{"paper_id":"2965c24d-1010-4ebc-be46-15a456bc4a33","body_text":"Cre-driven tdTomato expression unexpectedly confers lupus resistance in dLckCre 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 Article Cre-driven tdTomato expression unexpectedly confers lupus resistance in dLckCre mice Zhenghao Xu, Ning Han, Keer Wang, Xiaoxiao Hou, Mingxuan Han, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5142419/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 The Cre-driven tdTomato reporter system is widely used to visualize gene expression and cellular dynamics. Here we tested the impact of the tdTomato reporter system on the progression of both central and peripheral manifestations of systemic lupus erythematosus (SLE) in dLckCre mice. We crossed dLckCre mice with ROSA26 floxed-Stop-tdTomato mice to generate the conditional dLck-driven tdTomato reporter mice (DT mice). Then, SLE models were induced by pristane injection in DT mice or by crossing DT mice with B6/ lpr mice. Central and peripheral manifestations of SLE were tested by. Surprisingly, we found DT mice exhibited a significant reduction in the progression of peripheral manifestations of SLE, evidenced by decreased severity of lymph node and splenic lesions, and improved renal pathology. However, DT mice showed similar central manifestations of SLE as control mice, evidenced by similar behavioral performance, CD4 + T cell infiltration in the choroid plexus, and microglial activation in the hippocampus. Flow cytometry revealed a significant reduction in the proportion of double-negative (DN) T cells in the peripheral blood of the DT mice. Thus, conditional tdTomato expression alleviated peripheral but not central manifestations of SLE in dLckCre mice, suggesting a diverse role of dLck-labeled T lymphocytes in SLE development. Our results also provide additional evidence supporting the existence of a distinct separation mechanism between peripheral and central manifestations of SLE. Biological sciences/Immunology/Immunological disorders/Autoimmune diseases/Systemic lupus erythematosus Biological sciences/Developmental biology/Disease model Systemic lupus erythematosus Peripheral manifestations Central manifestations T Cell Labeling ROSA26floxed-Stop-tdTomato Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Highlight dLck-driven tdTomato expression did not affect the baseline central and peripheral manifestations in mice. dLck-driven tdTomato expression alleviates peripheral but not central symptoms in lupus model. Peripheral blood DNT cell numbers in mice were significantly decreased by the dLck-driven tdTomato expression, which may be a contributor in the altered progression of peripheral indicators of lupus. 1. Introduction Systemic lupus erythematosus (SLE) is a multifaceted autoimmune disorder characterized by a variety of clinical manifestations affecting various organ systems 1,2 . Peripheral symptoms such as rash, lymphadenopathy, splenomegaly, and renal impairment are particularly prevalent 3–5 . In addition, SLE frequently affects the central nervous system, leading to neuropsychiatric systemic lupus erythematosus (NPSLE), with clinical manifestations such as depression, cognitive impairment, seizures, and stroke 6–10 . The pathogenesis of SLE remains incompletely understood. Existing research suggests that SLE pathogenesis involves a complex interplay of aberrant autoimmune responses, production of autoantibodies, and immune-mediated damage to self-tissues 11 . However, the multifaceted nature of this disease remains a significant challenge in its study. Given the high incidence of SLE and the substantial difficulties associated with its treatment, there is a critical need for further investigation to elucidate the underlying pathophysiological mechanisms of SLE and to devise more efficacious therapeutic strategies. In recent years, the pivotal role of T lymphocytes in SLE has increasingly come under intensive investigation. Numerous studies have documented substantial alterations in the number and function of T lymphocytes in SLE patients 12–15 . For example, the proportion of double-negative T cells (DNT) is notably elevated in SLE patients and correlates strongly with the severity of lupus nephritis 15–17 . Additionally, CD4 + T cells, another critical subset of T cells, play a pivotal role in the pathogenesis of NPSLE 18,19 . Our previous research demonstrated that the presence of ectopic CD4 + T cells in the choroid plexus can trigger microglial activation through interferon signaling, ultimately leading to NPSLE-like symptoms in mice 20 . Increasing evidence highlights the importance of T cells in the pathology of peripheral and central SLE. However, research on different subsets of T cells in SLE still faces several limitations and challenges. The Cre-loxP system, widely applied in genetically engineered animal models 21–23 . The Cre-driven tdTomato reporter system is widely used to visualize gene expression and cellular dynamics. The distal lymphocyte-specific protein tyrosine kinase (dLck) gene, which is critical for T cell selection and maturation, is specifically expressed in T cells 24,25 . The dLckCre recombinase system has been extensively utilized to study T cell dynamics 25,26 . However, the use of dLck-driven T cell-specific fluorescence reporter systems has been rarely reported in SLE animal models, and their influence on the SLE models remains unclear. Therefore, here we crossed dLckCre mice with ROSA26 floxed − Stop−tdTomato mice to generate dLck-driven tdTomato reporter mice (DT mice). We further established two lupus models in DT mice by injection of pristane or crossed them with B6/ lpr mice. We unexpectedly found that these DT mice exhibit notable resistance to peripheral but not central lupus manifestations in both models of SLE. 2. Methods 2.1 Animals In this study, we utilized the distal Lck promoter-driven Cre (dLckCre) mice [B6.Cg-Tg(Lck-icre)3779Nik/J; Cat. No. NM-KI-225042, Shanghai Model Organisms Center, Inc.], B6.Cg Gt(ROSA)26Sor tm14(CAG−tdTomato)Hze/J mice [The Jackson Laboratory stock no. 007914], and B6/ lpr mice [The Jackson Laboratory stock no. 007914]. To generate dLckCre tdTomato mice, dLckCre mice were bred with ROSA26 flox − stop−tdTomato mice. Progeny harboring the dLckCre allele (Cre-/+) and expressing the tdTomato reporter were classified as the dLck-driven tdTomato reporter (DT) group (dLckCre tdTomato ). Littermates that did not carry the dLckCre allele (Cre-/-) were used as controls (Non-DT groups). To generate dLckCretdTomato B6/lpr mice, B6/lpr mice were crossed with dLckCretdTomato mice. Offspring possessing both the lpr mutation and the dLckCretdTomato transgene were selected as the experimental group (dLckCre tdTomato B6/ lpr ). Littermate controls, which carried only the lpr mutation without the dLckCre tdTomato transgene, were designated as the control group (B6/ lpr ). A littermate control design was implemented to reduce the confounding effects of genetic background and maternal influence on the experimental outcomes. Animals in both the experimental and control groups were randomly selected from the same litter, ensuring consistency in genotype and early environmental conditions. By controlling for these variables, this approach effectively mitigates potential biases arising from genetic or maternal factors. Each group consisted of 12 animals (n = 12), ensuring adequate statistical power for robust analysis. This design significantly enhances the validity and reproducibility of the experimental findings. Given the significant sex differences in the incidence of systemic lupus erythematosus (SLE), with estrogen playing a crucial role in the disease’s pathogenesis, this study utilizes female mice as the experimental model. All mice were housed at 24 ± 1°C with 50–55% humidity and a 12-hour light/dark cycle (light from 08:00 to 20:00), with ad libitum access to water and food. The study was approved by the Institutional Animal Care and Use Committee of Zhejiang Chinese Medical University (Approval No. IACUC-20220718-01) and followed the guidelines of the Animal Advisory Committee. 2.2 Pristane-induced lupus To establish the lupus model, 0.5 mL of pristane (99% pure, Sigma Aldrich Co., St. Louis, MO, USA) was administered intraperitoneally to non-DT and DT mice, following the method described by Satoh and Reeves 27 . In contrast, control group animals were administered 0.9% saline intraperitoneally. Mice were monitored for 8 months. Subsequently, the mice were humanely sacrificed, and their spleen, lymph nodes, and kidneys were harvested. 2.3 Flow cytometry Single-cell suspensions were prepared from the peripheral blood of mice following a standard protocol. Prior to antibody (Ab) staining, cells were preincubated with Fc receptor blocker (BD, catalog number 101312, USA). The complete list of antibodies and reagents used is as follows: Live-APC-A750 (Invitrogen, catalog number 65-0865-14, USA), CD3-FITC (BioLegend, catalog number 100203, USA), CD4-PerCP (BioLegend, catalog number 100539, USA), CD8-KO525 (BioLegend, catalog number 100752, USA). Data were collected via FACS analysis using the FC 500 MC system (Beckman Coulter, Fullerton, CA, USA) and were subsequently analyzed using FlowJo version 10 software (Tree Star, Inc., Ashland, OR, USA). 2.4 Renal histopathological analysis 2.4.1 Hematoxylin and Eosin (H&E) and Periodic Acid-Schiff (PAS) Staining Mouse kidney tissues were initially fixed in 4% paraformaldehyde (PFA) at 4℃ for 48 hours. The samples were then embedded in paraffin and sectioned at a thickness of 5 µm. Histological analysis was performed by staining the sections with Hematoxylin-Eosin (H&E) and Periodic Acid-Schiff (PAS) stains. Imaging analysis of the stained sections was carried out using an Olympus VS120 microscope and xvViewer software. 2.4.2 IgG Immunofluorescence Staining Kidney tissues were fixed in 4% PFA, followed by dehydration in 30% sucrose. Cryosections of 6 µm thickness were prepared for immunofluorescence staining. The sections were first blocked with 5% bovine serum albumin for 1 hour, then incubated with anti-IgG antibody (Proteintech, Cat No. 66,467-1-Ig, diluted 1:1000). Stained sections were imaged using the Olympus VS120 microscope and analyzed with xvViewer software. 2.4.3 Pathological Evaluation Glomerular damage was evaluated using a semi-quantitative scoring system ranging from 0 to 6, based on the examination of five randomly selected glomeruli per specimen from H&E-stained sections 28 . Cell counts were also performed for the same glomeruli. To quantify mesangial matrix expansion, the glomerular tuft area (GTA) and PAS-positive area (PTA) were measured. The percentage of PTA within the GTA was calculated using Image-Pro Plus 6.0 software. The area of IgG fluorescence was also quantified using Image-Pro Plus 6.0, with the average fluorescence area derived from five glomeruli to represent each sample. This method ensures a comprehensive and rigorous analysis of kidney pathology and IgG deposition.Simultaneously, cell counts were performed in the same glomeruli for each mouse. 2.5 Organ weight index Mice were euthanized with an overdose of 1% pentobarbital sodium at a dosage of 150 mg/kg, ensuring humane euthanasia. Death was confirmed by the cessation of respiratory and cardiac activity. The cervical, anterior axillary, posterior (near the brachial plexus), and inguinal lymph nodes, along with the spleen, were weighed and photographed. The organ weight index was calculated as follows: organ weight (g) / body weight (g) × 100. 2.6 Behavioral tests Neuropsychiatric symptoms were evaluated using various behavioral tests, as outlined in our previous research 20 . 2.6.1 Open Field Test (OFT) Spontaneous locomotion was assessed in a 40cm × 40cm × 40cm open field arena over 15 minutes. A central zone measuring 13.3cm × 13.3cm was used to evaluate the distance traveled within this area. Total distance traveled and distance within the central zone were recorded with Any-Maze Software (Version 7.1, Stoelting Co., Wood Dale, IL, USA). 