CD138 promotes the accumulation and activation of autoreactive T cells in autoimmune MRL/lpr mice
- Authors:
- Published online on: October 24, 2023 https://doi.org/10.3892/etm.2023.12267
- Article Number: 568
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Copyright: © Xie et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
Abstract
Introduction
Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the production of multiple autoantibodies, including anti-nuclear antibodies and anti-double-stranded (ds)DNA antibodies (1,2). The generation of these autoantibodies was reported to have a detrimental effect on multiple tissues and organs, such as the skin, joints and kidney (3-5). Previous studies suggested that SLE is a complex disease involving the participation of both T and B cells in its progression (6-8).
Activated B cells differentiate into plasma cells to secrete antibodies in response to an antigen. CD138 is a marker of plasma cells (9,10). CD138+ T cells, which express both CD3 and CD138 on their surface, were previously reported to be associated with plasmablastic B-cell neoplasms in clinical cases (11) and were also identified in SLE murine models (12-14). CD138+ T cells were reported to significantly accumulate in Fas-deficient lupus mice (12-14). Furthermore, previous studies demonstrated that double negative (DN) T cells serve an important role in the progression of lupus and significantly contribute to tissue injury in SLE (6,15,16). Plasma cell accumulation was reported to be a hallmark feature of SLE (17,18). Our previous study demonstrated that the majority of CD138+ cells in SLE murine models were CD138+ T cells (19). A previous study also reported that CD4+CD138+ T cells could significantly promote autoantibody production both in vivo and in vitro (12). This suggests that CD138+ T cells may be autoreactive and accumulate in Murphy Roths Large lymphoproliferative (MRL/lpr) mice to induce an autoimmune response. Therefore, CD138+ T cells may aid in elucidating the underlying mechanism of SLE progression. In the present study, the mechanism by which CD138+ T cells are involved in the progression of SLE in MRL/lpr mice was evaluated.
Phorbol 12-myristate 13-acetate (PMA) and ionomycin (PI) are commonly used to induce in vitro cellular activation (20,21). A previous study also demonstrated that T cell receptor (TCR) activation negatively regulates CD138 protein expression in DN T cells (14). PMA and PI are commonly used to activate T cells and promote cytokine secretion in T cells (22,23). In the present study, the effect of PI stimulation on CD138+ T cell accumulation in splenocytes of MRL/lpr mice was assessed. Furthermore, the function of CD138 protein expression in CD3+CD138- T cells and its resulting accumulation in MRL/lpr mice was evaluated.
Materials and methods
Experimental animals
Female Murphy Roths Large (MRL/MPJ-Fas+/+; n=8; age, 4-week-old; weight, 20-25 g) and Murphy Roths Large lymphoproliferative (MRL/lpr-Fas-/-; n=8; age, 4 weeks; weight, 20-25 g) mice were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. Mice were housed at 22±1˚C with a relative humidity of 50-60% and a 12-h light/dark cycle. The mice were allowed free access to water and food. All animal experiments were compliant with the institutional ethical guidelines of Beijing Institute of Chinese Medicine (Beijing, China).
Cell culture
At 17-18 weeks of age, mice that did not undergo intervention or treatment were anesthetized using an intraperitoneal injection of 1% sodium pentobarbital (80 mg/kg) to harvest blood for serum samples. Mice were then euthanized by cervical dislocation and subsequently the spleen was harvested. Single-cell suspensions of splenocytes were then obtained via filtration through a 70-µm cell strainer (BD Biosciences). Splenocytes with 5x10^6 cells in each experiment were then cultured in RPMI-1640 medium (HyClone; Cytiva) with 10% fetal bovine serum, (Gibco; Thermo Fisher Scientific, Inc.) at 37˚C with 5% CO2 or in the presence of PMA (50 ng/ml; Thermo Fisher Scientific, Inc.) and PI (1 µg/ml; Thermo Fisher Scientific, Inc.) to stimulate the splenocytes (24-26).
