Cistanche Improves Kidney Infection Due To Systemic Lupus Erythematosus
Mar 13, 2022
Nuclear antigen–reactive CD4+ cells expand in active systemic lupus erythematosus, produce effector cytokines, and infect the kidneys
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Dimas Abdirama , Sebastian Tesch and etc
Systemic lupus erythematosus is a systemic and chronic autoimmune disease characterized by a loss of tolerance towards nuclear antigens with autoreactive CD4+ T cells implicated in disease pathogenesis. However, very little is known about their receptor specificity since the detection of human autoantigen-specific CD4+ T cells has been extremely challenging. Here we present an analysis of CD4+ T cells reactive to nuclear antigens using two complementary methods: T cell libraries and antigen-reactive T cell enrichment. The frequencies of nuclear antigen-specific CD4+ T cells correlated with disease severity. These autoreactive T cells produce effector cytokines such as interferon-g, interleukin-17, and interleukin-10. Compared to blood, these cells were enriched in the urine of patients with active lupus nephritis, suggesting an infiltration of the infected kidneys. Thus, these previously unrecognized characteristics support a role for nuclear antigen-specific A CD4+ T cells in systemic lupus erythematosus.
Copyright ª 2020, International Society of Nephrology. Published by Elsevier Inc. This is an open-access article under the CC BY-NC-ND license
KEYWORDS: lupus erythematosus, kidney,
Systemic lupus erythematosus is a systemic and chronic autoimmune disease characterized by loss of tolerance toward nuclear antigens due to defective disposal of biological waste such as apoptotic material containing ribonucleoproteins and nucleosomes.1 Autoantibody directed against nuclear antigens is characteristic of systemic lupus erythematosus and usually occurs before the overt disease. In addition, autoreactive CD4+ T cells are implicated in the pathogenesis of systemic lupus erythematosus by promoting autoantibody production by B cells and directly propagating organ damage in infected target organs. Although the detection of nuclear antigen–reactive T cells has been reported previously, the evidence for their existence remains circumferential until now and it is presently unclear how they interplay in the pathogenesis. Understanding how these cells participate in autoimmunity will be critical to the design of effective treatment.
The detection of autoantigen-specific T cells is hindered by their extraordinarily low frequencies in circulating blood. Previous studies of human naive CD4+ T-cell repertoires have used tetramer-based technology to effectively provide precise information on T-cell responses to specific antigen epitopes; however, this method requires prior knowledge of the human leukocyte antigen types of the donor. Alternative techniques to simultaneously detect and enumerate rare T-cell populations with reactivity to different antigens without prior knowledge of human leukocyte antigen types were developed by using libraries of amplified polyclonal T cells and by enrichment of CD154-expressing T cells after stimulation with an antigen termed ARTE (antigen-reactive Tcell enrichment).
Systemic lupus erythematosus is harmful to kidneys, but cistanche can improve kidney functions.
CD4+ T cells mainly exert their function through secretion of cytokines upon antigenic activation16 and by propagating tissue inflammation such as in lupus nephritis which represents one of the most serious complications of systemic lupus erythematosus. Analysis of the T-cell repertoire present in kidney biopsies from patients with lupus nephritis revealed oligoclonality of kidney-infiltrating CD4+ T cells, indicating accumulation of antigen-specific T cells in infected kidneys. Our previous study revealed urinary T cells in patients with active lupus nephritis as a precise biomarker that resembles the phenotype of intrarenal cells. Although these cells are enriched for CXC chemokine receptor 3 and CC chemokine receptor 5 expressing T cells indicative of a T helper cell 1 (Th1) cell population and contain CD154+ T cells suggestive of recent antigen encounter, their antigen specificity toward nuclear antigens remains unclear.
In this study, we present an analysis of CD4+ T cells reactive to nuclear antigens in a total of 17 healthy individuals, 12 subjects with inactive systemic lupus erythematosus, and 20 subjects with active systemic lupus erythematosus by using T-cell libraries and ARTE techniques to analyze the frequencies of autoreactive CD4+ T cells in peripheral blood and urine of patients with lupus nephritis as well as the cytokine production.