2.6.2 Novel Object Recognition (NOR) Mice were first habituated to a 40 × 40 × 40 cm arena for 10 minutes. During the training phase, they were exposed to two identical objects in the arena for 10 minutes, followed by a 30-minute interval in their home cages. In the testing phase, mice were reintroduced to the arena for 5 minutes, where one of the familiar objects was replaced with a novel object. Object exploration was quantified by recording the time spent sniffing, touching, or approaching each object within ≤ 1cm. The discrimination ratio (DR) was calculated as DR = N / (N + F), where N represents the time spent exploring the novel object and F represents the time spent exploring the familiar object. A DR greater than 50% indicates a preference for the novel object, reflecting intact memory, while a DR of 50% or less suggests no significant preference, potentially indicating memory deficits. 2.6.3 Novel Object Location (NOL) Mice were first habituated to a 40 × 40 × 40 cm arena for 10 minutes. During the training phase, they were exposed to two identical objects within the arena for 10 minutes, followed by a 30-minute inter-trial interval in their home cages. In the subsequent testing phase, mice were reintroduced to the arena for 5 minutes, during which one of the familiar objects was relocated to a new position. Object exploration was quantified by measuring the time spent within a 1cm radius of each object, encompassing behaviors such as sniffing, touching, or approaching. Exploration time of the object in the new location is denoted as N, while exploration time of the object in the original location is denoted as F. The discrimination ratio (DR) is computed using the formula DR = N / (N + F). 2.6.4 Tail Suspension Test (TST) Mice were individually suspended by their tails using adhesive tape from a vertical aluminum bar positioned 36.6 cm above the floor, with partitions to prevent visual contact between them. Each mouse was suspended for 6 minutes in a quiet environment. Behavior was recorded using Any-Maze software (Stoelting Co., Wood Dale, IL, USA), and the duration of immobility was quantified. Immobility was defined as the absence of active movements, except for minimal adjustments to maintain balance. The immobility duration was analyzed as an indicator of depressive-like behavior. All procedures complied with institutional animal care guidelines. 2.6.5 Forced swim test (FST) Each mouse was placed individually in a transparent cylindrical tank filled with water at 27°C to a depth of 20cm. The tank was 30cm high and 15cm in diameter. The test consisted of a 2-minute acclimation period followed by a 4-minute test session. Behavior was recorded using Any-Maze software, and the total time spent immobile during the test was measured. Immobility was defined as the absence of movement, with only minor adjustments to keep the head above water. Immobility time was analyzed using automated tracking software as an indicator of depressive-like behavior. All procedures adhered to institutional animal care guidelines. 2.7. Immunofluorescent staining Immunofluorescence staining was performed on 16 µm-thick coronal brain tissue sections to assess microglial and CD4 + T cell populations. Primary antibodies used were anti-rabbit Iba1 (1:400; Wako, catalog no. 019-19741, Japan) to label microglia, and anti-mouse CD4 (1:200; Abcam, catalog no. ab33775, Cambridge, UK) to detect CD4 + T cells. Following incubation with the primary antibodies at 4℃ overnight, sections were washed and incubated with species-appropriate secondary antibodies. Alexa Fluor 488-conjugated secondary antibody (1:400; Abcam, catalog no. ab150081, Cambridge, UK) was used for Iba1 detection, and Alexa Fluor 647-conjugated secondary antibody (1:400; Abcam, catalog no. ab150115, Cambridge, UK) was used for CD4 detection. Nuclei were counterstained with DAPI (Solarbio, catalog no. MA0222, Beijing, China). Stained sections were imaged using an Olympus VS120-S6-W microscope under consistent exposure settings across all samples to ensure reliable comparison. Microglial morphological analysis was conducted using ImageJ and Fiji-ImageJ software, focusing on key metrics including microglial cell density, total branch length (skeletonized microglial processes), branch point number (indicating cellular complexity), and cell body area. For skeletonization, Z-stack images were processed using the \"Analyze Skeleton\" plugin to quantify the structural features of individual microglia. Quantification of microglia was carried out across randomly selected fields of view per section. In each sample, eight representative microglia were analyzed per field, with the final data presented as averages per sample. The analysis followed established protocols as outlined in previous studies 29,30 , ensuring consistency and reproducibility. Quantitative analysis of CD4 + T cells was conducted to determine the area of CD4 + positive regions within the choroid plexus in each brain section. For each sample, the area of CD4 + positive regions was measured across eight distinct choroid plexus regions, and the mean value was calculated to represent the CD4 + positive area for that sample. ImageJ software was used to perform threshold-based segmentation of the CD4 + immunoreactivity, ensuring accurate delineation of positive regions. The analysis followed the methodological guidelines outlined in Wang et al. (2024) 20 , ensuring consistency and comparability with previous studies. 2.8. Statistics Statistical analyses were performed using GraphPad Prism software (version 8.0.2). Normality of the data was assessed using the Shapiro-Wilk test before applying parametric or non-parametric tests. For comparisons between two groups, the Mann-Whitney U test was applied for non-normally distributed data, while the unpaired Student’s t-test was used for normally distributed data. For comparisons involving more than two groups, one-way analysis of variance (ANOVA) followed by Fisher’s Least Significant Difference (LSD) post hoc test was conducted. Data are presented as mean ± standard error of the mean (SEM), and statistical significance was defined as a two-tailed P-value < 0.05. All behavioral assessments, histopathological evaluations, and image analyses were conducted in a double-blinded manner to minimize bias. Comprehensive statistical results are provided in Supplementary Table 1. 3. Results 3.1 dLCK-driven tdTomato expression did not affect the baseline central and peripheral manifestations in mice. We generated dLckCre tdTomato mice (DT mice) by crossing dLckCre mice with ROSA26 flox-stop-tdTomato mice (Fig. 1A). Flow cytometric analysis confirmed that over 90% of CD3 + T lymphocytes in DT mice expressed tdTomato, whereas non-DT mice showed negligible red fluorescence ( P < 0.01, Fig. 1C). Additionally, both DT and non-DT mice were aged to 40 weeks to match the age of lupus model mice for subsequent experiments (Fig. 1B). We initially assessed central manifestations in DT mice relative to non-DT mice using a series of behavioral tests. In the open field test, no significant differences were observed between DT and non-DT mice in total distance traveled or the proportion of distance traveled in the central area, indicating similar locomotor activity and anxiety levels between the two groups ( P > 0.05 for all, Fig. 1D-G). Similarly, the novel object location and novel object recognition tests showed no significant differences in exploration times of new objects or their spatial placements, suggesting comparable learning and memory abilities ( P > 0.05 for all, Fig. 1H-K). Furthermore, depressive-like behaviors, as measured by the tail suspension and forced swim tests, showed similar immobility times in both groups, indicating no significant differences in depressive-like phenotypes ( P > 0.05 for all, Fig. 1L-O). Overall, these results suggest that DT mice exhibit no significant baseline differences from non-DT mice in locomotion, anxiety, learning, memory, or depressive-like behaviors. Thus, DT mice provide a reliable baseline model for further studies, particularly those related to NPSLE. Subsequently, we evaluated peripheral manifestations in DT mice. Baseline spleen and lymph node indices were comparable between DT mice and their non-DT littermates ( P > 0.05 for all, Fig. 1P-S). Similarly, baseline kidney pathology, assessed by hematoxylin-eosin (HE) staining, periodic acid-Schiff (PAS) staining, and IgG immunofluorescence staining, showed no significant differences between the two groups ( P > 0.05 for all, Fig. 1T-X). These findings indicate that DT mice exhibit peripheral features, including lupus-related markers, similar to those of non-DT mice. Therefore, DT mice provide a reliable baseline model for further investigations into peripheral lupus pathology. 3.2 dLCK-driven tdTomato expression alleviates peripheral but not central symptoms in pristane-induced lupus model After confirming that conditional tdTomato expression had no effect on central and peripheral manifestations in dLckCre mice under non-lupus conditions, we established the lupus model in DT mice via intraperitoneal injection of pristane (Fig. 2A). To assess central lupus manifestations, we performed several behavioral tests. Compared to normal control mice, the injection of pristane induces NPSLE-like behavioral alterations in both DT and non-DT mice. These changes encompass a decrease in the total travel distance and the percentage of distance traversed in the central area during the open field test ( P < 0.001 for total travel distance and P < 0.05 for the percentage of distance traversed in the central area; Fig. 2B-D) , poorer performance in both the novel object recognition and novel object location tests ( P < 0.05 for novel object recognition and P < 0.001 for novel object location; Fig. 2E-F), as well as an increase in the duration of immobility observed in the tail suspension and forced swim tests ( P < 0.0001 for both; Fig. 2G-H). However, no significant differences for all of these tests were observed between the two lupus model groups ( P > 0.05 for all, Fig. 2B-H). For peripheral manifestations, an unexpected finding emerged. Pristane injection resulted in weaker peripheral lupus symptoms in DT mice than that in non-DT mice, including the decreased splenomegaly ( P < 0.0001; Fig. 2I, J), reduced lymphadenopathy ( P < 0.001; Fig. 2K, L), and milder renal pathology (Fig. 2M; P < 0.001 for renal pathology scores, Fig. 2N; P < 0.001 for glomerular cell proliferation, Fig. 2O; P < 0.0001 for PAS-positive areas, Fig. 2P; P < 0.0001 for IgG deposition, Fig. 2Q). In conclusion, conditional tdTomato expression significantly attenuates the severity of peripheral lupus symptoms in the pristane-induced dLckCre lupus model, although it does not impact the progression of NPSLE. 