em\u003e2007, \u003cstrong\u003e26\u003c/strong\u003e(6):678-689.\u003c/li\u003e\n\u003cli\u003eMandal D, Lahiry L, Bhattacharyya A, Bhattacharyya S, Sa G, Das T: \u003cstrong\u003eTumor-induced thymic involution via inhibition of IL-7R alpha and its JAK-STAT signaling pathway: protection by black tea\u003c/strong\u003e. \u003cem\u003eInt Immunopharmacol \u003c/em\u003e2006, \u003cstrong\u003e6\u003c/strong\u003e(3):433-444.\u003c/li\u003e\n\u003cli\u003eChung B, Montel-Hagen A, Ge S, Blumberg G, Kim K, Klein S, Zhu Y, Parekh C, Balamurugan A, Yang OO, Crooks GM: \u003cstrong\u003eEngineering the human thymic microenvironment to support thymopoiesis in vivo\u003c/strong\u003e. \u003cem\u003eStem Cells \u003c/em\u003e2014, \u003cstrong\u003e32\u003c/strong\u003e(9):2386-2396.\u003c/li\u003e\n\u003cli\u003eGulla S, Reddy MC, Reddy VC, Chitta S, Bhanoori M, Lomada D: \u003cstrong\u003eRole of thymus in health and disease\u003c/strong\u003e. \u003cem\u003eInt Rev Immunol \u003c/em\u003e2023, \u003cstrong\u003e42\u003c/strong\u003e(5):347-363.\u003c/li\u003e\n\u003cli\u003eReina M, Espel E: \u003cstrong\u003eRole of LFA-1 and ICAM-1 in Cancer\u003c/strong\u003e. \u003cem\u003eCancers (Basel) \u003c/em\u003e2017, \u003cstrong\u003e9\u003c/strong\u003e(11).\u003c/li\u003e\n\u003cli\u003eWalling BL, Kim M: \u003cstrong\u003eLFA-1 in T Cell Migration and Differentiation\u003c/strong\u003e. \u003cem\u003eFront Immunol \u003c/em\u003e2018, \u003cstrong\u003e9\u003c/strong\u003e:952.\u003c/li\u003e\n\u003cli\u003eHogg N, Patzak I, Willenbrock F: \u003cstrong\u003eThe insider\u0026apos;s guide to leukocyte integrin signalling and function\u003c/strong\u003e. \u003cem\u003eNat Rev Immunol \u003c/em\u003e2011, \u003cstrong\u003e11\u003c/strong\u003e(6):416-426.\u003c/li\u003e\n\u003cli\u003eBivona TG, Doebele RC: \u003cstrong\u003eA framework for understanding and targeting residual disease in oncogene-driven solid cancers\u003c/strong\u003e. \u003cem\u003eNat Med \u003c/em\u003e2016, \u003cstrong\u003e22\u003c/strong\u003e(5):472-478.\u003c/li\u003e\n\u003cli\u003eNiu T, Li Z, Huang Y, Ye Y, Liu Y, Ye Z, Jiang L, He X, Wang L, Li J: \u003cstrong\u003eLFA-1 knockout inhibited the tumor growth and is correlated with treg cells\u003c/strong\u003e. \u003cem\u003eCell Commun Signal \u003c/em\u003e2023, \u003cstrong\u003e21\u003c/strong\u003e(1):233.\u003c/li\u003e\n\u003cli\u003eThakur V, Kutty RV: \u003cstrong\u003eRecent advances in nanotheranostics for triple negative breast cancer treatment\u003c/strong\u003e. \u003cem\u003eJ Exp Clin Cancer Res \u003c/em\u003e2019, \u003cstrong\u003e38\u003c/strong\u003e(1):430.\u003c/li\u003e\n\u003cli\u003eSanchez JJ, Sanchez JE, Noor S, Ruffaner-Hanson CD, Davies S, Wagner CR, Jantzie LL, Mellios N, Savage DD, Milligan ED: \u003cstrong\u003eTargeting the \u0026beta;2-integrin LFA-1, reduces adverse neuroimmune actions in neuropathic susceptibility caused by prenatal alcohol exposure\u003c/strong\u003e. \u003cem\u003eActa Neuropathol Commun \u003c/em\u003e2019, \u003cstrong\u003e7\u003c/strong\u003e(1):54.\u003c/li\u003e\n\u003cli\u003eBarnett JB: \u003cstrong\u003eConsequences of Blocking the Choreography of Double Negative Thymocyte Maturation\u003c/strong\u003e. In: \u003cem\u003eSignaling Mechanisms Regulating T Cell Diversity and Function.\u003c/em\u003e edn. Edited by Soboloff J, Kappes DJ. Boca Raton (FL): CRC Press/Taylor \u0026amp; Francis\u0026copy; 2017 Taylor \u0026amp; Francis Group, LLC.; 2018: 1-16.