CRL4b Inhibition Ameliorates Experimental Autoimmune Encephalomyelitis Progression

Multiple sclerosis (MS) is an autoimmune disease of the CNS, and experimental autoimmune encephalomyelitis (EAE) is a clinically relevant animal model of MS (1). It is estimated that up to 3 million people live with MS worldwide (2). Its onset is typically in adults with peak age at onset between 20 and 40 y. There is a female predominance of up to 3:1 (3, 4). Although there is no cure for MS, there are treatments that can slow the progression of the disease and also reduce the frequency and severity of relapses. The therapeutic efficacy of available drugs is variable, and many are associated with risk for adverse events (1). Thus, there is a strong need to develop new therapeutic agents to manage MS and other autoimmune diseases.

EAE is an experimentally induced autoimmune model in which immunization with myelin oligodendrocyte glycoprotein (MOG) peptides elicits a strong T cell response (1). Upon entering the CNS, T cells are reactivated by local and infiltrating APCs (5), resulting in pathogenic immune-mediated demyelination and axonal damage. T cells have been shown to drive these pathogenic events by direct mechanisms such as cytokine-induced damage, granzyme-mediated killing, and glutamate-induced neurotoxicity, or through indirect mechanisms such as activation of innate cells such as macrophages or neutrophils (6, 7). Considering the pathogenic role of T cells in MS and EAE, therapeutic targeting of T cells would likely be an effective strategy for improving symptoms. Indeed, two of the most commonly used drugs for treating MS, that is, IFN-β and glatiramer acetate, can increase regulatory T cells (Tregs) and IL-10, thereby suppressing proinflammatory T cell cytokine production (8). More deliberate strategies to manipulate T cells involve either targeting proinflammatory T cells or their effector function or boosting the function or numbers of Tregs (9). Ag-activated T cells have the most rapid division rates among all mammalian cell types and are thus predisposed to replication stress-induced DNA damage (10, 11). Thus, therapeutic targeting of DNA damage response pathways could specifically result in the elimination of pathogenic T cells during autoimmune disease flares while having little or no effect on nondividing T cells.

We recently revealed that the Cullin-RING E3 ubiquitin ligase 4b (CRL4b) promotes T cell proliferation, survival, and differentiation (12, 13). Cullin 4b (Cul4b) functions as a scaffold to assemble the multisubunit CRL4b complex that in addition to Cul4b contains the RING finger protein RBX1/ROC1, damaged DNA binding protein 1 (DDB1) as an adaptor, as well as DDB1- and CUL4-associated factor (DCAF) as a substrate receptor (13, 14). CRL4 is activated by Nedd8. Cul4b shares 82% sequence similarity with Cul4a (14). Given their high homology and usage of the same adaptor protein, Cul4a and Cul4b were initially thought to have mostly redundant functions. However, studies have revealed both distinct and overlapping functions (15). Loss-of-function mutations in Cul4b, but not Cul4a, have been identified in patients with cerebral cortical malformations and X-linked mental retardation (16–19), providing support that these two closely related proteins are not redundant. Both Cul4a and Cul4b are able to promote cancer progression (20). Elevated expression of these proteins has been observed in many types of tumors, and high expression correlates with alterations in tumor suppressor and cell cycle regulator genes (21–23). In cancer cells, Cul4a and Cul4b have been shown to regulate proliferation, DNA damage and repair, cell cycle progression, DNA methylation and histone acetylation, as well as cell signaling (24–28). Cul4b deficiency resulted in a nearly complete collapse in effector and memory differentiation of CD8⁺ T cells shortly after viral infection. This collapse was associated with a substantial increase in DNA damage. At the mechanistic level, Cul4b-deficient CD8⁺ T cells accumulated high levels of p21 and cyclin E2, fueling replication stress and triggering genomic instability (13). In CD4⁺ T cells, Cul4b loss decreased their proliferation, survival, and pathogenicity by inducing genomic instability (12). Manipulating the DNA damage–response signaling in lymphocytes has been shown to display therapeutic potential for the treatment of immune-mediated diseases (11). Based on this, we posited that inhibiting CRL4b in T cells would increase genomic stress, particularly in proliferating T cells, and thus CRL4b inhibitors could be useful in treating autoimmune disease.