RESULTS
Nuclear antigen–reactive CD4+ T cells are expanded inactive systemic lupus erythematosus as determined using the T-cell library and ARTE
with active systemic lupus erythematosus. These cells were also detectable in 5 of 6 patients with inactive systemic lupus erythematosus, while only 3 of 6 healthy controls presented circulating autoreactive T cells. Interestingly, patients with active systemic lupus erythematosus also showed a broader variety of autoreactive T-cell responses, with each patient having reactive T cells against at least 3 nuclear antigens. In comparison, the target portfolio was less variable in patients with inactive systemic lupus erythematosus and healthy controls. Taken together, circulating nuclear antigen–reactive T cells are not exclusive for systemic lupus erythematosus, yet inactive systemic lupus erythematosus, their number is greater and their target structures are more variable.
Because autoreactive T cells play a role in propagating autoimmune responses, we compared the number of autoreactive T cells with disease severity, assessed by the systemic lupus erythematosus disease activity index (systemic lupus erythematosus DAI) score. Only the frequencies of CD4+ T cells reactive to RNP70, Ro, and La positively correlated with systemic lupus erythematosus DAI (RNP70: P ¼ 0.04, r ¼ 0.4865; Ro: P ¼ 0.005, r ¼ 0.6299; La: P ¼ 0.01, r ¼ 0.5651) (Figure 1d). In contrast, the frequency of T cells reactive to transthyretin was found at a fairly similar level across all 3 groups (Figure 1e), thus dismissing a possible experimental bias due to general non–antigen-specific T-cell hyperreactivity inactive systemic lupus erythematosus.
T-cell libraries offer a series of advantages including high sensitivity to detect very rare cells within a low number of input cells. However, this technique relies on the expansion of cells, which may alter the cell composition and provides only a limited possibility to phenotypically characterize the reactive cells. Therefore, we validated our observations with the ARTE method. The ARTE protocol requires larger amounts of input cells, which is especially challenging in patients with systemic lupus erythematosus who often present themselves with lymphopenia and anemia. Because of this limitation and also to increase the expected readout of reactive cells, we pooled all 5 canonical nuclear antigens and used this pool to stimulate peripheral blood mononuclear cells (PBMCs) before enrichment of CD154-expressing cells (n ¼ 15 patients with systemic lupus erythematosus; n ¼ 6 healthy controls). Using a combination of the surface markers CD154 and CD69, T cells reactive to systemic lupus erythematosus-associated nuclear antigens could be identified within a background population (Figure 1f). Although background expression of CD154 and CD69 was abundantly observed in all individuals, patients with active systemic lupus erythematosus had significantly increased frequencies of CD154+CD69+ T cells when stimulated with nuclear antigens compared to background stimulation (P ¼ 0.008), whereas healthy individuals and patients with inactive systemic lupus erythematosus had similar frequencies between antigen-stimulated and nonstimulated cells (healthy individuals: P ¼ 0.44; patients with inactive systemic lupus erythematosus: P ¼ 0.56) (Figure 1g). Furthermore, by subtracting the background frequencies, the frequencies of nuclear antigen– reactive T cells were higher in patients with active disease than in healthy individuals and patients with inactive disease (P ¼ 0.02 and P ¼ 0.11, respectively) (Figure 1h). The frequency of background-subtracted nuclear antigen–reactive T cells thereby positively correlated with disease activity (P ¼ 0.004, r ¼ 0.5978), suggesting their potential role in systemic lupus erythematosus.