\u003c/li\u003e\n\u003cli\u003eTakaba H, Takayanagi H: \u003cstrong\u003eThe Mechanisms of T Cell Selection in the Thymus\u003c/strong\u003e. \u003cem\u003eTrends Immunol \u003c/em\u003e2017, \u003cstrong\u003e38\u003c/strong\u003e(11):805-816.\u003c/li\u003e\n\u003cli\u003eStarr TK, Jameson SC, Hogquist KA: \u003cstrong\u003ePositive and negative selection of T cells\u003c/strong\u003e. \u003cem\u003eAnnu Rev Immunol \u003c/em\u003e2003, \u003cstrong\u003e21\u003c/strong\u003e:139-176.\u003c/li\u003e\n\u003cli\u003eEvans R, Patzak I, Svensson L, De Filippo K, Jones K, McDowall A, Hogg N: \u003cstrong\u003eIntegrins in immunity\u003c/strong\u003e. \u003cem\u003eJ Cell Sci \u003c/em\u003e2009, \u003cstrong\u003e122\u003c/strong\u003e(Pt 2):215-225.\u003c/li\u003e\n\u003cli\u003eWoska JR, Jr., Shih D, Taqueti VR, Hogg N, Kelly TA, Kishimoto TK: \u003cstrong\u003eA small-molecule antagonist of LFA-1 blocks a conformational change important for LFA-1 function\u003c/strong\u003e. \u003cem\u003eJ Leukoc Biol \u003c/em\u003e2001, \u003cstrong\u003e70\u003c/strong\u003e(2):329-334.\u003c/li\u003e\n\u003cli\u003eCargnello M, Roux PP: \u003cstrong\u003eActivation and function of the MAPKs and their substrates, the MAPK-activated protein kinases\u003c/strong\u003e. \u003cem\u003eMicrobiol Mol Biol Rev \u003c/em\u003e2011, \u003cstrong\u003e75\u003c/strong\u003e(1):50-83.\u003c/li\u003e\n\u003cli\u003eKeshet Y, Seger R: \u003cstrong\u003eThe MAP kinase signaling cascades: a system of hundreds of components regulates a diverse array of physiological functions\u003c/strong\u003e. \u003cem\u003eMethods Mol Biol \u003c/em\u003e2010, \u003cstrong\u003e661\u003c/strong\u003e:3-38.\u003c/li\u003e\n\u003cli\u003eSabio G, Davis RJ: \u003cstrong\u003eTNF and MAP kinase signalling pathways\u003c/strong\u003e. \u003cem\u003eSemin Immunol \u003c/em\u003e2014, \u003cstrong\u003e26\u003c/strong\u003e(3):237-245.\u003c/li\u003e\n\u003cli\u003ePlotnikov A, Zehorai E, Procaccia S, Seger R: \u003cstrong\u003eThe MAPK cascades: signaling components, nuclear roles and mechanisms of nuclear translocation\u003c/strong\u003e. \u003cem\u003eBiochim Biophys Acta \u003c/em\u003e2011, \u003cstrong\u003e1813\u003c/strong\u003e(9):1619-1633.\u003c/li\u003e\n\u003cli\u003eWang HX, Pan W, Zheng L, Zhong XP, Tan L, Liang Z, He J, Feng P, Zhao Y, Qiu YR: \u003cstrong\u003eThymic Epithelial Cells Contribute to Thymopoiesis and T Cell Development\u003c/strong\u003e. \u003cem\u003eFront Immunol \u003c/em\u003e2019, \u003cstrong\u003e10\u003c/strong\u003e:3099.\u003c/li\u003e\n\u003cli\u003eTakahama Y: \u003cstrong\u003eJourney through the thymus: stromal guides for T-cell development and selection\u003c/strong\u003e. \u003cem\u003eNat Rev Immunol \u003c/em\u003e2006, \u003cstrong\u003e6\u003c/strong\u003e(2):127-135.\u003c/li\u003e\n\u003cli\u003ePeschon JJ, Morrissey PJ, Grabstein KH, Ramsdell FJ, Maraskovsky E, Gliniak BC, Park LS, Ziegler SF, Williams DE, Ware CB\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eEarly lymphocyte expansion is severely impaired in interleukin 7 receptor-deficient mice\u003c/strong\u003e. \u003cem\u003eJ Exp Med \u003c/em\u003e1994, \u003cstrong\u003e180\u003c/strong\u003e(5):1955-1960.