Measurement of serum cytokine levels using the Luminex™ platform
Serum levels of IFN-γ, IFN-α, tumor necrosis factor, IL-6, IL-10, IL-17, IL-21 and IL-2 were measured using 8-Plex ProcartaPlex Panel (cat. no. PPX-08; Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions. Diluted serum samples with universal assay buffer (from the Luminex kit) were added onto 96-well plates coated with magnetic beads and incubated at room temperature for 120 min after vortexing. The beads were then washed with wash buffer, and the 50X detection antibody mixture was diluted to 1X with detection antibody diluent, and then added and incubated for an additional 30 min at room temperature. After incubation and plate washing with wash buffer, the samples were analyzed on the Luminex 200 platform.
Flow cytometry
Splenocytes were incubated on ice with CD16/CD32 monoclonal antibodies (cat. no. 14-0161-85; eBioscience™; Thermo Fisher Scientific, Inc.) for 15 min, and then red blood cells were lysed using lysis buffer (BD Biosciences). Cells were subsequently incubated with the following antibodies (100 µl/test as recommended by the manufacturer for flow cytometry) at room temperature for 30 min for flow cytometric analysis: Anti-CD3 phycoerythrin (PE)-cyanine7 (cy7) (cat. no. 25-0032-82; eBioscience; Thermo Fisher Scientific, Inc.); anti-CD3 allophycocyanin (APC)-cy7 (cat. no. 47-0032-82; eBioscience; Thermo Fisher Scientific, Inc.); anti-CD4 fluorescein isothiocyante (FITC) (cat. no. 53-0041-82; eBioscience; Thermo Fisher Scientific, Inc.); anti-CD8 peridinin-chlorophyll-protein (PerCP) (cat. no. 45-0081-82; eBioscience; Thermo Fisher Scientific, Inc.); anti-CD8 APC (cat. no. 17-0081-82; eBioscience; Thermo Fisher Scientific, Inc.); anti-CD19 APC-cy7 (cat. no. 47-0193-82; eBioscience; Thermo Fisher Scientific, Inc.); anti-CD138 PE (cat. no. 142504; BioLegend, Inc.); anti-CD69 PE (cat. no. 104508; BioLegend, Inc.); anti-CD69 APC (cat. no. 17-0691-82; eBioscience; Thermo Fisher Scientific, Inc.); anti-CD25 APC (cat. no. 17-0251-82; eBioscience; Thermo Fisher Scientific, Inc.); anti-FasL APC (cat. no. 17-5911-82; eBioscience; Thermo Fisher Scientific, Inc.); anti-B220 PerCP (cat. no. 45-0452-82; eBioscience; Thermo Fisher Scientific, Inc.); and anti-B220 PE-cy7 (cat. no. 25-0452-82; eBioscience; Thermo Fisher Scientific, Inc.). Annexin V-FITC and 7-aminoactinomycin D (7-AAD) PerCP (cat. no. 35-6410 KIT; Tonbo Biosciences, Inc.) were utilized for staining, performed according to the manufacturer's instructions. Cells were incubated with annexin V conjugate for 15 min at room temperature and 7-AAD staining solution was added and incubated at room temperature for 5 min before flow cytometric analysis. Stained cells were analyzed using BD FACSVerse (BD Biosciences). Data were analyzed using FlowJo software (version 10.6 for PC; Tree Star, Inc.).
Immunofluorescence
Frozen tissue sections (6 µm thick) were used for immunofluorescence assays. They were embedded in optimal cutting temperature compound (cat. no. 4583; Sakura Finetek), fixed with acetone cooled to 4˚C for 5 min, blocked with 5% donkey serum (cat. no. S9100; Solarbio Co., Ltd., China) at room temperature for 1 h and then stained with primary antibodies at room temperature for 1 h, namely anti-CD3 antibody (1:100 dilution; cat. no. ab33429; Abcam) and anti-CD138 antibody (10 µg/ml; cat. no. AF3190; R&D Systems), as previously described (27). Sections were visualized using donkey anti-goat IgG H&L (Alexa Fluor® 488) (1:200 dilution; cat. no. ab150129; Abcam) and donkey anti-rat IgG H&L (Alexa Fluor® 594) (1:200 dilution; cat. no. ab150156; Abcam) secondary antibodies at room temperature for 30 min. Immunofluorescence images were obtained and analyzed using ZEN Blue lite 2.3 software (Zeiss GmbH). Representative imaging data were obtained at identical settings with the ZEN Blue lite software and all assays included negative controls, where the primary antibodies were omitted (Fig. S1A).