3.3 dLCK-driven tdTomato expression alleviates peripheral but not central symptoms in the B6/ lpr lupus model To confirm the findings in pristane-induced lupus model, we employed an additional lupus model, the B6/ lpr transgenic mouse model. DT mice were crossed with B6/ lpr mice to generate spontaneous lupus DT B6/ lpr mice (Fig. 3A, B), and marker expression was confirmed by flow cytometry (Fig. 3C). To evaluate central lupus manifestations, we conducted a series of behavioral tests. Compared to normal control mice, both non-DT B6/ lpr and DT B6/ lpr mice exhibited NPSLE-like behavioral changes. These changes included a reduction in total travel distance and the percentage of distance traversed in the central area during the open field test ( P < 0.001 for total travel distance and P < 0.01 and P < 0.001 for the percentage of distance traversed in the central area; Fig. 3E, F). Additionally, both groups showed decreased exploration time of novel objects and new locations in the novel object recognition and novel object location tests ( P < 0.05 for novel object recognition and P < 0.01 and P < 0.001 for novel object location; Fig. 3G, H). Increased immobility duration was also observed in the tail suspension and forced swim tests ( P < 0.0001; Fig. 3I, J). For peripheral manifestations, a significant difference was still observed. Compared to non-DT B6/ lpr mice, DT B6/ lpr mice exhibited milder peripheral lupus symptoms, including reduced splenomegaly ( P < 0.001; Fig. 3K), decreased lymphadenopathy ( P < 0.0001; Fig. 3L), and attenuated renal pathology (Fig. 3M; P < 0.0001, Fig. 3N; P < 0.001 for glomerular cell proliferation, Fig. 3O; P < 0.0001 for PAS-positive area, Fig. 3P; P < 0.0001 for IgG deposition, Fig. 3Q). In summary, the central and peripheral manifestations in DT B6/ lpr mice indicate that the reduced severity of systemic lupus symptoms due to the conditional tdTomato reporter gene expression is significant. This specific genetic modification seems to delay the progression of lupus symptoms in peripheral tissues, though its impact on the central nervous system appears to be less pronounced. 3.4. dLCK-driven tdTomato expression alters T lymphocyte subset proportions in peripheral Blood To further explore the mechanisms related to the unexpected resistance to peripheral lupus exhibited by DT mice, we conducted a comprehensive analysis using peripheral blood flow cytometry. When comparing non-DT and DT mice, a statistically significant elevation ( P < 0.001, Fig. 4E) in the proportion of CD4 + CD8 - T cells was observed, whereas no significant changes were detected in the other T cell subsets analyzed (Fig. 4B-D). When comparing non-DT and non-DT B6/ lpr , we found that non-DT B6/ lpr showed a decrease of CD4 - CD8 + ( P < 0.001), CD4 + CD8 - ( P < 0.0001) cells and an increase of DNT ( P < 0.0001) cells. When further comparing non-DT B6/ lpr and DT B6/ lpr mice, we found that DT B6/ lpr mice showed a large decrease in CD4 - CD8 - double-negative T (DNT) cells ( P < 0.001) and significant increases in CD4 - CD8 + ( P < 0.05), CD4 + CD8 - (P < 0.01), and CD4 + CD8 + double-positive T (DPT) cells ( P < 0.001). 3.5. dLCK-driven tdTomato expression does not affect microglial phenotype and CD4 + T cell accumulation in the choroid plexus The excessive activation of microglia and the extensive infiltration of CD4 + T cells in the choroid plexus have been widely recognized as key drivers of neuroinflammation in CNS in SLE, contributing to the onset and progression of NPSLE. Therefore, in addition to observing behavioral outcomes, we conducted an in-depth analysis of microglial activation and CD4 + T cell-related markers in each group of mice to further investigate the role of DT in NPSLE. Immunofluorescence staining results showed significant increases in microglial numbers within the hippocampus in both group of lupus mice ( P < 0.0001; Fig. 5A, B), Additionally, these microglia in lupus mice exhibited increased microglial branch number ( P < 0.0001; Fig. 5C), branch length ( P < 0.0001; Fig. 5D), and cell body size ( P < 0.0001; Fig. 5E). However, no significant differences were observed between non-DT and DT, both in in normal or lupus state ( P > 0.05, Fig. A-E). Similarly, CD4 + T cell accumulation in the choroid plexus were found in both group of lupus mice; However no significant differences were observed between non-DT and DT, both in in normal or lupus state ( P > 0.05, Fig. 5F, G). Discussion SLE is an autoimmune disease characterized by severe inflammation and has widespread impact on both central and peripheral manifestations 11,31 . In this study, by using two lupus model. we unexpectedly revealed several key findings by utilizing the conditional tdTomato reporter system in dLckCre mice: (1) dLCK-driven tdTomato expression had no impact on baseline central or peripheral manifestations in normal mice. (2) dLCK-driven tdTomato expression unexpectedly induces peripheral lupus resistance in two SLE mouse models; (3) dLCK-driven tdTomato expression did not affect the central lupus manifestations in two SLE mouse models. Taken together, our results highlight the dLCK-driven tdTomato expression mainly affects the development of peripheral manifestations rather than central manifestations of SLE, indicating a distinction in a distinct role for T cells in peripheral and central lupus pathogenesis. Previous studies have indicated that fluorescence reporter systems, such as tdTomato reporter gene system, can potentially alter the function of labeled cells 32-34 . For instance, insertion of the tdTomato reporter gene at the Puma locus was associated with a reduction in TRP53-induced apoptosis, thus modifying apoptotic responses 35 . In the present study, we employed the dLck-driven tdTomato reporter system to specifically tag dLck-positive T cells, allowing for their identification and tracking within the context of our experiments. Unexpectedly, by using two SLE mouse models (pristane-induced and B6/ lpr transgenic models), we found this approach significantly attenuated the progression of peripheral lupus pathology in these DT mice. Specifically, compared with non-DT control groups, we observed a decrease in lymphadenopathy, splenomegaly, and renal pathology severity in DT groups. The pristane-induced model predominantly triggers an excessive type I interferon response 36-38 , whereas the B6/ lpr model is characterized by marked lymphadenopathy and abnormal T cell proliferation 39-41 . Thus, our findings suggest that the dLck-driven tdTomato expression may have a unique modulatory effect on peripheral lupus progression, highlighting dLckCre-positive T cells as a potential therapeutic target for peripheral lupus. This underscores the importance of further exploring the differential mechanisms underlying central and peripheral manifestations of lupus. Numerous studies have highlighted the pivotal role of T cells in the peripheral manifestations of lupus 42-46 . Our flow cytometric analysis demonstrated that the dLck-driven tdTomato expression significantly altered T cell subsets in lupus mice, notably leading to a reduction in DNT cells. In SLE, DNT cells exhibit profound phenotypic and functional abnormalities and make up a disproportionately large fraction of the T cell population compared to their minimal presence in healthy individuals. The increased abundance of DNT cells is believed to worsen the disease by impairing normal T cell functions and promoting autoimmunity 43,47-49 . In this study, we observed that dLck-driven tdTomato expression led to a reduction in the number of DNT cells. This change corresponded with an alleviation of peripheral symptoms in these DT lupus mice. Our findings further support the notion that targeting dysregulated DNT cell subsets can effectively intervene in the progression of lupus. Moreover, this highlights a novel and promising T-cell modification strategy that could be leveraged for therapeutic intervention in lupus. NPSLE is characterized by a wide spectrum of neurological and psychiatric symptoms, including depression and cognitive impairment, and represents a severe and potentially life-threatening manifestation of SLE. One of the key mechanisms driving NPSLE pathology is aberrant microglial activation 18,50 , which contributes to increased neuroinflammation, potentially leading to neuronal damage and neuropsychiatric symptoms 50-52 . Recent studies have also implicated CD4 + T cells in the choroid plexus as crucial players in the development of NPSLE 20 . In this study, we conducted a thorough investigation into the central nervous system manifestations in our lupus-modeled DT mice using a series of behavioral assays. Our results demonstrated that the dLck-driven tdTomato expression did not significantly impact the progression of NPSLE. Behavioral assessments showed no meaningful differences in motor function, anxiety, cognitive performance, or depressive behaviors across the tested groups. Additionally, the reporter system did not influence microglial activation in the hippocampus or the accumulation of CD4 + T cells in the choroid plexus, despite more than 90% of T cells in the choroid plexus co-expressing tdTomato. Interestingly, our preliminary data indicate that intracerebroventricular injection of CD4 + T cells isolated from DT mice is sufficient to induce NPSLE-like symptoms in non-DT mice. These findings suggest that while the dLck-driven tdTomato expression does not alter the pathological role of T cells in central lupus manifestations, these mice could serve as a valuable model for studying NPSLE with mild peripheral lupus pathology. This model offers a promising platform for future mechanistic studies and therapeutic drug screening. In summary, our study successfully utilized the Cre-driven tdTomatoreporter system to label most T cells in mice. However, we unexpectedly found that dLck-driven tdTomato expressionexerts a significant protective effect on peripheral lupus but not central lupus in two lupus-like mouse models. Our findings highlight a clear separation between peripheral and central pathological mechanisms in lupus mice. dLck-driven tdTomato expressionretard the development of peripheral lupus maybe by reducing the population of DNT cells, while it did not affect the central lupus maybe because of the unchanged CD4 + T cells in the choroid plexus. Overall, our results provide significant insight into the immunological mechanisms underlying SLE and serve as crucial sources of information for future research and therapeutic strategies. Declarations Acknowledgments We would like to appreciate the technical support from the Public Platform of Medical Research Center, Academy of Chinese Medical Science, Zhejiang Chinese Medical University. Author contributions All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Zhenghao Xu had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Study conception and design. Ning Han, Keer Wang, Zhenghao Xu. Acquisition of data. Ning Han, Mingxuan Han,Dan Yang. Analysis and interpretation of data. Ning Han, Zhenghao Xu, Xiaoxiao Hou. Funding This work was supported by National Natural Science Foundation of China: (82174005), Natural Science Foundation of Zhejiang: (LY22H280007), Zhejiang TCM Science and Technology Plan: (GZY-ZJ-KJ-24066) and Foundation of Zhejiang Chinese Medical University: (BZXCG-2022-20) (2023JKZKTS02). Competing interests The authors have no competing financial interests. Patient and public involvement Patients and/or the public were not involved in the design, or conduct, or reporting, or dissemination plans of this research. Ethics approval For the animal experiments was granted by the Institutional Animal Care and Use Committee of Zhejiang Chinese Medical University (Approval No. IACUC-20220718-01). Data availability Data are available upon reasonable request. Other data are available in the main text or the supplementary materials and are available upon reasonable request to the corresponding author. References Kiriakidou, M. & Ching, C. L. Systemic Lupus Erythematosus. Annals of internal medicine 172 , Itc81-itc96, doi:10.7326/aitc202006020 (2020). Li, M. et al. 2020 Chinese Guidelines for the Diagnosis and Treatment of Systemic Lupus Erythematosus. Rheumatology and immunology research 1 , 5-23, doi:10.2478/rir-2020-0009 (2020). Lu, Q. et al. Guideline for the diagnosis, treatment and long-term management of cutaneous lupus erythematosus. 