T-cell libraries offer a series of advantages including high sensitivity to detect very rare cells within a low number of input cells. However, this technique relies on the expansion of cells, which may alter the cell composition and provides only a limited possibility to phenotypically characterize the reactive cells. Therefore, we validated our observations with the ARTE method. The ARTE protocol requires larger amounts of input cells, which is especially challenging in patients with systemic lupus erythematosus who often present themselves with lymphopenia and anemia. Because of this limitation and also to increase the expected readout of reactive cells, we pooled all 5 canonical nuclear antigens and used this pool to stimulate peripheral blood mononuclear cells (PBMCs) before enrichment of CD154-expressing cells (n ¼ 15 patients with systemic lupus erythematosus; n ¼ 6 healthy controls). Using a combination of the surface markers CD154 and CD69, T cells reactive to systemic lupus erythematosus-associated nuclear antigens could be identified within a background population (Figure 1f). Although background expression of CD154 and CD69 was abundantly observed in all individuals, patients with active systemic lupus erythematosus had significantly increased frequencies of CD154+CD69+ T cells when stimulated with nuclear antigens compared to background stimulation (P ¼ 0.008), whereas healthy individuals and patients with inactive systemic lupus erythematosus had similar frequencies between antigen-stimulated and nonstimulated cells (healthy individuals: P ¼ 0.44; patients with inactive systemic lupus erythematosus: P ¼ 0.56) (Figure 1g). Furthermore, by subtracting the background frequencies, the frequencies of nuclear antigen– reactive T cells were higher in patients with active disease than in healthy individuals and patients with inactive disease (P ¼ 0.02 and P ¼ 0.11, respectively) (Figure 1h). The frequency of background-subtracted nuclear antigen–reactive T cells thereby positively correlated with disease activity (P ¼ 0.004, r ¼ 0.5978), suggesting their potential role in systemic lupus erythematosus pathogenesis (Figure 1i). Moreover, CD4+ T cells reactive to a recall antigen Candida albicans MP65 demonstrated similar frequencies across the donors (Figure 1j), excluding the possibility of a general bias toward higher frequencies of antigen-reactive T cells in patients with active systemic lupus erythematosus.
Collectively, our data generated using the T-cell library and ARTE methods show expansion of autoantigen-specific CD4+T cells in patients with active systemic lupus erythematosus. Both techniques are comparable as indicated by similar median stimulation index values between antigen-stimulated and nonstimulated T-cell responses (median stimulation index-T-cell library: 1.857; ARTE: 1.614; P ¼ 0.33), although the ARTE technique revealed higher frequencies of nuclear antigen–reactive CD4+ T cells than did the T-cell library technique (Figure 1k).
Cistanche can improve the infection of the kidney.
Nuclear antigen–reactive T cells in healthy individuals are bona fife autoreactive CD4+ T cells
Both T-cell library and ARTE techniques demonstrated the detection of autoreactive T cells in healthy individuals, which represent a possible origin for the development of human autoimmunity. However, their number was not significantly different from the background frequencies, leaving it uncertain whether healthy subjects indeed have to circulate nuclear antigen–reactive CD4+T cells. To unravel the antigen-specificity of autoreactive T cells in healthy subjects, we generated single T-cell clones from CD154+CD69+ CD4+Tcell precursors isolated from healthy individuals after stimulation of PBMCs with individual nuclear antigens and a control antigen derived from Aspergillus fumigatus lysate. A considerable number of systemic lupus erythematosus-associated autoantigen-specific T-cell clones responded to their corresponding antigens after antigen restimulation as highlighted by the coexpression of IFN-g with CD154 (number of specific clones—Aspergillus fumigatus: 5 of 5; SmD1: 4 of 8; RNP70: 3 of 11; histone: 5 of 5; Ro: 4 of 9; La: 5 of 7) (Supplementary Figure S2A and B). Moreover, the antigen response (representatively shown for SmD1) was dose-dependent, emphasizing that nuclear antigen–reactive T cells in healthy individuals are bona fife autoreactive CD4+ T cells (Supplementary Figure S2C). Thus, we confirmed the existence of nuclear antigen–reactive T cells in healthy subjects within the background signal of the applied detection methods.