Polymerase chain reaction (PCR)
To detect the genotype of the fas gene in MRL/lpr mice, total genomic DNA was extracted from the kidney tissue from all the eight MRL/MPJ and eight MRL/lpr mice using the TIANamp Genomic DNA Kit (cat. no. DP304; Tiangen Biotech Co., Ltd.) according to the manufacturer's instructions. The PCR containing 1.1X CataAmp Taq Plus PCR Mix (cat. no. C105; Beijing Catascis Biotech. Co. Ltd.) was performed according to genotyping protocols provided by the Jackson Laboratory (Jax Lab). The primer sequences for PCR were as follows: olMR1678/olMR1680 forward, 5'-GTAAATAATTGTGCTTCGTCAG-3'; olMR1678 reverse, 5'-TAGAAAGGTGCACGGGTGTG-3'; and olMR1680 reverse, 5'-CAAATCTAGGCATTAACAGTG-3'. The PCR protocol was executed as follows: 94˚C for 3 min for initial denaturation, then 94˚C for 10 sec for denaturation, 65˚C (initially, then decreasing by 0.5˚C per cycle) for 10 sec for annealing and 68˚C for 30 sec for extension, repeated for 10 cycles, next 72˚C for 10 sec, 60˚C for 10 sec and 72˚C for 30 sec for 28 cycles. This was followed by 72˚C for 60 sec for final extension and a hold at 10˚C. The PCR products were electrophoresed in a 3% agarose gel (cat. no. 1110GR100; BioFroxx) at a voltage of 80V. The agarose gel was stained with GelRed for 15 min. An image of the agarose gel was then obtained by Amersham ImageQuant 800 (Amersham Pharmacia Biotech, Inc.). The amplified products were analyzed and revealed a fragment length of 217 bp for the mutant and 179 bp for the wild-type (WT).
Statistical analysis
Data from all experiments are representative of 2-3 independent experiments showing reproducibility and are expressed as mean ± standard deviation. Data were analyzed using SPSS 17.0 (SPSS, Inc.). Comparisons between groups were performed using the Student's t-test, while comparisons among ≥3 groups were performed using one-way ANOVA followed by Tukey's post-hoc test. P<0.05 was considered to indicate a statistically significant difference.
Results
CD138 protein expression in T cells leads to defective apoptosis of T cells in MRL/lpr mice
Results of PCR showed all MRL/MPJ mice had homozygous WT fas genes and all MRL/lpr mice had homozygous mutant fas gene (Fig. S1B). It demonstrated that the MRL/lpr mice we used were fas-deficient and SLE murine models were absolutely established in the present study (Fig. S1B). CD138 protein expression levels in CD3+ T cells were negligible in splenocytes of MRL/MPJ mice, compared with the significant accumulation of abnormal (CD138+) T cells in the splenocytes of Fas-deficient MRL/lpr mice (Figs. 1A and S1C). In addition, it was demonstrated that CD138+ T cells infiltrated the kidneys of MRL/lpr mice, but is negligible in the kidneys of MRL/MPJ mice (Fig. 1B). CD138+ T cells in MRL/MPJ mice also had significantly higher levels of apoptosis, compared with MRL/lpr mice, which were found to have low levels (Fig. 1C). This indicated that CD138+ T cell apoptosis in mice was Fas-dependent and Fas deficiency in MRL/lpr mice led to the accumulation of CD138+ T cells. However, it was demonstrated that CD138+ T cells in the splenocytes of MRL/lpr mice were associated with a significant decrease in the number of apoptotic cells and a significant increase in the number of live cells compared with CD3+CD138- T cells (Fig. 1D).
