The 8–10–week-old MRL/lpr (stock # 000485) and MRL/+ (stock # 000486) mice were purchased from The Jackson Laboratories (Bar Harbor, ME, USA). Female mice were used unless otherwise specified. NPSLE manifestations are absent in the congenic MRL/+ strain and more prominent in female than in male MRL/lpr mice (18, 19), and CP infiltrating T cells were found to be rare or diminished in the non-autoimmune control MRL/+ strain and in age matched male MRL/lpr mice, respectively (see below). Hence, MRL/+ or male MRL/lpr mice were used as controls in some experiments. Mice were housed in the animal facility of Albert Einstein College of Medicine until they were 16–18 weeks of age, at which time the MRL/lpr strain exhibits a profound neurobehavioral phenotype including cognitive deficits and depressive like behavior (20–22). All animal studies were performed under protocols approved by the Institutional Animal Care and Use Committee of the Albert Einstein College of Medicine.

Spleens and brains were harvested from mice after transcardial perfusion with ice cold HBSS (Cellgro, Manassas, VA, USA). Single cell suspensions of spleens were prepared by mechanical disruption, and residual red blood cells were lysed using ACK lysis buffer (Quality Biologicals, Gaithersburg, MD, USA) for 5 min at room temperature. The CP was isolated from the brain by careful dissection and the tissue was dissociated in 0.25% trypsin–2.21 mM EDTA (Cellgro) for 30 min at 37°C. Cells were washed twice with ice cold HBSS supplemented with 2% heat inactivated fetal bovine serum (GIBCO, Auckland, New Zealand) and then used for downstream applications.

Brain tissue devoid of CP [ex-choroid plexus (ex-CP)] was dissociated in a digestion buffer containing Liberase TL (3.25 U/ml; Sigma, St. Louis, MO, USA), DNase I (0.1 mg/ml; Sigma), and BSA (1%; Sigma) in HBSS (with Ca²⁺ and Mg²⁺; GIBCO) for 30 min at 37°C. EDTA (1 mM; Sigma) was added to the solution and the cell suspension was filtered through a 40 μm filter (BD, San Diego, CA, USA) and centrifuged at 1,500 rpm for 15 min at 4°C. Isotonic Percoll (30%) (GE Healthcare, Uppsala, Sweden) was added to the pellet, and the suspension carefully layered onto 70% of isotonic Percoll. The gradient was centrifuged for 30 min at 20°C and the cells at the 70–30% interphase were collected, washed, and used for downstream applications.

Formalin fixed paraffin embedded sections were deparaffinized in xylene and rehydrated in graded ethanol concentrations. Sections were blocked in 20% normal horse serum in PBS and incubated in 1:100 rat antimouse CD4 in 2% normal horse serum in PBS (eBioscience) overnight at room temperature. Sections were washed in PBS followed by staining with secondary donkey antirat for 1 h at room temperature. After washing, sections were counter stained with DAPI and mounted with fluoromount-G. Slides were then visualized with a Thermo Scientific EvosFL Auto 2.

For surface staining, Fc receptors (FcRs) were blocked using an antimouse CD16/32 antibody (BD) for 15 min on ice, and standard multiparameter flow cytometric analyzes was performed by staining cells with the antibodies listed in Table 1. Data were collected using FACS Diva software on a BD LSR II analyzer (BD), and analyzed using FlowJo_V10 software (TreeStar, Ashland, OR, USA). Doublet discrimination was performed and viable cells were analyzed using either 7AAD (Invitrogen, Carlsbad, CA, USA) or the fixable viability dye (Biolegend, San Diego, CA, USA) exclusion method. For intracellular cytokine staining, cells were initially stimulated with a cell activation cocktail (Biolegend) (6 h, 37°C, 5% CO₂) and staining for IFN-γ, IL-21, IL-17, and IL-4 was performed using the BD CytoFix/Perm Kit following the manufacturer’s instructions. Staining for intracellular IL-21 was done using an IL-21R/Fc chimera (R&D Systems) and PE-conjugated F(ab′)2 fragment of goat anti–human Fcγ antibody (anti-Fc PE; Jackson ImmunoResearch, West Grove, PA, USA). For all intracellular staining, unstained cells (permeabilized as well as non-permeabilized) as well as fluorescence-minus-one were used to set gates and as negative controls. Staining for transcription factors including FoxP3, Tbet, and Bcl6 was done using the FoxP3 staining kit from eBiosciences (ThermoFisher, Waltham, MA, USA).

Analysis of proliferating CD4⁺ T cells was done using Ki-67 staining, with minor modifications. Briefly, cells were surface stained with an anti-mouse CD4 antibody and fixed by adding 70–80% ice-cold ethanol (dropwise with continuous vortexing). Cells were incubated at −20°C overnight, washed, and stained with either PE-labeled Ki-67 antibody or isotype control IgG1κ antibody (BD). Cells were incubated for 30 min in the dark at room temperature, washed, and analyzed by flow cytometry.