\u003c/li\u003e\n\u003cli\u003eGascoigne NR, Rybakin V, Acuto O, Brzostek J: \u003cstrong\u003eTCR Signal Strength and T Cell Development\u003c/strong\u003e. \u003cem\u003eAnnu Rev Cell Dev Biol \u003c/em\u003e2016, \u003cstrong\u003e32\u003c/strong\u003e:327-348.\u003c/li\u003e\n\u003cli\u003eFehling HJ, Krotkova A, Saint-Ruf C, von Boehmer H: \u003cstrong\u003eCrucial role of the pre-T-cell receptor alpha gene in development of alpha beta but not gamma delta T cells\u003c/strong\u003e. \u003cem\u003eNature \u003c/em\u003e1995, \u003cstrong\u003e375\u003c/strong\u003e(6534):795-798.\u003c/li\u003e\n\u003cli\u003ePalmer E: \u003cstrong\u003eNegative selection--clearing out the bad apples from the T-cell repertoire\u003c/strong\u003e. \u003cem\u003eNat Rev Immunol \u003c/em\u003e2003, \u003cstrong\u003e3\u003c/strong\u003e(5):383-391.\u003c/li\u003e\n\u003cli\u003eZiegler SF: \u003cstrong\u003eFOXP3: of mice and men\u003c/strong\u003e. \u003cem\u003eAnnu Rev Immunol \u003c/em\u003e2006, \u003cstrong\u003e24\u003c/strong\u003e:209-226.\u003c/li\u003e\n\u003cli\u003eDutta A, Zhao B, Love PE: \u003cstrong\u003eNew insights into TCR \u0026beta;-selection\u003c/strong\u003e. \u003cem\u003eTrends Immunol \u003c/em\u003e2021, \u003cstrong\u003e42\u003c/strong\u003e(8):735-750.\u003c/li\u003e\n\u003cli\u003eMichie AM, Z\u0026uacute;\u0026ntilde;iga-Pfl\u0026uuml;cker JC: \u003cstrong\u003eRegulation of thymocyte differentiation: pre-TCR signals and beta-selection\u003c/strong\u003e. \u003cem\u003eSemin Immunol \u003c/em\u003e2002, \u003cstrong\u003e14\u003c/strong\u003e(5):311-323.\u003c/li\u003e\n\u003cli\u003eMallis RJ, Bai K, Arthanari H, Hussey RE, Handley M, Li Z, Chingozha L, Duke-Cohan JS, Lu H, Wang JH\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003ePre-TCR ligand binding impacts thymocyte development before \u0026alpha;\u0026beta;TCR expression\u003c/strong\u003e. \u003cem\u003eProc Natl Acad Sci U S A \u003c/em\u003e2015, \u003cstrong\u003e112\u003c/strong\u003e(27):8373-8378.\u003c/li\u003e\n\u003cli\u003eMallis RJ, Arthanari H, Lang MJ, Reinherz EL, Wagner G: \u003cstrong\u003eNMR-directed design of pre-TCR\u0026beta; and pMHC molecules implies a distinct geometry for pre-TCR relative to \u0026alpha;\u0026beta;TCR recognition of pMHC\u003c/strong\u003e. \u003cem\u003eJ Biol Chem \u003c/em\u003e2018, \u003cstrong\u003e293\u003c/strong\u003e(3):754-766.\u003c/li\u003e\n\u003cli\u003eDas DK, Mallis RJ, Duke-Cohan JS, Hussey RE, Tetteh PW, Hilton M, Wagner G, Lang MJ, Reinherz EL: \u003cstrong\u003ePre-T Cell Receptors (Pre-TCRs) Leverage V\u0026beta; Complementarity Determining Regions (CDRs) and Hydrophobic Patch in Mechanosensing Thymic Self-ligands\u003c/strong\u003e. \u003cem\u003eJ Biol Chem \u003c/em\u003e2016, \u003cstrong\u003e291\u003c/strong\u003e(49):25292-25305.\u003c/li\u003e\n\u003cli\u003eBrady BL, Steinel NC, Bassing CH: \u003cstrong\u003eAntigen receptor allelic exclusion: an update and reappraisal\u003c/strong\u003e. \u003cem\u003eJ Immunol \u003c/em\u003e2010, \u003cstrong\u003e185\u003c/strong\u003e(7):3801-3808.\u003c/li\u003e\n\u003cli\u003eWoska JR, Jr., Last-Barney K, Rothlein R, Kroe RR, Reilly PL, Jeanfavre DD, Mainolfi EA, Kelly TA, Caviness GO, Fogal SE\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eSmall molecule LFA-1 antagonists compete with an anti-LFA-1 monoclonal antibody for binding to the CD11a I domain: development of a flow-cytometry-based receptor occupancy assay\u003c/strong\u003e. \u003cem\u003eJ Immunol Methods \u003c/em\u003e2003, \u003cstrong\u003e277\u003c/strong\u003e(1-2):101-115.