CD69 and CD25 are markers of early and late active T cells (12). In the present study, it was demonstrated that CD138+ T cells in fresh splenocytes of MRL/lpr mice had a significant increase in CD69+ cell frequency with a simultaneous significant decrease in CD25+ cell frequency, compared with CD138- T cells in fresh splenocytes of MRL/lpr mice (Fig. 1E). Furthermore, CD138 protein expression significantly increased Fas ligand (FasL) protein expression in CD3+ T cells of MRL/lpr mice, compared with CD3- T cells (Fig. 1E). CD138 protein expression also significantly increased FasL protein expression in CD138+ DN T cells of MRL/lpr mice, compared with CD138- DN T cells (Fig. 1F). Therefore, it was concluded that CD138 protein expression in CD3+ T cells led to defective apoptosis of CD3+ T cells but prompted activation of CD3+ T cells in Fas-deficiency MRL/lpr mice.
CD4+ T cells and DN T cells express CD138 in MRL/lpr mice
The majority of CD138+ T cells in MRL/lpr mice were demonstrated to be CD4 and CD8 double negative T cells, with a small proportion of CD138+ T cells expressing CD4 and a negligible proportion of cells expressing CD8 (Fig. S1D). A significantly lower frequency of CD138+ cells was demonstrated in CD8+ T cells, compared with CD4+ T cells and DN T cells (Fig. 1G). There was also a significantly greater frequency of CD138+ cells in DN T cells than in CD4+ T cells (Fig. 1G). This indicated that the majority of CD138+ T cells in MRL/lpr mice were derived from CD138- DN T cells in MRL/lpr mice that had an increased accumulation of DN T cells. Additionally, it was demonstrated that CD4+ T cells in MRL/lpr mice were comprised of two subsets, CD4 hi T cells and CD4 int T cells (Fig. 1H). CD4 int T cells had reduced expression of CD4 but increased expression of B220, compared with CD4 hi T cells (Fig. 1H). CD4+CD138+ T cells simultaneously expressed B220 with a significant downregulation of CD4 protein expression compared with CD4+CD138- T cells (Fig. 1I and J).
CD138 protein expression improves defective activation of CD138- T cells in MRL/lpr mice
Multiple cytokines were found in the serum of MRL/lpr mice, of which interferon-γ, tumor necrosis factor (TNF), interleukin (IL)-6, IL-10, IL-17, and IL-2 were significantly increased compared with those in MRL/MPJ mice (Fig. 2A). However, there were no significant changes of IFN-α and IL-21 levels in the serum of MRL/lpr mice, compared with those in MRL/MPJ mice (Fig. 2A). This suggested that MRL/lpr mice were undergoing an inflammatory response (Fig. 2A). Unstimulated CD138- T cells derived from fresh splenocytes of MRL/lpr mice had a significantly increased frequency of CD69+ cells compared with that in CD138- T cells from fresh splenocytes of MRL/MPJ mice (Fig. 2B). However, the frequency of CD69+ cells in CD138- T cells of MRL/MPJ mice was significantly increased compared with that in CD138- T cells from MRL/lpr mice after 5 h of in vitro stimulation of the fresh splenocytes by PI (Fig. 2C). Furthermore, both CD69 and FasL protein expression levels in CD138- T cells were not significantly increased after in vitro stimulation of splenocytes with 2 or 4 h PI stimulation compared with the CD138- T cells without PI stimulation (Fig. 2D). These results demonstrated a defect in CD138- T cell activation in MRL/lpr mice.
CD138+ T cells from fresh splenocytes of MRL/lpr mice had significantly increased CD69+ cell frequencies (Figs. 1E and 2B) and FasL protein expression (Fig. 1E) compared with CD138- T cells. In addition, CD138+ T cells showed a significantly increased CD69+ cell frequency after in vitro 5 h PI stimulation, as well as significantly increased FasL protein expression compared with that in CD138- T cells in splenocytes from MRL/lpr mice (Fig. 2C and E). These results suggested that CD138 protein expression improved the defective activation of CD138- T cells in MRL/lpr mice.