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(n=5/group). \\u003cstrong\\u003e(D) \\u003c/strong\\u003eSchematic of the open field test (OFT). \\u003cstrong\\u003e(E) \\u003c/strong\\u003eRepresentative trajectories observed during the OFT. \\u003cstrong\\u003e(F) \\u003c/strong\\u003eTotal distance traveled in the OFT (n = 8/group). \\u003cstrong\\u003e(G)\\u003c/strong\\u003e Distance traveled by mice in the central area (purple region in B) during the OFT (n = 8/group). \\u003cstrong\\u003e(H) \\u003c/strong\\u003eSchematic representation of the Novel Object Recognition (NOR) test. \\u003cstrong\\u003e(I)\\u003c/strong\\u003e Discrimination Ratio (DR) in the NOR test (n = 8/group). \\u003cstrong\\u003e(J)\\u003c/strong\\u003e Schematic of the Novel Object Location (NOL) test. \\u003cstrong\\u003e(K)\\u003c/strong\\u003e DR in the NOL test (n = 8/group). \\u003cstrong\\u003e(L) \\u003c/strong\\u003eSchematic of the Tail Suspension Test (TST). \\u003cstrong\\u003e(M)\\u003c/strong\\u003e Immobility time in the TST (n = 8/group). \\u003cstrong\\u003e(N)\\u003c/strong\\u003e Schematic of the Forced Swim Test (FST). \\u003cstrong\\u003e(O)\\u003c/strong\\u003e Immobility time in the FST (n = 8/group). \\u003cstrong\\u003e(P)\\u003c/strong\\u003e Representative images of spleens from non-DT and DT mice. \\u003cstrong\\u003e(Q) \\u003c/strong\\u003estatistical data of spleen indices (Bar=5mm, n=12/group). \\u003cstrong\\u003e(R)\\u003c/strong\\u003e Representative images of cervical lymph nodes from non-DT and DT mice. \\u003cstrong\\u003e(S)\\u003c/strong\\u003e statistical data of lymph node indices (right) (Bar=5mm, n=12/group). \\u003cstrong\\u003e(T)\\u003c/strong\\u003e Representative images of glomeruli showing HE staining (left), PAS staining (center), and IgG immunofluorescence staining (right) (Bar=50μm, 20μm, 10μm). \\u003cstrong\\u003e(U)\\u003c/strong\\u003e Renal pathology severity score (6-point scale) (n=8/group). \\u003cstrong\\u003e(V)\\u003c/strong\\u003e Normalized glomerular cell proliferation statistics (n=8/group). \\u003cstrong\\u003e(W)\\u003c/strong\\u003e Normalized glomerular mesangial expansion level statistics (n=8/group). \\u003cstrong\\u003e(X) \\u003c/strong\\u003eNormalized IgG fluorescence area deposition in glomeruli statistics (n=6/group). **\\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.01.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"1.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5142419/v1/8210e11f23e6983c8a6671ab.png\"},{\"id\":71161554,\"identity\":\"cfee47d6-a729-4f44-a948-360a8d569c8e\",\"added_by\":\"auto\",\"created_at\":\"2024-12-11 16:38:10\",\"extension\":\"png\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":1185911,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eComparative analysis of central and peripheral manifestations in non-DT and DT Pristane-induced mice.\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e(A) \\u003c/strong\\u003eSchematic diagram of the experimental flow for Pristane-induced mouse model and behavioral tests. \\u003cstrong\\u003e(B) \\u003c/strong\\u003eRepresentative trajectories observed during the OFT. \\u003cstrong\\u003e(C)\\u003c/strong\\u003e Total distance traveled in the OFT (n = 8/group). \\u003cstrong\\u003e(D)\\u003c/strong\\u003eDistance traveled by mice in the central area during the OFT (n = 8/group). \\u003cstrong\\u003e(E) \\u003c/strong\\u003eDiscrimination Ratio (DR) in the NOR test (n = 8/group). \\u003cstrong\\u003e(F)\\u003c/strong\\u003e DR in the NOL test (n = 8/group). \\u003cstrong\\u003e(G) \\u003c/strong\\u003eImmobility time in the TST (n = 8/group). \\u003cstrong\\u003e(H)\\u003c/strong\\u003e Immobility time in the FST (n = 8/group). *\\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.05, **\\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.01, ***\\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.001, ****\\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.0001. \\u003cstrong\\u003e(I)\\u003c/strong\\u003eRepresentative images of spleens from non-DT Pristane-induced and DT Pristane-induced mice. \\u003cstrong\\u003e(J) \\u003c/strong\\u003eStatistical data of spleen indices (Bar=5mm, n=12/group). \\u003cstrong\\u003e(K)\\u003c/strong\\u003eRepresentative images of cervical lymph nodes from non-DT Pristane-induced and DT Pristane-induced mice. \\u003cstrong\\u003e(L)\\u003c/strong\\u003e Statistical data of lymph node indices (Bar=5mm, n=12/group). \\u003cstrong\\u003e(M)\\u003c/strong\\u003e Representative images of glomeruli showing HE staining (left), PAS staining (center), and IgG immunofluorescence staining (right) (Bar=50μm, 20μm, 10μm). \\u003cstrong\\u003e(N)\\u003c/strong\\u003e Renal pathology severity score (6-point scale) (n=8/group). \\u003cstrong\\u003e(O)\\u003c/strong\\u003e Normalized glomerular cell proliferation statistics (n=8/group). \\u003cstrong\\u003e(P)\\u003c/strong\\u003e Normalized glomerular mesangial expansion level statistics (n=8/group). \\u003cstrong\\u003e(Q) \\u003c/strong\\u003eNormalized IgG fluorescence area deposition in glomeruli statistics (n=6/group). ***\\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.001, ****\\u003cem\\u003eP\\u003c/em\\u003e\\u0026lt; 0.0001.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"2.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5142419/v1/cc2fa3e6a204138282bf7f6e.png\"},{\"id\":71161559,\"identity\":\"bec7f4ad-c324-44d2-bbea-4fb17142b2d0\",\"added_by\":\"auto\",\"created_at\":\"2024-12-11 16:38:10\",\"extension\":\"png\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":644800,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eComparative analysis of central and peripheral manifestations in non-DT B6/\\u003c/strong\\u003e\\u003cem\\u003e\\u003cstrong\\u003elpr\\u003c/strong\\u003e\\u003c/em\\u003e\\u003cstrong\\u003e and DT B6/\\u003c/strong\\u003e\\u003cem\\u003e\\u003cstrong\\u003elpr\\u003c/strong\\u003e\\u003c/em\\u003e\\u003cstrong\\u003e mice.\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e(A) \\u003c/strong\\u003eSchematic representation of the construction strategy for dLckCre\\u003csup\\u003etdTomato \\u003c/sup\\u003eB6/\\u003cem\\u003elpr\\u003c/em\\u003e (DT B6/\\u003cem\\u003elpr\\u003c/em\\u003e) mice. \\u003cstrong\\u003e(B) \\u003c/strong\\u003eSchematic representation of the experimental procedures. \\u003cstrong\\u003e(C)\\u0026nbsp; \\u003c/strong\\u003eFlow cytometry validation of fluorescence labeling in DT B6/\\u003cem\\u003elpr\\u003c/em\\u003e mice. (n=5/group) .\\u0026nbsp; \\u003cstrong\\u003e(D) \\u003c/strong\\u003eRepresentative trajectories observed during the OFT. \\u003cstrong\\u003e(E)\\u003c/strong\\u003e Total distance traveled in the OFT (n = 8/group). \\u003cstrong\\u003e(F)\\u003c/strong\\u003e Distance traveled by mice in the central area during the OFT (n = 8/group). \\u003cstrong\\u003e(G) \\u003c/strong\\u003eDiscrimination Ratio (DR) in the NOR test (n = 8/group). \\u003cstrong\\u003e(H)\\u003c/strong\\u003e DR in the NOL test (n = 8/group). \\u003cstrong\\u003e(I) \\u003c/strong\\u003eImmobility time in the TST (n = 8/group). \\u003cstrong\\u003e(J)\\u003c/strong\\u003e Immobility time in the FST (n = 8/group). *\\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.05, **\\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.01, ***\\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.001, ****\\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.0001. \\u003cstrong\\u003e(K)\\u003c/strong\\u003e Representative images of spleens from non-DT B6/\\u003cem\\u003elpr\\u003c/em\\u003e and DT B6/\\u003cem\\u003elpr\\u003c/em\\u003e mice. Statistical data of spleen indices (right) (Bar=5mm, n=12/group). \\u003cstrong\\u003e(L)\\u003c/strong\\u003e Representative images of cervical lymph nodes from non-DT B6/\\u003cem\\u003elpr\\u003c/em\\u003e and DT B6/\\u003cem\\u003elpr\\u003c/em\\u003e mice (left) , with statistical data of lymph node indices (right) (Bar=5mm, n=12/group). \\u003cstrong\\u003e(M)\\u003c/strong\\u003e Representative images of glomeruli showing HE staining (left), PAS staining (center), and IgG immunofluorescence staining (right) (Bar=50μm, 20μm, 10μm). \\u003cstrong\\u003e(N)\\u003c/strong\\u003e Renal pathology severity score (6-point scale) (n=8/group). \\u003cstrong\\u003e(O)\\u003c/strong\\u003e Normalized glomerular cell proliferation statistics (n=8/group). \\u003cstrong\\u003e(P)\\u003c/strong\\u003e Normalized glomerular mesangial expansion level statistics (n=8/group). \\u003cstrong\\u003e(Q) \\u003c/strong\\u003eNormalized IgG fluorescence area deposition in glomeruli statistics (n=6/group). ***\\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.001, ****\\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.0001.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"3.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5142419/v1/90791a1b04c915321f6584f6.png\"},{\"id\":71162662,\"identity\":\"80c55b9a-bcb3-4b14-9b9d-eca15e3fcb7f\",\"added_by\":\"auto\",\"created_at\":\"2024-12-11 16:46:10\",\"extension\":\"png\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":370292,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eFlow cytometry results of blood from experimental mice.\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e(A)\\u003c/strong\\u003e Representative flow cytometry plot for non-DT group, DT group, non-DT B6/\\u003cem\\u003elpr\\u003c/em\\u003e group and DT B6/\\u003cem\\u003elpr\\u003c/em\\u003e group.\\u0026nbsp;\\u003cstrong\\u003e(B)\\u003c/strong\\u003e Percentage change of CD4\\u003csup\\u003e-\\u003c/sup\\u003e CD8\\u003csup\\u003e+\\u003c/sup\\u003e cells in CD3\\u003csup\\u003e+\\u003c/sup\\u003e ( n\\u0026nbsp;=\\u0026nbsp;4/group). \\u003cstrong\\u003e(C) \\u003c/strong\\u003ePercentage change of DPT cells in CD3\\u003csup\\u003e+\\u003c/sup\\u003e ( n\\u0026nbsp;=\\u0026nbsp;4/group). \\u003cstrong\\u003e(D) \\u003c/strong\\u003ePercentage change of DNT cells in CD3\\u003csup\\u003e+\\u003c/sup\\u003e ( n\\u0026nbsp;=\\u0026nbsp;4/group).\\u0026nbsp;\\u003cstrong\\u003e(E)\\u003c/strong\\u003e Percentage change of CD4\\u003csup\\u003e+\\u003c/sup\\u003e CD8\\u003csup\\u003e-\\u003c/sup\\u003e cells in CD3\\u003csup\\u003e+\\u003c/sup\\u003e ( n\\u0026nbsp;=\\u0026nbsp;4/group). *\\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.05, **\\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.01, ***\\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.001,****\\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.0001.\\u0026nbsp;\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"4.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5142419/v1/4a5016cb586a05d825d476ee.png\"},{\"id\":71162661,\"identity\":\"2295f80c-2494-42b5-a331-c886c556edc3\",\"added_by\":\"auto\",\"created_at\":\"2024-12-11 16:46:10\",\"extension\":\"png\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":559729,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eNo significant changes in microglia and CD4\\u003c/strong\\u003e\\u003csup\\u003e\\u003cstrong\\u003e+\\u003c/strong\\u003e\\u003c/sup\\u003e\\u003cstrong\\u003e cell accumulation in DT and lupus-modeled DT mice compared to controls.\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u0026nbsp;(A)\\u003c/strong\\u003e Representative immunohistochemical images of microglia.(Bar = 200μm, 50μm, 10μm, n = 12/group). \\u003cstrong\\u003e(B) \\u003c/strong\\u003eNormalized Iba1+ cell count in the hippocampus (n = 12/group). \\u003cstrong\\u003e(C)\\u003c/strong\\u003e Normalized total branch number of microglia in the hippocampus (n = 12/group). \\u003cstrong\\u003e(D)\\u003c/strong\\u003e Normalized total branch length of microglia in the hippocampus (n = 12/group). \\u003cstrong\\u003e(E)\\u003c/strong\\u003e Normalized soma size of microglia in the hippocampus (n = 12/group). \\u003cstrong\\u003e(F) \\u003c/strong\\u003eRepresentative immunohistochemical images of CD4\\u003csup\\u003e+\\u003c/sup\\u003e (Bar = 100μm, n = 12/group). \\u003cstrong\\u003e(G) \\u003c/strong\\u003eNormalized area of CD4\\u003csup\\u003e+\\u003c/sup\\u003e positive regions in the choroid plexus (n = 12/group). **** denotes significant differences between the non-DT Pristane group and the non-DT Control group, as well as between the non-DT B6/\\u003cem\\u003elpr\\u003c/em\\u003e group and the non-DT Control group (\\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.0001). ####denotes significant differences between the DT Pristane group and the DT Control group, as well as between the DT B6/lpr group and the DT Control group (\\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.0001). There are no significant differences (\\u003cem\\u003eP\\u003c/em\\u003e \\u0026gt; 0.05) between the non-DT and DT conditions within the Control, Pristane, and B6/\\u003cem\\u003elpr\\u003c/em\\u003e groups. Data are presented as mean ± SEM. Individual data points are shown.