\u003c/li\u003e\n\u003cli\u003eLi D, Molldrem JJ, Ma Q: \u003cstrong\u003eLFA-1 regulates CD8+ T cell activation via T cell receptor-mediated and LFA-1-mediated Erk1/2 signal pathways\u003c/strong\u003e. \u003cem\u003eJ Biol Chem \u003c/em\u003e2009, \u003cstrong\u003e284\u003c/strong\u003e(31):21001-21010.\u003c/li\u003e\n\u003cli\u003eGermain RN: \u003cstrong\u003eT-cell development and the CD4-CD8 lineage decision\u003c/strong\u003e. \u003cem\u003eNat Rev Immunol \u003c/em\u003e2002, \u003cstrong\u003e2\u003c/strong\u003e(5):309-322.\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":"LFA-1, Transgenic mice, T cells differentiation, CD4+ T cells, CD8+ T cells, ERK Signaling Pathway","lastPublishedDoi":"10.21203/rs.3.rs-4337853/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4337853/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eT cell precursors from fetal liver bone marrow migrate into the thymus to initiate their development, including double-negative selection, double-positive selection, and single-positive selection. Subsequently, fully matured single-positive CD4\u003csup\u003e+\u003c/sup\u003e T cells or CD8\u003csup\u003e+\u003c/sup\u003e T cells traverse the bloodstream to the peripheral tissues, executing immune functions. Lymphocyte function-associated antigen-1 (LFA-1) is invovuled with thymic cortical epithelial cells facilitate positive selection. But LFA-1 mediates signaling pathways in thymic keep unknown. Here, Knockout LFA-1 displayed thymic atrophy and aberrant structural alterations in the cortical and medullary of the thymus in mice. And the cells populations of thymocytes during the positive and negative selection process was observed, characterized by CD4\u003csup\u003e+\u003c/sup\u003e T cells increased and CD8\u003csup\u003e+\u003c/sup\u003e T cells decreased. Furthermore, LFA-1 inhibitor also impact on thymic development. A significant downregulation of pERK1/2 in MAPK signaling pathway. The thymus gland medullary atrophy still was observed in LFA-1 knockout mice with tail vein tumor metastasis, along with CD4\u003csup\u003e+\u003c/sup\u003e T lymphocytes increased and a reduced CD8\u003csup\u003e+\u003c/sup\u003e T cells. The Genome Databases revealed that mutations in LFA-1 in clinical patients, suggesting that LFA-1 mutation individuals maybe affect the CD8+ T cells function. This study indicated that LFA-1 regulates the differentiation of CD4\u003csup\u003e+\u003c/sup\u003e T and CD8\u003csup\u003e+\u003c/sup\u003e T cells in the thymus, implying that LFA-1 mutation in health individuals may influence the tumor immunity or therapy when they get tumor.\u003c/p\u003e","manuscriptTitle":"LFA-1 Knockout Leads to CD4 + and CD8 + T Cells Differentiation Disorder in Thymus Gland and is Related with ERK Signaling Pathway in Mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-10 20:45:46","doi":"10.21203/rs.3.rs-4337853/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":"e60d6eb7-6e29-4353-a7b1-0bac8b685f16","owner":[],"postedDate":"May 10th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-05-10T20:45:48+00:00","versionOfRecord":[],"versionCreatedAt":"2024-05-10 20:45:46","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4337853","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4337853","identity":"rs-4337853","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}