PI stimulation may significantly prevent CD138+ T cell accumulation
Splenocytes from MRL/lpr mice were stimulated by PI in order to demonstrate changes in CD138+ T cell frequency. In vitro culture of unstimulated splenocytes from MRL/lpr mice for 4 h resulted in a significant increase in CD138+ T cell frequency and CD138 protein expression in CD138+ T cells, compared with that at 0 h (Fig. 3A and D). However, CD138+ T cell frequencies progressively and significantly reduced after 2 and 4 h PI stimulation (Fig. 3A). PI stimulation (both 2 and 4 h) also significantly reduced CD138+ cell frequencies in DN T cells and CD4+ T cells compared with the 4 h DN T cells and CD4+ T cells without PI stimulation (Fig. 3B and C). CD138 protein expression in CD138+ T cells was also significantly and progressively downregulated after 2 and 4 h of PI stimulation, compared with the 4 h DN T cells and CD4+ T cells without PI stimulation (Fig. 3D). These results demonstrated that PI stimulation prevented CD138+ T cell accumulation in splenocytes from MRL/lpr mice.
CD138 protein expression in T cells contributes to DN T cell accumulation in MRL/lpr mice
Four-hour PI stimulation significantly reduced CD138+ T cell frequency in splenocytes (Fig. 3A), as well as significantly reduced both CD3+ T cell and DN T cell frequencies in splenocytes from MRL/lpr mice (Fig. 3E). However, 4-h PI stimulation failed to decrease CD4+CD138- and CD8+ T cell frequencies in splenocytes, conversely significantly increasing CD8+ T cell frequencies (Fig. S1E). Splenocytes from MRL/lpr mice were then cultured without any stimulation for 24 h. CD138+ T cell frequency in splenocytes was significantly increased after 24 h, compared with at 0 h (Fig. 3F). Simultaneously, CD3+ T cell frequency and DN T cell accumulation were significantly increased at 24 h, compared with at 0 h (Fig. 3F). Conversely, 24-h in vitro culture without any stimulation significantly decreased the frequencies of CD4+CD138- and CD8+ T cells in splenocytes (Fig. S1F). These results demonstrated that CD138 protein expression significantly contributed to the increase in CD3+ T cell frequency and DN T cell accumulation in splenocytes from MRL/lpr mice.
Stimulation with PI induces specific apoptosis of CD138+ T cells
The mechanism by which PI stimulation prevented the accumulation of CD138+ T cells in MRL/lpr mice was assessed. Splenocytes stimulated with 4-h PI demonstrated a significantly increased number of apoptotic cells and a decreased frequency of live cells in CD138+ T cells, compared with controls (Fig. 4A). However, PI stimulation did not affect the number of apoptotic cells in CD3+CD138- T cells, compared with controls (Fig. 4A). Conversely, although not significantly, PI stimulation even increased the frequency of live cells, compared with the controls (Fig. 4A). Furthermore, PI stimulation significantly decreased apoptotic cell numbers and simultaneously and significantly increased live cell numbers in CD3-CD138+ plasma cells, compared with the controls (Fig. 4B). This indicated that PI stimulation specifically induced cellular apoptosis in CD3+CD138+ T cells, but not in CD3+CD138- T cells and CD3-CD138+ plasma cells. Moreover, the increased levels of apoptosis in CD138+ T cells induced by PI stimulation were not caused by the cytotoxic effect of PI (28,29).
Stimulation with PI promotes the activation of T cells with CD138 expression
PI stimulation was demonstrated to have significantly increased the activation levels of CD3+ T cells from MRL/lpr mice, with a significant increase in CD69+ cell frequency and FasL protein expression, compared with controls (Fig. 4C). CD4+ and DN T cells demonstrated significantly increased frequencies of CD69+ cells after 4-h PI stimulation, compared with controls (Fig. 4D). CD69+ cell frequency and FasL protein expression in CD138+ T cells from MRL/lpr mice were also significantly increased after 4 h of PI stimulation, compared with the CD138+ T cells without PI stimulation (Fig. 4E and F). Both CD138+ DN and CD4+CD138+ T cells demonstrated significantly increased FasL protein expression after 4-h PI stimulation, compared with controls (Fig. 4F). However, PI stimulation failed to significantly promote the activation of CD138- T cells in MRL/lpr mice (Fig. 2D). This indicated that PI stimulation resulted in increased apoptosis levels of CD138+ T cells, whilst simultaneously and significantly activating CD138+ T cells but not CD138- T cells in splenocytes from MRL/lpr mice.