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"5.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5142419/v1/b7433bcd41f7f84f53b6ac51.png\"},{\"id\":81136910,\"identity\":\"66ca0ef9-1086-461b-a2fe-58c160aa45a7\",\"added_by\":\"auto\",\"created_at\":\"2025-04-22 15:50:16\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":4717289,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5142419/v1/d88e389b-d43b-4902-87ae-b468f87ad7a0.pdf\"}],\"financialInterests\":\"There is \\u003cb\\u003eNO\\u003c/b\\u003e Competing Interest.\",\"formattedTitle\":\"\\u003cp\\u003e\\u003cstrong\\u003eCre-driven tdTomato expression unexpectedly confers lupus resistance in dLckCre mice\\u003c/strong\\u003e\\u003c/p\\u003e\",\"fulltext\":[{\"header\":\"Highlight \",\"content\":\"\\u003col\\u003e\\n \\u003cli\\u003edLck-driven tdTomato expression did not affect the baseline central and peripheral manifestations in mice.\\u003c/li\\u003e\\n \\u003cli\\u003edLck-driven tdTomato expression alleviates peripheral but not central symptoms in lupus model.\\u003c/li\\u003e\\n \\u003cli\\u003ePeripheral blood DNT cell numbers in mice were significantly decreased by the dLck-driven tdTomato expression, which may be a contributor in the altered progression of peripheral indicators of lupus.\\u003c/li\\u003e\\n\\u003c/ol\\u003e\"},{\"header\":\"1. Introduction\",\"content\":\"\\u003cp\\u003eSystemic lupus erythematosus (SLE) is a multifaceted autoimmune disorder characterized by a variety of clinical manifestations affecting various organ systems\\u003csup\\u003e1,2\\u003c/sup\\u003e. Peripheral symptoms such as rash, lymphadenopathy, splenomegaly, and renal impairment are particularly prevalent\\u003csup\\u003e3\\u0026ndash;5\\u003c/sup\\u003e. In addition, SLE frequently affects the central nervous system, leading to neuropsychiatric systemic lupus erythematosus (NPSLE), with clinical manifestations such as depression, cognitive impairment, seizures, and stroke\\u003csup\\u003e6\\u0026ndash;10\\u003c/sup\\u003e. The pathogenesis of SLE remains incompletely understood. Existing research suggests that SLE pathogenesis involves a complex interplay of aberrant autoimmune responses, production of autoantibodies, and immune-mediated damage to self-tissues\\u003csup\\u003e11\\u003c/sup\\u003e. However, the multifaceted nature of this disease remains a significant challenge in its study. Given the high incidence of SLE and the substantial difficulties associated with its treatment, there is a critical need for further investigation to elucidate the underlying pathophysiological mechanisms of SLE and to devise more efficacious therapeutic strategies.\\u003c/p\\u003e \\u003cp\\u003eIn recent years, the pivotal role of T lymphocytes in SLE has increasingly come under intensive investigation. Numerous studies have documented substantial alterations in the number and function of T lymphocytes in SLE patients\\u003csup\\u003e12\\u0026ndash;15\\u003c/sup\\u003e. For example, the proportion of double-negative T cells (DNT) is notably elevated in SLE patients and correlates strongly with the severity of lupus nephritis\\u003csup\\u003e15\\u0026ndash;17\\u003c/sup\\u003e. Additionally, CD4\\u003csup\\u003e+\\u003c/sup\\u003e T cells, another critical subset of T cells, play a pivotal role in the pathogenesis of NPSLE\\u003csup\\u003e18,19\\u003c/sup\\u003e. Our previous research demonstrated that the presence of ectopic CD4\\u0026thinsp;+\\u0026thinsp;T cells in the choroid plexus can trigger microglial activation through interferon signaling, ultimately leading to NPSLE-like symptoms in mice\\u003csup\\u003e20\\u003c/sup\\u003e. Increasing evidence highlights the importance of T cells in the pathology of peripheral and central SLE. However, research on different subsets of T cells in SLE still faces several limitations and challenges.\\u003c/p\\u003e \\u003cp\\u003eThe Cre-loxP system, widely applied in genetically engineered animal models\\u003csup\\u003e21\\u0026ndash;23\\u003c/sup\\u003e. The Cre-driven tdTomato reporter system is widely used to visualize gene expression and cellular dynamics. The distal lymphocyte-specific protein tyrosine kinase (dLck) gene, which is critical for T cell selection and maturation, is specifically expressed in T cells\\u003csup\\u003e24,25\\u003c/sup\\u003e. The dLckCre recombinase system has been extensively utilized to study T cell dynamics \\u003csup\\u003e25,26\\u003c/sup\\u003e. However, the use of dLck-driven T cell-specific fluorescence reporter systems has been rarely reported in SLE animal models, and their influence on the SLE models remains unclear. Therefore, here we crossed dLckCre mice with ROSA26\\u003csup\\u003efloxed\\u0026thinsp;\\u0026minus;\\u0026thinsp;Stop\\u0026minus;tdTomato\\u003c/sup\\u003e mice to generate dLck-driven tdTomato reporter mice (DT mice). We further established two lupus models in DT mice by injection of pristane or crossed them with B6/\\u003cem\\u003elpr\\u003c/em\\u003e mice. We unexpectedly found that these DT mice exhibit notable resistance to peripheral but not central lupus manifestations in both models of SLE.\\u003c/p\\u003e\"},{\"header\":\"2. Methods\",\"content\":\"\\u003cdiv id=\\\"Sec3\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.1 Animals\\u003c/h2\\u003e \\u003cp\\u003eIn this study, we utilized the distal Lck promoter-driven Cre (dLckCre) mice [B6.Cg-Tg(Lck-icre)3779Nik/J; Cat. No. NM-KI-225042, Shanghai Model Organisms Center, Inc.], B6.Cg Gt(ROSA)26Sor\\u003csup\\u003etm14(CAG\\u0026minus;tdTomato)Hze/J\\u003c/sup\\u003e mice [The Jackson Laboratory stock no. 007914], and B6/\\u003cem\\u003elpr\\u003c/em\\u003e mice [The Jackson Laboratory stock no. 007914].\\u003c/p\\u003e \\u003cp\\u003eTo generate dLckCre\\u003csup\\u003etdTomato\\u003c/sup\\u003e mice, dLckCre mice were bred with ROSA26\\u003csup\\u003eflox\\u0026thinsp;\\u0026minus;\\u0026thinsp;stop\\u0026minus;tdTomato\\u003c/sup\\u003e mice. Progeny harboring the dLckCre allele (Cre-/+) and expressing the tdTomato reporter were classified as the dLck-driven tdTomato reporter (DT) group (dLckCre\\u003csup\\u003etdTomato\\u003c/sup\\u003e). Littermates that did not carry the dLckCre allele (Cre-/-) were used as controls (Non-DT groups).\\u003c/p\\u003e \\u003cp\\u003eTo generate dLckCretdTomato B6/lpr mice, B6/lpr mice were crossed with dLckCretdTomato mice. Offspring possessing both the lpr mutation and the dLckCretdTomato transgene were selected as the experimental group (dLckCre\\u003csup\\u003etdTomato\\u003c/sup\\u003e B6/\\u003cem\\u003elpr\\u003c/em\\u003e). Littermate controls, which carried only the lpr mutation without the dLckCre\\u003csup\\u003etdTomato\\u003c/sup\\u003e transgene, were designated as the control group (B6/\\u003cem\\u003elpr\\u003c/em\\u003e).\\u003c/p\\u003e \\u003cp\\u003eA littermate control design was implemented to reduce the confounding effects of genetic background and maternal influence on the experimental outcomes. Animals in both the experimental and control groups were randomly selected from the same litter, ensuring consistency in genotype and early environmental conditions. By controlling for these variables, this approach effectively mitigates potential biases arising from genetic or maternal factors. Each group consisted of 12 animals (n\\u0026thinsp;=\\u0026thinsp;12), ensuring adequate statistical power for robust analysis. This design significantly enhances the validity and reproducibility of the experimental findings.\\u003c/p\\u003e \\u003cp\\u003eGiven the significant sex differences in the incidence of systemic lupus erythematosus (SLE), with estrogen playing a crucial role in the disease\\u0026rsquo;s pathogenesis, this study utilizes female mice as the experimental model. All mice were housed at 24\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1\\u0026deg;C with 50\\u0026ndash;55% humidity and a 12-hour light/dark cycle (light from 08:00 to 20:00), with ad libitum access to water and food. The study was approved by the Institutional Animal Care and Use Committee of Zhejiang Chinese Medical University (Approval No. IACUC-20220718-01) and followed the guidelines of the Animal Advisory Committee.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec4\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.2 Pristane-induced lupus\\u003c/h2\\u003e \\u003cp\\u003eTo establish the lupus model, 0.5 mL of pristane (99% pure, Sigma Aldrich Co., St. Louis, MO, USA) was administered intraperitoneally to non-DT and DT mice, following the method described by Satoh and Reeves\\u003csup\\u003e27\\u003c/sup\\u003e. In contrast, control group animals were administered 0.9% saline intraperitoneally. Mice were monitored for 8 months. Subsequently, the mice were humanely sacrificed, and their spleen, lymph nodes, and kidneys were harvested.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec5\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.3 Flow cytometry\\u003c/h2\\u003e \\u003cp\\u003eSingle-cell suspensions were prepared from the peripheral blood of mice following a standard protocol. Prior to antibody (Ab) staining, cells were preincubated with Fc receptor blocker (BD, catalog number 101312, USA). The complete list of antibodies and reagents used is as follows: Live-APC-A750 (Invitrogen, catalog number 65-0865-14, USA), CD3-FITC (BioLegend, catalog number 100203, USA), CD4-PerCP (BioLegend, catalog number 100539, USA), CD8-KO525 (BioLegend, catalog number 100752, USA). Data were collected via FACS analysis using the FC 500 MC system (Beckman Coulter, Fullerton, CA, USA) and were subsequently analyzed using FlowJo version 10 software (Tree Star, Inc., Ashland, OR, USA).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec6\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.4 Renal histopathological analysis\\u003c/h2\\u003e \\u003cdiv id=\\\"Sec7\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003e2.4.1 Hematoxylin and Eosin (H\\u0026amp;E) and Periodic Acid-Schiff (PAS) Staining\\u003c/h2\\u003e \\u003cp\\u003eMouse kidney tissues were initially fixed in 4% paraformaldehyde (PFA) at 4℃ for 48 hours. The samples were then embedded in paraffin and sectioned at a thickness of 5 \\u0026micro;m. Histological analysis was performed by staining the sections with Hematoxylin-Eosin (H\\u0026amp;E) and Periodic Acid-Schiff (PAS) stains. Imaging analysis of the stained sections was carried out using an Olympus VS120 microscope and xvViewer software.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec8\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003e2.4.2 IgG Immunofluorescence Staining\\u003c/h2\\u003e \\u003cp\\u003eKidney tissues were fixed in 4% PFA, followed by dehydration in 30% sucrose. Cryosections of 6 \\u0026micro;m thickness were prepared for immunofluorescence staining. The sections were first blocked with 5% bovine serum albumin for 1 hour, then incubated with anti-IgG antibody (Proteintech, Cat No. 66,467-1-Ig, diluted 1:1000). Stained sections were imaged using the Olympus VS120 microscope and analyzed with xvViewer software.