Cytokine production of nuclear antigen–reactive CD4+ T cells
We next examined the cytokine production of nuclear antigen–reactive CD4+ T cells. CD154-expressing cells were stained intracellularly for effector cytokines such as IFN-g, interleukin (IL)-17, IL-4, and IL-10 after stimulation with a pool of systemic lupus erythematosus-associated nuclear antigens (Figure 2a). Frequencies of IFN-g–, IL-17–, and IL-10–producing CD154+CD4+ T cells were significantly increased in patients with active systemic lupus erythematosus (all cytokines: P ¼ 0.004), whereas the frequency of IL-4–producing autoreactive T cells remained indistinguishable from the background frequency (P ¼ 0.125) (Figure 2b). Moreover, absolute numbers of IFN-g– and IL-10–producing autoreactive T cells were higher in patients with active systemic lupus erythematosus than in healthy individuals and patients with inactive systemic lupus erythematosus (Figure 2b). By subtracting the background frequencies, patients with active systemic lupus erythematosus had significantly higher frequencies of IFN-g– and IL-10–producing autoreactive T cells than did healthy individuals (IFN-g: P ¼ 0.003; IL-10: P ¼ 0.004) as well as of IFN-g–, IL-17–, and IL-10–producing autoreactive T cells than did patients with inactive systemic lupus erythematosus (IFN-g: P ¼ 0.002; IL-17: P ¼ 0.002; IL-10: P ¼ 0.005) (Figure 2c). In addition, the frequency of IFN-g– and IL-10– but not of IL-17– and IL-4–producing nuclear antigen– reactive T cells correlated with disease activity. However, only the frequency of IL-4–producing cells showed less correlation with disease activity (IFN-g: P < 0.0001, r ¼ 0.7574; IL-17: P ¼ 0.005, r ¼ 0.5889; IL-4: P ¼ 0.14, r ¼ 0.3717; IL- 10: P ¼ 0.0004, r ¼ 0.6977) (Figure 2d). Judged by the frequency of IFN-g producers, antigen-reactive CD4+ T cells were mainly of the Th1 lineage. In contrast, IL-17, as well as IL-10 producers, were detected only at lower frequencies.26 Compared to autoantibody levels, the frequencies of cytokine-producing CD4+ T cells did not correlate with the concentration of anti-double-stranded DNA and with the serum titer of antinuclear antibodies (Supplementary Figure S3A and B). No other correlation between nuclear antigen–reactive CD4+ T cells and autoantibodies was observed except for La-reactive CD4+ T cells toward La autoantibodies (Supplementary Figure S3C).
Detection of nuclear antigen–reactive CD4+ T cells in the urine of patients with active systemic lupus erythematosus and lupus nephritis
The population of nuclear antigen–reactive Th1 cells is suspected to invade the kidney tissue, where they might encounter their almost omnipresent cognate antigen. By investigating the T-cell receptor (TCR)-b repertoire of CD4+T cells isolated from peripheral blood compared with those isolated from the urine of 5 patients with systemic lupus erythematosus and active lupus nephritis, we observed an oligoclonal skewed TCR repertoire of urine-derived cells (Figure 3a). The cumulative frequency of the 20 most abundant T-cell clones was significantly higher in urine corresponding to a lower diversity index (Supplementary Figure S4A). To test whether these cells also demonstrate reactivity to a set of systemic lupus erythematosus-associated nuclear antigens, we generated libraries consisting of urinary CD4+ T cells isolated from 3 patients with systemic lupus erythematosus and active lupus nephritis (Supplementary Figure S4B). Urinary CD4+ T cells were metabolically and functionally active cells indicated by the ability to proliferate upon stimulation with SEB but with signs of exhaustion shown by a reduced proliferation capacity after polyclonal activation (Supplementary Figure S4C and D). Because of these circumstances, only microcultures with a SEB stimulation index of >4 were included for the enumeration of urinary CD4+T cells specific to the individual nuclear antigen. In parallel to urinary CD4+T cells, the frequency of nuclear antigen–reactive CD4+T cells in peripheral blood was determined, allowing a direct comparison of the respective frequencies within the same individuals (Figure 3b). We found that inactive lupus nephritis, the frequencies of urinary nuclear antigen–reactive CD4+T cells were higher than those of peripheral blood. To confirm this observation, we labeled urinary cells isolated from 4 active lupus nephritis donors with carboxyfluorescein succinimidyl ester and mixed the labeled cells with donor-matched PBMCs to allow a direct comparison of antigen-reactive cells in 1 assay using the ARTE technique. The frequency of CD154+CD69+ T cells in stimulated and nonstimulated samples of donor-matched urinary and peripheral blood cells was used to assess whether nuclear antigen–reactive T cells are enriched in urine as a proxy for the kidney by calculating the stimulation index (Figure 3c). Although the urinary T-cell library and ARTE methods differed in their median stimulation index to the nuclear antigens (stimulation index—urinary T-cell library: 17; ARTE: 2.79; P ¼ 0.4) (Figure 3d), analysis of autoreactivity in peripheral and urinary CD4+ T cells from both methods (in total n ¼ 7) demonstrated an increased nuclear antigen-specific stimulation index in urine (P ¼ 0.0156) (Figure 3e). These observations suggest that nuclear antigen–reactive CD4+T cells invade and accumulate in the infected kidneys. Thus, local tissue inflammation in systemic lupus erythematosus might be an antigen-specific phenomenon and directly modulated by the infiltrating autoreactive T cells.
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