The frequency of CD138+ T cells is inversely associated with the activation levels of CD138+ T cells
Fresh splenocytes were isolated and cultured from MRL/lpr mice without any stimulation. CD138+ T cell frequency significantly increased during the 48-h in vitro culture (Fig. 5A) and was accompanied by significantly reduced CD69+ cell frequency in CD3+ and CD138+ T cells, when compared with those at 0 h (Fig. 5B-D). Subsequently, the CD138+ T cell frequency significantly decreased (Fig. 5A) after 72 h of in vitro culture, accompanied by a significant increase in activation of CD3+ and CD138+ T cells, compared with that after 48 h (Fig. 5B-D). This indicated that CD138+ T cell frequency was inversely associated with the activation levels of CD3+ and CD138+ T cells in MRL/lpr mice, suggesting that PI stimulation may have promoted the activation of CD138+ T cells to induce specific apoptosis of CD138+ T cells, which prevented CD138+ T cell accumulation.
CD69 protein expression in CD138+ T cells results in a significant increase in apoptosis of CD138+ T cells
The authors hypothesized that the activation of CD138+ T cells, stimulated by PI, induced their apoptosis. CD69+ cells in CD138+ T cells of MRL/lpr mice were demonstrated to have had a significant increase in apoptosis levels compared with CD69- cells in CD138+ T cell populations (Fig. 5E). However, CD69 protein expression failed to significantly increase apoptosis levels of CD138- T cells in MRL/lpr mice (Fig. 5F). These results suggested that CD69 protein expression in CD138+ T cells may have promoted its specific apoptosis. The increase in specific apoptosis in CD138+ T cells was demonstrated to be due to the activation of CD138+ T cells via the increase in CD69 protein expression.
Discussion
The present study demonstrated that CD138+ T cell apoptosis in MRL/MPJ mice operates via a Fas-dependent pathway. CD138+ T cells were found to accumulate in MRL/lpr mice and CD138 protein expression in CD3+ T cells significantly prevented the apoptosis of CD3+ T cells. This contributed to the accumulation of CD3+ T and DN T cell subsets whilst simultaneously promoting CD3+ T cell activation in Fas-deficient MRL/lpr mice.
CD138 protein expression may also have increased FasL protein expression in DN T cells to promote their cytotoxicity. PI stimulation prevented CD138+ T cell accumulation and decreased CD138+ cell frequencies in DN and CD4+ T cells by inducing specific apoptosis of CD138+ T cells. The specific apoptosis was caused by the activation of CD138+ T cells via an increase in CD69 protein expression.
Fas (CD95) is a member of the TNF receptor family and interacts with FasL after TCR activation to induce apoptosis (16). Fas deficiency leads to DN T cell accumulation in MRL/lpr mice resulting in lymphadenectasis and splenomegaly (30,31). Results from the present study demonstrated that apoptosis of CD138+ T cells was via a Fas-dependent pathway. A previous study reported that Fas deficiency in MRL/lpr mice led to the accumulation of CD138+ T cells as they aged and lupus developed (12). Moreover, the majority of CD138+ T cells were demonstrated to be CD4- and CD8- DN T cells in the present study. DN T cells had been commonly regarded as the abnormal T cells closely related to SLE. However, most of the DN T cells are also the CD138+ T cells. It is interesting that these two subjects are related to each other and suggests CD138 expression in T cells would implicate in SLE.
CD138 protein expression was demonstrated to have significantly increased FasL protein expression in CD3+ T cells and their subsets, including DN T cells. However, DN T cells in MRL/lpr mice were reported to upregulate FasL and be strongly cytotoxic. This results in autoimmune injuries to multiple tissues and organs that express small amounts of the Fas receptor (6,16). In the present study, however, the results showed CD138 protein expression significantly increased the cytotoxicity of DN T cells, which in turn promotes lupus development and tissue injury in MRL/lpr mice.