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec9\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003e2.4.3 Pathological Evaluation\\u003c/h2\\u003e \\u003cp\\u003eGlomerular damage was evaluated using a semi-quantitative scoring system ranging from 0 to 6, based on the examination of five randomly selected glomeruli per specimen from H\\u0026amp;E-stained sections\\u003csup\\u003e28\\u003c/sup\\u003e. Cell counts were also performed for the same glomeruli. To quantify mesangial matrix expansion, the glomerular tuft area (GTA) and PAS-positive area (PTA) were measured. The percentage of PTA within the GTA was calculated using Image-Pro Plus 6.0 software. The area of IgG fluorescence was also quantified using Image-Pro Plus 6.0, with the average fluorescence area derived from five glomeruli to represent each sample. This method ensures a comprehensive and rigorous analysis of kidney pathology and IgG deposition.Simultaneously, cell counts were performed in the same glomeruli for each mouse.\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec10\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.5 Organ weight index\\u003c/h2\\u003e \\u003cp\\u003eMice were euthanized with an overdose of 1% pentobarbital sodium at a dosage of 150 mg/kg, ensuring humane euthanasia. Death was confirmed by the cessation of respiratory and cardiac activity. The cervical, anterior axillary, posterior (near the brachial plexus), and inguinal lymph nodes, along with the spleen, were weighed and photographed. The organ weight index was calculated as follows: organ weight (g) / body weight (g) \\u0026times; 100.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec11\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.6 Behavioral tests\\u003c/h2\\u003e \\u003cp\\u003eNeuropsychiatric symptoms were evaluated using various behavioral tests, as outlined in our previous research\\u003csup\\u003e20\\u003c/sup\\u003e.\\u003c/p\\u003e \\u003cdiv id=\\\"Sec12\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003e2.6.1 Open Field Test (OFT)\\u003c/h2\\u003e \\u003cp\\u003eSpontaneous locomotion was assessed in a 40cm \\u0026times; 40cm \\u0026times; 40cm open field arena over 15 minutes. A central zone measuring 13.3cm \\u0026times; 13.3cm was used to evaluate the distance traveled within this area. Total distance traveled and distance within the central zone were recorded with Any-Maze Software (Version 7.1, Stoelting Co., Wood Dale, IL, USA).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec13\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003e2.6.2 Novel Object Recognition (NOR)\\u003c/h2\\u003e \\u003cp\\u003eMice were first habituated to a 40 \\u0026times; 40 \\u0026times; 40 cm arena for 10 minutes. During the training phase, they were exposed to two identical objects in the arena for 10 minutes, followed by a 30-minute interval in their home cages. In the testing phase, mice were reintroduced to the arena for 5 minutes, where one of the familiar objects was replaced with a novel object. Object exploration was quantified by recording the time spent sniffing, touching, or approaching each object within \\u0026le;\\u0026thinsp;1cm.\\u003c/p\\u003e \\u003cp\\u003eThe discrimination ratio (DR) was calculated as DR\\u0026thinsp;=\\u0026thinsp;N / (N\\u0026thinsp;+\\u0026thinsp;F), where N represents the time spent exploring the novel object and F represents the time spent exploring the familiar object. A DR greater than 50% indicates a preference for the novel object, reflecting intact memory, while a DR of 50% or less suggests no significant preference, potentially indicating memory deficits.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec14\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003e2.6.3 Novel Object Location (NOL)\\u003c/h2\\u003e \\u003cp\\u003eMice were first habituated to a 40 \\u0026times; 40 \\u0026times; 40 cm arena for 10 minutes. During the training phase, they were exposed to two identical objects within the arena for 10 minutes, followed by a 30-minute inter-trial interval in their home cages. In the subsequent testing phase, mice were reintroduced to the arena for 5 minutes, during which one of the familiar objects was relocated to a new position. Object exploration was quantified by measuring the time spent within a 1cm radius of each object, encompassing behaviors such as sniffing, touching, or approaching.\\u003c/p\\u003e \\u003cp\\u003eExploration time of the object in the new location is denoted as N, while exploration time of the object in the original location is denoted as F. The discrimination ratio (DR) is computed using the formula DR\\u0026thinsp;=\\u0026thinsp;N / (N\\u0026thinsp;+\\u0026thinsp;F).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec15\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003e2.6.4 Tail Suspension Test (TST)\\u003c/h2\\u003e \\u003cp\\u003eMice were individually suspended by their tails using adhesive tape from a vertical aluminum bar positioned 36.6 cm above the floor, with partitions to prevent visual contact between them. Each mouse was suspended for 6 minutes in a quiet environment. Behavior was recorded using Any-Maze software (Stoelting Co., Wood Dale, IL, USA), and the duration of immobility was quantified. Immobility was defined as the absence of active movements, except for minimal adjustments to maintain balance. The immobility duration was analyzed as an indicator of depressive-like behavior. All procedures complied with institutional animal care guidelines.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec16\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003e\\u003cb\\u003e2.6.5 Forced swim test (FST)\\u003c/b\\u003e\\u003c/h2\\u003e \\u003cp\\u003eEach mouse was placed individually in a transparent cylindrical tank filled with water at 27\\u0026deg;C to a depth of 20cm. The tank was 30cm high and 15cm in diameter. The test consisted of a 2-minute acclimation period followed by a 4-minute test session. Behavior was recorded using Any-Maze software, and the total time spent immobile during the test was measured. Immobility was defined as the absence of movement, with only minor adjustments to keep the head above water. Immobility time was analyzed using automated tracking software as an indicator of depressive-like behavior. All procedures adhered to institutional animal care guidelines.\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec17\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.7. Immunofluorescent staining\\u003c/h2\\u003e \\u003cp\\u003eImmunofluorescence staining was performed on 16 \\u0026micro;m-thick coronal brain tissue sections to assess microglial and CD4\\u003csup\\u003e+\\u003c/sup\\u003e T cell populations. Primary antibodies used were anti-rabbit Iba1 (1:400; Wako, catalog no. 019-19741, Japan) to label microglia, and anti-mouse CD4 (1:200; Abcam, catalog no. ab33775, Cambridge, UK) to detect CD4\\u003csup\\u003e+\\u003c/sup\\u003e T cells. Following incubation with the primary antibodies at 4℃ overnight, sections were washed and incubated with species-appropriate secondary antibodies. Alexa Fluor 488-conjugated secondary antibody (1:400; Abcam, catalog no. ab150081, Cambridge, UK) was used for Iba1 detection, and Alexa Fluor 647-conjugated secondary antibody (1:400; Abcam, catalog no. ab150115, Cambridge, UK) was used for CD4 detection. Nuclei were counterstained with DAPI (Solarbio, catalog no. MA0222, Beijing, China). Stained sections were imaged using an Olympus VS120-S6-W microscope under consistent exposure settings across all samples to ensure reliable comparison.\\u003c/p\\u003e \\u003cp\\u003eMicroglial morphological analysis was conducted using ImageJ and Fiji-ImageJ software, focusing on key metrics including microglial cell density, total branch length (skeletonized microglial processes), branch point number (indicating cellular complexity), and cell body area. For skeletonization, Z-stack images were processed using the \\\"Analyze Skeleton\\\" plugin to quantify the structural features of individual microglia. Quantification of microglia was carried out across randomly selected fields of view per section. In each sample, eight representative microglia were analyzed per field, with the final data presented as averages per sample. The analysis followed established protocols as outlined in previous studies\\u003csup\\u003e29,30\\u003c/sup\\u003e, ensuring consistency and reproducibility.\\u003c/p\\u003e \\u003cp\\u003eQuantitative analysis of CD4\\u003csup\\u003e+\\u003c/sup\\u003e T cells was conducted to determine the area of CD4\\u003csup\\u003e+\\u003c/sup\\u003e positive regions within the choroid plexus in each brain section. For each sample, the area of CD4\\u003csup\\u003e+\\u003c/sup\\u003e positive regions was measured across eight distinct choroid plexus regions, and the mean value was calculated to represent the CD4\\u003csup\\u003e+\\u003c/sup\\u003e positive area for that sample. ImageJ software was used to perform threshold-based segmentation of the CD4\\u003csup\\u003e+\\u003c/sup\\u003e immunoreactivity, ensuring accurate delineation of positive regions. The analysis followed the methodological guidelines outlined in Wang et al. (2024)\\u003csup\\u003e20\\u003c/sup\\u003e, ensuring consistency and comparability with previous studies.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec18\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.8. Statistics\\u003c/h2\\u003e \\u003cp\\u003eStatistical analyses were performed using GraphPad Prism software (version 8.0.2). Normality of the data was assessed using the Shapiro-Wilk test before applying parametric or non-parametric tests. For comparisons between two groups, the Mann-Whitney U test was applied for non-normally distributed data, while the unpaired Student\\u0026rsquo;s t-test was used for normally distributed data. For comparisons involving more than two groups, one-way analysis of variance (ANOVA) followed by Fisher\\u0026rsquo;s Least Significant Difference (LSD) post hoc test was conducted. Data are presented as mean\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;standard error of the mean (SEM), and statistical significance was defined as a two-tailed P-value\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05. All behavioral assessments, histopathological evaluations, and image analyses were conducted in a double-blinded manner to minimize bias. Comprehensive statistical results are provided in Supplementary Table\\u0026nbsp;1.\\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"3. Results\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003e3.1\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003edLCK-driven tdTomato expression\\u0026nbsp;did not affect the baseline central and peripheral manifestations in mice.\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eWe generated dLckCre\\u003csup\\u003etdTomato\\u003c/sup\\u003e mice (DT mice) by crossing dLckCre mice with ROSA26\\u003csup\\u003eflox-stop-tdTomato\\u003c/sup\\u003e mice (Fig. 1A). Flow cytometric analysis confirmed that over 90% of CD3\\u003csup\\u003e+\\u003c/sup\\u003e T lymphocytes in DT mice expressed tdTomato, whereas non-DT mice showed negligible red fluorescence (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026lt; 0.01, Fig. 1C). Additionally, both DT and non-DT mice were aged to 40 weeks to match the age of lupus model mice for subsequent experiments (Fig. 1B).