CD4⁺ T cells were sorted from spleens and CP in complete RPMI media on a BD FACS Aria. Sorted cells were counted and washed in complete RPMI media before plating (in duplicates or triplicates) at a density of 5 × 10⁴ − 2 × 10⁵ cells per well in a 96-well plate precoated with anti-CD3 (5 µg/ml; BD). Cells were suspended in complete RPMI-1640 medium (GE Healthcare Life Sciences, Logan, UT, USA) supplemented with FBS (10%), sodium pyruvate (Cellgro), MEM non-essential amino acids (Hyclone), penicillin–streptomycin (Cellgro), and 2-mercaptoethanol (55 µM; GIBCO). Anti-CD28 antibody (2.5 µg/ml; BD) was added to the cultures and incubated for 48 h at 37°C in the presence of 5% CO₂. Appropriate unstimulated controls were included as well. Supernatants were collected and analyzed for cytokine levels, whereas cells were harvested and stained for the T cell activation markers listed in Table 1.

Measurement of T helper (Th) cytokines was done in the culture supernatants obtained from in vitro cultures using the LEGENDplex Mouse Th cytokine panel (Biolegend). Using this multiplex bead based array, soluble analytes were quantified in the supernatants following the manufacturer’s instructions.

Statistical analysis was done using GraphPad Prism 7 Software. Values in the figures are depicted as mean ± SEM of n observations, where n is detailed in the figure legends. p-Values of ≤0.05 were considered significant.

Female MRL/lpr mice at ~16 weeks of age exhibit profound neurobehavioral deficits, including depression-like behavior and cognitive (memory) abnormalities that model key manifestations of human disease. MRL/+, the congenic strain, do not have significant neurologic deficits (19, 21, 23–25). Therefore, we began these studies using the congenic non-autoimmune Fas-sufficient MRL/+ strain as an appropriate and commonly used control for the lupus prone MRL/lpr mice. To identify the cells infiltrating the brain of lupus mice which are associated with neuropsychiatric disease, CPs from the brains of transcardially perfused 16–18-week-old female MRL/lpr and MRL/+ control mice were isolated and stained for the presence of T lymphocytes. There was extensive lymphocytic infiltration of the CP at this age in the lupus prone MRL/lpr strain, which was absent in age- and sex-matched MRL/+ control mice (Figure 1A). As seen by immunohistochemical staining, many of the infiltrating cells were T lymphocytes (Figure 1A). T cells were rare in other regions of the brain (data not shown). We confirmed these observations by analysis of single cell suspensions. We found that there was a significant population of CD4⁺ and CD8⁺ T cells in the CP of MRL/lpr mice, which was almost absent in MRL/+ controls (Figures 1B,C). As compared to the DN (CD4⁻CD8⁻) phenotype of T cells expanding in lymphoid tissue and responsible for the profound lymphadenopathy and splenomegaly exhibited by MRL/lpr mice (26), T cells infiltrating the CP were preferentially CD4⁺ or CD8⁺ single positive cells. Of these, more than 70% cells were CD4⁺ T cells. Further analysis of brain infiltrating CD4⁺ T cells revealed that they had a functional effector phenotype. In the CP of MRL/lpr mice there was a substantial fraction of effector CD4⁺ T cells (CD44⁺CD62L⁻), followed by a small fraction of central memory CD4⁺ T cells (CD44⁺CD62L⁺) (Figure 1D). Moreover, few naive CD4⁺ T cells (CD44⁻CD62L⁺) were present (Figure 1D). A similar trend was seen with respect to the proportions of CD8⁺ effector, central memory, and naive T cells infiltrating the CP (Figures S1A,B in Supplementary Material). The presence of increased effector CD4⁺ and CD8⁺ T cells indicates abnormal T cell activity and signaling in the CP of MRL/lpr mice with neuropsychiatric manifestations. While CD8⁺ T cells have also been implicated in SLE (27, 28), the subsequent studies are focused on functional and phenotypic characterization of the more abundant CD4⁺ T cells infiltrating the brains of MRL/lpr mice.

Next, to investigate whether brain infiltrating CD4⁺ T cells were localized primarily in the CP or are present as well elsewhere in the brain, we digested brain tissue of MRL/lpr mice following the removal of the CP (i.e., ex-CP) and analyzed this tissue by flow cytometry. Interestingly, we found that brain tissue devoid of CP showed only rare CD3⁺CD4⁺ or CD3⁺CD8⁺ T cells, despite the presence of CD45⁺ cells (Figure 1E). Taken together, these results indicate that T lymphocytes present in the brain of MRL/lpr lupus mice with neuropsychiatric disease are primarily localized to the CP. In addition, the infiltrating T cells are either CD4⁺ or CD8⁺ T cells displaying an effector phenotype.