The increase in CD138+ T cells significantly decreased the number of apoptotic cells in the present study, whilst simultaneously increasing the number of live cells compared with CD138- T cells. This demonstrated that CD138+ T cells had defective apoptosis in MRL/lpr mice. Moreover, previous studies reported that CD138+ T cells had a lower proliferation compared to CD138- T cell subsets (12,14). Based on these results, it was concluded that CD138+ T cell accumulation was mainly a result of their defective apoptosis. CD138 protein expression significantly contributed to the accumulation of T and DN T cells in MRL/lpr mice by reducing the number of apoptotic T and DN T cells.
Autoantibody production has a detrimental effect on multiple organs and serves a key role in SLE progression (32). Immature T cells undergo both positive and negative selection to become mature single positive T cells. During this process, T cells that recognize self-antigens are eliminated (33,34). Autoreactive T cells are usually eliminated by Fas-mediated apoptosis during negative selection in the thymus (35). Fas deficiency results in autoreactive T cells escaping negative selection (36,37). CD138+ T cells commonly express B220, which is also expressed on autoreactive T cells, such as non-selected CD8+ and DN T cells (35,38). CD4+CD138+ T cells were reported to be important for the generation of autoantibodies, such as anti-dsDNA and anti-smooth muscle antibodies (12). CD138+ T cells were also reported to promote tissue injury when self-antigens are exposed to the immune system (7,12). Results from the present study demonstrated that CD138+ T cells were autoreactive T cells that escaped Fas-dependent apoptosis during negative selection (12,14).
The present study demonstrated that CD138 protein expression in T cells served a key role in the progression of lupus in MRL/lpr mice. In addition to the significant decrease in apoptosis levels, CD138+ T cells were demonstrated to be activated more easily than CD138- T cells. This indicated that CD138 protein expression could promote the activation of CD138- T cells in MRL/lpr mice. However, the mechanism by which CD138 protein is expressed in these abnormal T cells was not elucidated. The results from the present study demonstrated that CD138- T cells in MRL/MPJ mice were more easily activated compared to CD138- T cells in MRL/lpr mice. PI stimulation failed to significantly activate CD3+CD138- T cells in MRL/lpr mice. These results indicated that CD138- T cells in MRL/lpr mice had defective activation. However, CD138 protein expression significantly reversed the defective activation of CD3+CD138- T cells and promoted the activation of CD3+ T cells in MRL/lpr mice.
PI stimulation could also significantly decrease CD138 protein expression in CD138+ T cells. PI stimulation has been used to activate T cells and promote cytokines secretion in T cells (22,23). However, in the present study, PI stimulation was able to prevent CD138+ T cell accumulation by inducing specific apoptosis of CD138+ T cells. Furthermore, PI stimulation could significantly reduce the frequency of CD138+ cells in the CD3+ T cell population and its cell subsets by increasing the activation levels of CD3+ T and CD138+ T cells. A previous study demonstrated that TCR activation downregulated CD138 protein expression in DN T cells (14). The results from the present study indicated that CD138 protein expression in CD3+ T cells could be prevented by PI stimulation to activate CD3+ T cells.
CD69 and CD25 are broadly known as markers for early and late active T cells (12). Results from the present study demonstrated that CD138+ T cells had a high level of CD69 protein expression and low levels of CD25 protein expression. In addition, it was demonstrated that CD69+ cells in the CD138+ T cell population had a significant increase in apoptosis levels compared to CD69- cells in the CD138+ T cell population. However, both PI stimulation and TCR activation could significantly activate T cells and subsequently increase the number of CD69+ cells in the T cell population (22,39). The present study demonstrated that the increased apoptosis of CD138+ T cells and subsequent decreased accumulation of CD138+ T cells were a result of the activation of CD138+ T cells via the increase of CD69 protein expression.
A previous study also reported that CD138+ T cells were autoreactive T cells that promoted autoantibody production when self-antigens were exposed to the immune system (12). The results from the present study demonstrated that CD138+ T cell frequency was inversely associated with the activation levels of CD3+ T and CD138+ T cells. CD3+CD138- T cells in MRL/lpr mice had defective activation, whilst CD138 protein expression significantly increased the activation of CD3+CD138- T cells. PI stimulation activated CD3+ T cells and simultaneously prevented CD138 protein expression in CD3+CD138- T cells. These results indicated that autoreactive CD3+CD138- T cells in MRL/lpr mice failed to be activated in the absence of self-antigen exposure, which may have promoted the upregulation of CD138 protein expression. CD138 protein expression in these T cells therefore promoted the accumulation of autoreactive T cells and the autoreactive response in lupus.