\\u003c/p\\u003e\\n\\u003cp\\u003eWe initially assessed central manifestations in DT mice relative to non-DT mice using a series of behavioral tests. In the open field test, no significant differences were observed between DT and non-DT mice in total distance traveled or the proportion of distance traveled in the central area, indicating similar locomotor activity and anxiety levels between the two groups (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026gt; 0.05 for all, Fig. 1D-G). Similarly, the novel object location and novel object recognition tests showed no significant differences in exploration times of new objects or their spatial placements, suggesting comparable learning and memory abilities (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026gt; 0.05 for all, Fig. 1H-K). Furthermore, depressive-like behaviors, as measured by the tail suspension and forced swim tests, showed similar immobility times in both groups, indicating no significant differences in depressive-like phenotypes (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026gt; 0.05 for all, Fig. 1L-O). Overall, these results suggest that DT mice exhibit no significant baseline differences from non-DT mice in locomotion, anxiety, learning, memory, or depressive-like behaviors. Thus, DT mice provide a reliable baseline model for further studies, particularly those related to NPSLE.\\u003c/p\\u003e\\n\\u003cp\\u003eSubsequently, we evaluated peripheral manifestations in DT mice. Baseline spleen and lymph node indices were comparable between DT mice and their non-DT littermates (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026gt; 0.05 for all, Fig. 1P-S). Similarly, baseline kidney pathology, assessed by hematoxylin-eosin (HE) staining, periodic acid-Schiff (PAS) staining, and IgG immunofluorescence staining, showed no significant differences between the two groups (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026gt; 0.05 for all, Fig. 1T-X). These findings indicate that DT mice exhibit peripheral features, including lupus-related markers, similar to those of non-DT mice. Therefore, DT mice provide a reliable baseline model for further investigations into peripheral lupus pathology.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e3.2\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003edLCK-driven tdTomato expression alleviates\\u0026nbsp;peripheral but not central symptoms in pristane-induced lupus model\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eAfter confirming that conditional tdTomato expression had no effect on central and peripheral manifestations in dLckCre mice under non-lupus conditions, we established the lupus model in DT mice via intraperitoneal injection of pristane (Fig. 2A).\\u003c/p\\u003e\\n\\u003cp\\u003eTo assess central lupus manifestations, we performed several behavioral tests. Compared to normal control mice, the injection of pristane induces NPSLE-like behavioral alterations in both DT and non-DT mice. These changes encompass a decrease in the total travel distance and the percentage of distance traversed in the central area during the open field test (\\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.001 for total travel distance and\\u003cem\\u003e\\u0026nbsp;P\\u003c/em\\u003e \\u0026lt; 0.05 for the percentage of distance traversed in the central area; Fig. 2B-D) , poorer performance in both the novel object recognition and novel object location tests (\\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.05 for novel object recognition and \\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.001 for novel object location; Fig. 2E-F), as well as an increase in the duration of immobility observed in the tail suspension and forced swim tests (\\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.0001 for both; Fig. 2G-H). However, no significant differences for all of these tests were observed between the two lupus model groups (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026gt; 0.05 for all, Fig. 2B-H).\\u003c/p\\u003e\\n\\u003cp\\u003eFor peripheral manifestations, an unexpected finding emerged. Pristane injection resulted in weaker peripheral lupus symptoms in DT mice than that in non-DT mice, including the decreased splenomegaly (\\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.0001; Fig. 2I, J), reduced lymphadenopathy (\\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.001; Fig. 2K, L), and milder renal pathology (Fig. 2M;\\u003cem\\u003e\\u0026nbsp;P\\u003c/em\\u003e \\u0026lt; 0.001 for renal pathology scores, Fig. 2N; \\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.001 for glomerular cell proliferation, Fig. 2O; \\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.0001 for PAS-positive areas, Fig. 2P; \\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.0001 for IgG deposition, Fig. 2Q). In conclusion, conditional tdTomato expression significantly attenuates the severity of peripheral lupus symptoms in the pristane-induced dLckCre lupus model, although it does not impact the progression of NPSLE.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e3.3\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003edLCK-driven tdTomato expression\\u0026nbsp;alleviates\\u0026nbsp;peripheral but not central symptoms in the B6/\\u003cem\\u003elpr\\u003c/em\\u003e lupus model\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eTo confirm the findings in pristane-induced lupus model, we employed an additional lupus model,\\u0026nbsp;the B6/\\u003cem\\u003elpr\\u003c/em\\u003e transgenic mouse model. DT mice were crossed with B6/\\u003cem\\u003elpr\\u003c/em\\u003e mice to generate spontaneous lupus DT B6/\\u003cem\\u003elpr\\u003c/em\\u003e mice (Fig. 3A, B), \\u0026nbsp;and marker expression was confirmed by flow cytometry (Fig. 3C).\\u003c/p\\u003e\\n\\u003cp\\u003eTo evaluate central lupus manifestations, we conducted a series of behavioral tests. Compared to normal control mice, both non-DT B6/\\u003cem\\u003elpr\\u003c/em\\u003e and DT B6/\\u003cem\\u003elpr\\u003c/em\\u003e mice exhibited NPSLE-like behavioral changes. These changes included a reduction in total travel distance and the percentage of distance traversed in the central area during the open field test (\\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.001 for total travel distance and \\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.01 and \\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.001 for the percentage of distance traversed in the central area; Fig. 3E, F). Additionally, both groups showed decreased exploration time of novel objects and new locations in the novel object recognition and novel object location tests (\\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.05 for novel object recognition and \\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.01 and \\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.001 for novel object location; Fig. 3G, H). Increased immobility duration was also observed in the tail suspension and forced swim tests (\\u003cem\\u003eP\\u0026nbsp;\\u003c/em\\u003e\\u0026lt; 0.0001; Fig. 3I, J).\\u003c/p\\u003e\\n\\u003cp\\u003eFor peripheral manifestations, a significant difference was still observed. Compared to non-DT B6/\\u003cem\\u003elpr\\u003c/em\\u003e mice, DT B6/\\u003cem\\u003elpr\\u003c/em\\u003e mice exhibited milder peripheral lupus symptoms, including reduced splenomegaly (\\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.001; Fig. 3K), decreased lymphadenopathy (\\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.0001; Fig. 3L), and attenuated renal pathology (Fig. 3M; \\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.0001, Fig. 3N; \\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.001 for glomerular cell proliferation, Fig. 3O; \\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.0001 for PAS-positive area, Fig. 3P; \\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.0001 for IgG deposition, Fig. 3Q).\\u003c/p\\u003e\\n\\u003cp\\u003eIn summary, the central and peripheral manifestations in DT B6/\\u003cem\\u003elpr\\u003c/em\\u003e mice indicate that the reduced severity of systemic lupus symptoms due to the conditional tdTomato reporter gene expression is significant. This specific genetic modification seems to delay the progression of lupus symptoms in peripheral tissues, though its impact on the central nervous system appears to be less pronounced.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e3.4.\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003edLCK-driven tdTomato expression\\u0026nbsp;alters T lymphocyte subset\\u0026nbsp;proportions\\u0026nbsp;in peripheral Blood\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eTo further explore the mechanisms related to the unexpected resistance to peripheral lupus exhibited by DT mice, we conducted a comprehensive analysis using peripheral blood flow cytometry. When comparing non-DT and DT mice, a statistically significant elevation (\\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.001, Fig. 4E) in the proportion of CD4\\u003csup\\u003e+\\u003c/sup\\u003eCD8\\u003csup\\u003e-\\u003c/sup\\u003e T cells was observed, whereas no significant changes were detected in the other T cell subsets analyzed (Fig. 4B-D). When comparing non-DT and non-DT B6/\\u003cem\\u003elpr\\u003c/em\\u003e, we found that non-DT B6/\\u003cem\\u003elpr\\u003c/em\\u003e showed a decrease of CD4\\u003csup\\u003e-\\u003c/sup\\u003eCD8\\u003csup\\u003e+\\u003c/sup\\u003e (\\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.001), CD4\\u003csup\\u003e+\\u003c/sup\\u003eCD8\\u003csup\\u003e-\\u003c/sup\\u003e (\\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.0001) cells and an increase of DNT (\\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.0001) cells. When further comparing non-DT B6/\\u003cem\\u003elpr\\u003c/em\\u003e and DT B6/\\u003cem\\u003elpr\\u003c/em\\u003e mice, we found that DT B6/\\u003cem\\u003elpr\\u003c/em\\u003e mice showed a large decrease in CD4\\u003csup\\u003e-\\u003c/sup\\u003eCD8\\u003csup\\u003e-\\u003c/sup\\u003e double-negative T (DNT) cells (\\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.001) and significant increases in CD4\\u003csup\\u003e-\\u003c/sup\\u003eCD8\\u003csup\\u003e+\\u003c/sup\\u003e (\\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.05), CD4\\u003csup\\u003e+\\u003c/sup\\u003eCD8\\u003csup\\u003e-\\u003c/sup\\u003e (P \\u0026lt; 0.01), and CD4\\u003csup\\u003e+\\u003c/sup\\u003eCD8\\u003csup\\u003e+\\u0026nbsp;\\u003c/sup\\u003edouble-positive T (DPT) cells (\\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.001).\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e3.5.\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003edLCK-driven tdTomato expression\\u0026nbsp;does not affect microglial phenotype and CD4\\u003csup\\u003e+\\u003c/sup\\u003e T cell accumulation in the choroid plexus\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe excessive activation of microglia and the extensive infiltration of CD4\\u003csup\\u003e+\\u003c/sup\\u003e T cells in the choroid plexus have been widely recognized as key drivers of neuroinflammation in CNS in SLE, contributing to the onset and progression of NPSLE. Therefore, in addition to observing behavioral outcomes, we conducted an in-depth analysis of microglial activation and CD4\\u003csup\\u003e+\\u003c/sup\\u003e T cell-related markers in each group of mice to further investigate the role of DT in NPSLE.\\u003c/p\\u003e\\n\\u003cp\\u003eImmunofluorescence staining results showed significant increases in microglial numbers within the hippocampus in both group of lupus mice (\\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.0001; Fig. 5A, B), Additionally, these microglia in lupus mice exhibited increased microglial branch number (\\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.0001; Fig. 5C), branch length (\\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.0001; Fig. 5D), and cell body size (\\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.0001; Fig. 5E). However, no significant differences were observed between non-DT and DT, both in in normal or lupus state (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026gt; 0.05, Fig. A-E). Similarly, CD4\\u003csup\\u003e+\\u003c/sup\\u003e T cell accumulation in the choroid plexus were found in both group of lupus mice; However no significant differences were observed between non-DT and DT, both in in normal or lupus state (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026gt; 0.05, Fig. 5F, G).