Next, we determined whether brain infiltrating CD4⁺ T cells in MRL/lpr mice have an activated phenotype. To address this, we sorted CD4⁺ T cells from CP tissue, and stained for the expression of T cell activation markers. We found that unstimulated brain derived CD4⁺ T cells from MRL/lpr mice had an enhanced activated phenotype as compared to MRL/+ controls, as evidenced by increased expression of CD25 and CD69 and decreased expression of CD62L (Figure 2A). Moreover, upon stimulation with anti-CD3 and anti-CD28, the expression of T cell activation markers in MRL/lpr mice was further significantly enhanced (Figure 2A). Thus, infiltrating CD4⁺ T cells from the CP of MRL/lpr mice have an inherently activated phenotype, which can be further enhanced by activating signals.

To further understand the functional potential of these T cells, we isolated CD4⁺ T cells from MRL/lpr CP, cultured them in vitro in the presence of anti-CD3 and anti-CD28 antibodies, and measured cytokine production. There was a significant increase in the production of IFN-γ and IL-2 in activated CD4⁺ isolated from MRL/lpr compared to those obtained from MRL/+ mice (Figure 2B). IL-4, IL-17, and IL-9 could not be detected. In addition, increased expression of TNF was also detected in MRL/lpr T cells, but at lower absolute concentrations (Figure 2B). These findings further support that brain infiltrating CD4⁺ T cells in MRL/lpr mice have a functionally activated phenotype.

Hyperactive lupus T cells have impaired tolerance due to altered intracellular signaling, leading to heightened proliferative potential (29). Therefore, we investigated whether the abnormal accumulation of brain infiltrating CD4⁺ T cells is due to enhanced proliferative capability. For examination of the proliferative potential, we stained CP CD4⁺ T cells for the expression of Ki67. To better display the findings in female MRL/lpr mice and further demonstrate the close association between the T cell phenotype and neuropsychiatric disease, the control strain used in the following experiments were male MRL/lpr mice, which display an attenuated neuropsychiatric phenotype (18). We found that besides being more extensively infiltrated by T cells, female MRL/lpr CP T cells showed significantly more expression of Ki67 as compared to male MRL/lpr mice (Figure 2C). This is consistent with our previous report that while male MRL/lpr mice do display a neuropsychiatric phenotype, it is delayed and less severe relative to female mice (18). It is important to emphasize that the expression of Ki67 was measured here without additional stimulation in vitro, supporting the notion that CP T cells in lupus receive substantial activation signals in vivo. To further correlate this observation with differences in the severity of systemic disease between female and male mice, we also compared Ki67 staining in spleen CD4⁺ T cells between the sexes and found similar results (Figure 2C). From these observations, we conclude that brain infiltrating CD4⁺ T cells in MRL/lpr mice not only have a functional activated phenotype, but also substantially increased proliferative capacity.

T cell signaling abnormalities account for the B cell hyperactivity that drives lupus manifestations (5). Th subsets such as Th1, Th17, and T_FH cells, secreting IFN-γ, IL-17, and IL-21, respectively, play an important role in the progression of SLE (30). To identify the specific Th subsets associated with neuropsychiatric lupus manifestations, we performed intracellular cytokine staining for IFN-γ (Th1), IL-17 (Th17), and IL-21(T_FH) on CD4⁺ T cells isolated from the CP of MRL/lpr and MRL/+ mice.

We found that the CD4⁺ T cells that infiltrate the brains of MRL/lpr mice contained significant populations of CD4⁺IFN-γ⁺ as well as CD4⁺IL-21⁺ cells (Figures 3A,B). In contrast, we could not identify any significant absolute numbers of IL-4 or IL-17 secreting CD4⁺ T cells in these mice (Figure S2 in Supplementary Material). Furthermore, as compared to MRL/+, MRL/lpr CP did not overexpress IL-4 or IL-17 by RNA-sequencing (data not shown). Therefore, we concluded that neuropsychiatric disease manifestations in MRL/lpr lupus mice are primarily associated with CP localization of IFN-γ and IL-21 positive CD4⁺ T cells. We further analyzed brain infiltrating CD4⁺ T cells by costaining for both IFN-γ and IL-21, and found that most cells were IL-21^hi/+, although IL-21^lo/− cells were present as well (Figure 3C). Interestingly, there were two distinct IL-21^hi/+ sub-populations of almost similar size, IL-21^hi/+ IFN-γ⁺ and IL-2^hi/+ IFN-γ⁻ (Figure 3C middle panel, 3D). These two subpopulations were also observed in IL-21^lo/− cells (Figure 3C right panel, 3D), but the absolute representation of both IL-21^lo/− IFN-γ⁺ and IL-21^lo/− IFN-γ⁻ T cells was small (Figure 3D). These findings lead us to conclude that neuropsychiatric manifestations are associated with a subset of CD4⁺ T cells that principally secretes IL-21 and IFN-γ.