The present study provided a novel insight into the mechanism of autoreactive T cell promotion in the progression of SLE. However, the findings were based on murine SLE models. Clinical studies are required to substantiate these findings to determine the specific marker expressed in human autoreactive T cells that serves a key role in promoting SLE development. In addition, the critical proteins that regulate CD138 protein expression in autoreactive T cells have remained elusive. In the future, more research needs to be conducted to explore the underlying mechanisms of the critical proteins regulating CD138 protein expression in autoreactive T cells.
In conclusion, CD138 protein expression in T cells was implicated in the progression of lupus in MRL/lpr mice. CD138 protein expression in CD3+ T cells prevented apoptosis of T cells in MRL/lpr mice, simultaneously contributed to CD3+ and DN T cell accumulation and promoted CD3+ T cell activation. CD138 protein expression in DN T cells significantly increased their FasL protein expression, enhancing the cytotoxicity of the DN T cells. Defective apoptosis, induced by CD138 protein expression in CD3+ T cells, resulted in CD138+ T cell accumulation in the spleens of MRL/lpr mice. Results from the present study indicated that activation of T cells prevented CD138+ T cell accumulation by increasing the specific apoptosis of CD138+ T cells.
Furthermore, the results demonstrated that CD138- T cells in MRL/lpr mice had defective activation. Increased activation levels of CD3+ T cells could prevent CD138 protein expression in CD3+ T cells of MRL/lpr mice. The results also suggested that CD138 protein expression in CD3+ T cells of Fas-deficiency MRL/lpr mice may be caused by the failure of activation in autoreactive T cells before self-antigen exposure to the immune system.
Supplementary Material
(A) Frozen kidney sections were stained with or without CD3 (red), or CD138 (green) and DAPI (blue) for negative control of Fig. 1B, where primary antibodies were omitted for immunofluorescence measurement (original magnification, x200). (B) Image of agarose gels showing the fragment length denoting the genotype of the fas gene in all of the MRL/MPJ and MRL/lpr mice used in the present study; mutant: 217 bp, WT: 179 bp. (C) Negative control for flow cytometry analyses of CD138+ T cells for figures in the main text. Abnormal T cells express both CD3 and CD138. These results demonstrate that CD138 protein expression in CD3+ T cells in flow cytometry was not the result of fluorescence artifacts or due to PE-cy7 degradation. (D) Flow cytometry analyses showing double negative, CD4+ and CD8+ T cell frequencies in CD138+ T cells of fresh splenocytes from MRL/lpr mice that were cultured in vitro for 0, 48 and 72 h, respectively. (E) Bar charts demonstrating CD4+ and CD8+ T cell frequencies in splenocytes from MRL/lpr mice with or without 4 h in vitro ionomycin stimulation. (F) Bar charts demonstrating CD4+ and CD8+ T cell frequencies in splenocytes from MRL/lpr mice that were cultured in vitro for 0 and 24 h without stimulation. n=4-5 per group/experiment. *P<0.05 and **P<0.01 by one-way analysis of variance. MRL/lpr, Murphy Roths Large lymphoproliferative; PI, ionomycin; FMO, fluorescence minus one.
Acknowledgements
Not applicable.
Funding
Funding: The present study was funded by the Beijing Postdoctoral Research Foundation (grant no. ZZ2019-23) and the MiaoPu Research Foundation of the Beijing Institute of Chinese Medicine (grant no. MP-2020-45).
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Authors' contributions
TX conceived the study and wrote the manuscript. TX and PL designed the experiments. TX and XL performed the laboratory work. TX and XL performed the data analysis. PL revised and edited the manuscript. All authors read and approved the final version of the manuscript. TX and XL confirm the authenticity of all the raw data.
Ethics approval and consent to participate
All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of the Beijing Institute of Chinese Medicine (approval no. 2021040202 and were performed according to institutional ethical guidelines.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
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