\\u003c/p\\u003e\"},{\"header\":\"Discussion\",\"content\":\"\\u003cp\\u003eSLE is an autoimmune disease characterized by severe inflammation and has widespread impact on both central and peripheral manifestations\\u003csup\\u003e11,31\\u003c/sup\\u003e. In this study, by using two lupus model. we unexpectedly revealed several key findings by utilizing the conditional tdTomato reporter system in dLckCre mice: (1) dLCK-driven tdTomato expression had no impact on baseline central or peripheral manifestations in normal mice. (2) dLCK-driven tdTomato expression unexpectedly induces peripheral lupus resistance in two SLE mouse models; (3) dLCK-driven tdTomato expression did not affect the central lupus manifestations in two SLE mouse models. Taken together, our results highlight the dLCK-driven tdTomato expression mainly affects the development of peripheral manifestations rather than central manifestations of SLE, indicating a distinction in a distinct role for T cells in peripheral and central lupus pathogenesis.\\u003c/p\\u003e\\n\\u003cp\\u003ePrevious studies have indicated that fluorescence reporter systems, such as tdTomato reporter gene system, can potentially alter the function of labeled cells \\u003csup\\u003e32-34\\u003c/sup\\u003e. For instance, insertion of the tdTomato reporter gene at the Puma locus was associated with a reduction in TRP53-induced apoptosis, thus modifying apoptotic responses\\u003csup\\u003e35\\u003c/sup\\u003e. In the present study, we employed the dLck-driven tdTomato reporter system to specifically tag dLck-positive T cells, allowing for their identification and tracking within the context of our experiments. Unexpectedly, by using two SLE mouse models (pristane-induced and B6/\\u003cem\\u003elpr\\u003c/em\\u003e transgenic models), we found this approach significantly attenuated the progression of peripheral lupus pathology in these DT mice. Specifically, compared with non-DT control groups, we observed a decrease in lymphadenopathy, splenomegaly, and renal pathology severity in DT groups. The pristane-induced model predominantly triggers an excessive type I interferon response\\u003csup\\u003e36-38\\u003c/sup\\u003e, whereas the B6/\\u003cem\\u003elpr\\u003c/em\\u003e model is characterized by marked lymphadenopathy and abnormal T cell proliferation\\u003csup\\u003e39-41\\u003c/sup\\u003e. Thus, our findings suggest that the dLck-driven tdTomato expression may have a unique modulatory effect on peripheral lupus progression, highlighting dLckCre-positive T cells as a potential therapeutic target for peripheral lupus. This underscores the importance of further exploring the differential mechanisms underlying central and peripheral manifestations of lupus.\\u003c/p\\u003e\\n\\u003cp\\u003eNumerous studies have highlighted the pivotal role of T cells in the peripheral manifestations of lupus\\u003csup\\u003e42-46\\u003c/sup\\u003e. Our flow cytometric analysis demonstrated that the dLck-driven tdTomato expression significantly altered T cell subsets in lupus mice, notably leading to a reduction in DNT cells. In SLE, DNT cells exhibit profound phenotypic and functional abnormalities and make up a disproportionately large fraction of the T cell population compared to their minimal presence in healthy individuals. The increased abundance of DNT cells is believed to worsen the disease by impairing normal T cell functions and promoting autoimmunity\\u003csup\\u003e43,47-49\\u003c/sup\\u003e. In this study, we observed that dLck-driven tdTomato expression led to a reduction in the number of DNT cells. This change corresponded with an alleviation of peripheral symptoms in these DT lupus mice. Our findings further support the notion that targeting dysregulated DNT cell subsets can effectively intervene in the progression of lupus. Moreover, this highlights a novel and promising T-cell modification strategy that could be leveraged for therapeutic intervention in lupus.\\u003c/p\\u003e\\n\\u003cp\\u003eNPSLE is characterized by a wide spectrum of neurological and psychiatric symptoms, including depression and cognitive impairment, and represents a severe and potentially life-threatening manifestation of SLE. One of the key mechanisms driving NPSLE pathology is aberrant microglial activation\\u003csup\\u003e18,50\\u003c/sup\\u003e, which contributes to increased neuroinflammation, potentially leading to neuronal damage and neuropsychiatric symptoms \\u003csup\\u003e50-52\\u003c/sup\\u003e. Recent studies have also implicated CD4\\u003csup\\u003e+\\u003c/sup\\u003e T cells in the choroid plexus as crucial players in the development of NPSLE\\u003csup\\u003e20\\u003c/sup\\u003e. In this study, we conducted a thorough investigation into the central nervous system manifestations in our lupus-modeled DT mice using a series of behavioral assays. Our results demonstrated that the\\u0026nbsp;dLck-driven tdTomato expression did not significantly impact the progression of NPSLE. Behavioral assessments showed no meaningful differences in motor function, anxiety, cognitive performance, or depressive behaviors across the tested groups. Additionally, the reporter system did not influence microglial activation in the hippocampus or the accumulation of CD4\\u003csup\\u003e+\\u003c/sup\\u003e T cells in the choroid plexus, despite more than 90% of T cells in the choroid plexus co-expressing tdTomato. Interestingly, our preliminary data indicate that intracerebroventricular injection of CD4\\u003csup\\u003e+\\u003c/sup\\u003e T cells isolated from DT mice is sufficient to induce NPSLE-like symptoms in non-DT mice. These findings suggest that while the dLck-driven tdTomato expression does not alter the pathological role of T cells in central lupus manifestations, these mice could serve as a valuable model for studying NPSLE with mild peripheral lupus pathology. This model offers a promising platform for future mechanistic studies and therapeutic drug screening.\\u003c/p\\u003e\\n\\u003cp\\u003eIn summary, our study successfully utilized the Cre-driven tdTomatoreporter system to label most T cells in mice. However, we unexpectedly found that dLck-driven tdTomato expressionexerts a significant protective effect on peripheral lupus but not central lupus in two lupus-like mouse models. Our findings highlight a clear separation between peripheral and central pathological mechanisms in lupus mice. dLck-driven tdTomato expressionretard the development of peripheral lupus maybe by reducing the population of DNT cells, while it did not affect the central lupus maybe because of the unchanged CD4\\u003csup\\u003e+\\u003c/sup\\u003e T cells in the choroid plexus. Overall, our results provide significant insight into the immunological mechanisms underlying SLE and serve as crucial sources of information for future research and therapeutic strategies.\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eAcknowledgments\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eWe would like to appreciate the technical support from the Public Platform of Medical Research Center, Academy of Chinese Medical Science, Zhejiang Chinese Medical University.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAuthor contributions\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eAll authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Zhenghao Xu had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.\\u003c/p\\u003e\\n\\u003cp\\u003eStudy conception and design. Ning Han, Keer Wang, Zhenghao Xu.\\u003c/p\\u003e\\n\\u003cp\\u003eAcquisition of data. Ning Han, Mingxuan Han,Dan Yang.\\u003c/p\\u003e\\n\\u003cp\\u003eAnalysis and interpretation of data. Ning Han, Zhenghao Xu, Xiaoxiao Hou.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eFunding\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThis work was supported by National Natural Science Foundation of China: (82174005), Natural Science Foundation of Zhejiang: (LY22H280007), Zhejiang TCM Science and Technology Plan: (GZY-ZJ-KJ-24066) and Foundation of Zhejiang Chinese Medical University: (BZXCG-2022-20) (2023JKZKTS02).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eCompeting interests\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe authors have no competing financial interests.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003ePatient and public involvement\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003ePatients and/or the public were not involved in the design, or conduct, or reporting, or dissemination plans of this research.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eEthics approval\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eFor the animal experiments was granted by the Institutional Animal Care and Use Committee of Zhejiang Chinese Medical University (Approval No. IACUC-20220718-01).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eData availability\\u003c/strong\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eData are available upon reasonable request. Other data are available in the main text or the supplementary materials and are available upon reasonable request to the corresponding author.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\n \\u003cli\\u003eKiriakidou, M. \\u0026amp; Ching, C. L. 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Here we tested the impact of the tdTomato reporter system on the progression of both central and peripheral manifestations of systemic lupus erythematosus (SLE) in dLckCre mice. We crossed dLckCre mice with ROSA26\\u003csup\\u003efloxed-Stop-tdTomato\\u003c/sup\\u003e mice to generate the conditional dLck-driven tdTomato reporter mice (DT mice). Then, SLE models were induced by pristane injection in DT mice or by crossing DT mice with B6/\\u003cem\\u003elpr\\u003c/em\\u003e mice. Central and peripheral manifestations of SLE were tested by. Surprisingly, we found DT mice exhibited a significant reduction in the progression of peripheral manifestations of SLE, evidenced by decreased severity of lymph node and splenic lesions, and improved renal pathology. However, DT mice showed similar central manifestations of SLE as control mice, evidenced by similar behavioral performance, CD4\\u003csup\\u003e+\\u003c/sup\\u003e T cell infiltration in the choroid plexus, and microglial activation in the hippocampus. Flow cytometry revealed a significant reduction in the proportion of double-negative (DN) T cells in the peripheral blood of the DT mice. Thus, conditional tdTomato expression alleviated peripheral but not central manifestations of SLE in dLckCre mice, suggesting a diverse role of dLck-labeled T lymphocytes in SLE development. Our results also provide additional evidence supporting the existence of a distinct separation mechanism between peripheral and central manifestations of SLE.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Cre-driven tdTomato expression unexpectedly confers lupus resistance in dLckCre mice\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2024-12-11 16:38:06\",\"doi\":\"10.21203/rs.3.rs-5142419/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"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\":\"16c3252a-d226-496a-bcd4-4122e1b0f95e\",\"owner\":[],\"postedDate\":\"December 11th, 2024\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"posted\",\"subjectAreas\":[{\"id\":41416838,\"name\":\"Biological sciences/Immunology/Immunological disorders/Autoimmune diseases/Systemic lupus erythematosus\"},{\"id\":41416839,\"name\":\"Biological sciences/Developmental biology/Disease model\"}],\"tags\":[],\"updatedAt\":\"2025-04-22T15:42:07+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2024-12-11 16:38:06\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-5142419\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-5142419\",\"identity\":\"rs-5142419\",\"version\":[\"v1\"]},\"buildId\":\"qtupq5eGEP_6zYnWcrvyt\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}