Both IL-21 and IFN-γ have been implicated in lupus pathogenesis (31, 32). IL-21 is primarily secreted by T_FH cells that are phenotypically defined as CD4⁺ICOS⁺PD1⁺CXCR5⁺ (33). It has also been shown in inflammatory bowel disease that T_FH cells coproduce IFN-γ in addition to IL-21 (34). Moreover, excessive production of IFN-γ promotes accumulation of T_FH cells and the formation of germinal centers (35). Therefore, we investigated whether the CD4⁺ T cells infiltrating the brain of MRL/lpr mice have a T_FH phenotype. We found that a major subset of CP CD4⁺ T cells had upregulated ICOS (Figures 4A,B) and that CD4⁺ICOS⁺ cells also had significant expression of PD1 and CXCR5, thereby qualifying them as T_FH cells (Figures 4A,B).

To further confirm that neurological manifestations in MRL/lpr mice are accompanied by the accumulation of IFN-γ and IL-21 producing CD4⁺ T_FH cells, we analyzed the expression of these cytokines along with the signature T_FH markers. We found that CD4⁺ICOS⁺PD1⁺CXCR5⁺ cells had significant expression of IFN-γ and IL-21 (Figure 4C; Figure S3 in Supplementary Material). To further establish that these cells are bona-fide CD4⁺ T_FH cells and not Th1 cells, we studied the expression of the transcription factors Bcl6 and Tbet. We found that CD4⁺ICOS⁺PD1⁺CXCR5⁺ cells expressed Bcl6 as well as Tbet (Figure 4D). Although the difference between male and female mice was not significant, this experiment does clearly demonstrate a cell population expressing Bcl6 without Tbet coexpression infiltrating the CP of MRL/lpr mice. The prominent T_FH phenotype present in the CP of MRL/lpr mice was supported by RNA-sequencing studies, which demonstrated highly significant differential expression of IL-21, ICOS, PD1, and CXCR5 (data not shown). We also studied GATA3 and RORγt, other key transcription factors in T cell subsets, but these were negative, both by intracellular staining and by RNA-sequencing (data not shown). These observations lead to a conclusion that a major subset of CP infiltrating T cells are IFN-γ and IL-21 secreting CD4⁺ T_FH cells.

Systemic lupus erythematosus has been linked to abnormally downregulated regulatory control that can be attributed to reduced percentages of regulatory T cells (Tregs) (36). Tregs are classically defined as CD4⁺CD127⁻CD25⁺FoxP3⁺ cells that can secrete IL-10 and TGF-β. Moreover, Tregs reciprocally regulate pathogenic IFN-γ+ populations (37, 38). To establish whether reduced numbers of CD4⁺ Tregs might be contributing to the pathogenesis of murine NPSLE, we stained CP derived T cells from age-matched male and female MRL/lpr mice for the expression of CD4⁺CD127⁻CD25⁺FoxP3⁺. We found a significant reduction in CD4⁺CD127⁻FoxP3⁺ cells in the CP of female mice (Figure 5A, upper right panel). For comparison, we also stained spleens of the same male and female MRL/lpr mice for the presence of Tregs. Spleens from sick female MRL/lpr mice also had decreased Treg numbers (Figure 5A, upper left panel), in line with previous studies indicating that the T regulatory axis is defective in lupus (39, 40).

Using an alternative gating strategy, we found significant expression of FoxP3 on CD4⁺ T cells alone (Figure 5B left panel). This led us to speculate that there may be another FoxP3 mediated regulatory population that is involved. It is well known that the CD4⁺ T_FH mediated GC reaction is controlled by a suppressive population of cells known as Tfr (41, 42). Tfr cells are defined as CD4⁺ PD1⁺CXCR5⁺ FoxP3⁺ cells that can coexpress Bcl6. In addition, IL-21 aids in B cell stimulation and GC formation by mediating the suppression of Tfr (43). Moreover, altered T_FH:Tfr ratios are implicated in several autoimmune diseases (44, 45). We did identify Tfr cells among brain infiltrating T cells, as demonstrated by ICOS, PD1, and CXCR5 positivity in CD4⁺FoxP3⁺ cells (Figure 5B; middle and right panel). Moreover, there were some differences between male and female mice in marker distribution (Figure 5B). However, since the absolute number of Tfr cells in each sex was very small, no firm conclusion can be reached at this time regarding a possible contribution of abnormalities in Tfr numbers to the CP T cell or neuropsychiatric disease phenotype.