A selective small-molecule modulator of CRL4b function would allow us to address mechanistic and phenotypic questions about its potential to act as a DNA damage response modulator. To date, there are no reported small-molecule lead compounds specifically targeting the function of Cul4b. However, recent studies showed that binding of compound KH-4-43 to CRL4 inhibits its ability to support ubiquitination in vitro. KH-4-43 was shown to exhibit improved antitumor efficacy in an AML MV4-11 xenograft model (29). T cells express copious levels of Cul4b constituting ∼95% of total Cul4 proteins (12, 13). Furthermore, Cul4a did not rescue the Cul4b loss in T cells, and thus Cul4b-deficient T cells were unable to maintain genome stability, proliferation, or survival (12, 13). We posited that inhibition of CRL4b with KH-4-43 would be a logical strategy to manipulate the DNA damage response of lymphocytes for treatment of autoimmune diseases.

In this study, we employed the EAE model to investigate whether deleting Cul4b in T cells impacted T cell activation and autoimmune disease progression. We found that mice lacking Cul4b in T cells showed lower clinical scores after EAE induction. Consistent with this, we observed a significant reduction in immune cells in the CNS. When the immune cell infiltrates were analyzed, we found fewer T cells, particularly cytokine-producing T cells, as well as fewer innate immune cells such as neutrophils. These data supported our prior findings that Cul4b is required for T cell proliferation/expansion. We found that treatment of T cells in vitro with CRL4 inhibitor KH-4-43 resulted in the accumulation of DNA damage, increased apoptosis, and reduced proliferation. To assess the therapeutic potential for CRL4b inhibition in autoimmune disease, we induced wild-type (WT) mice with EAE and then treated them with KH-4-43. The inhibitor treatment stabilized symptoms of EAE and significantly decreased disease severity. These data highlight the requirement for CRL4b in CD4⁺ and CD8⁺ T cell expansion and effector function during the development of T cell–mediated autoimmune disease and support that targeting CRL4 might be a viable treatment option for MS and other autoimmune diseases.

C57BL/6J (strain 000664), CD45.1 (strain 002014), and CD4-Cre (strain 022071) mice were purchased from The Jackson Laboratory. Cul4b^fl/fl mice were generated using CRISPR/Cas9 as described previously (12). Male and female mice with conditional deletion of Cul4b are referred to as Cul4b^fl/flCD4Cre or Cul4b^cKO. All mice were bred in-house under specific pathogen-free conditions in the animal facility at the Children’s Hospital of Philadelphia (CHOP). The mice were housed at 18–23°C with 40–60% humidity, with 12-h light/12-h dark cycles. All mice, unless stated otherwise, were 8–12 wk of age, and both sexes were used without randomization or blinding. Animal housing, care, and experimental procedures were performed in compliance with the CHOP Institutional Animal Care and Use Committee.

C57BL/6 mice (control and Cul4b^cKO) were injected s.c. with 200 μg of MOG_35–55 emulsified in 5 mg/ml CFA (BD Biosciences). On days 0 and 2, animals received i.p. injections of 200 ng of pertussis toxin (Sigma-Aldrich). Mice were then examined daily for the clinical signs of EAE in a blinded fashion. The disease severity of EAE was scored using the standard clinical score as previously described (30): 0, no disease; 0.5, distal limp tail; 1, compete limp tail; 1.5, limp tail and hindlimb weakness; 2, unilateral partial hindlimb paralysis; 2.5, bilateral partial hindlimb paralysis; 3, complete bilateral hindlimb paralysis; 3.5 complete bilateral hindlimb paralysis and partial forelimb paralysis; 4, moribund (completely paralyzed); 5, death.

Mice were humanely euthanized and perfused with PBS through the left ventricle of the heart using a 25G butterfly needle attached to a 10-ml syringe. The spinal cord, brain, and brain stem were removed by dissection. All of the CNS tissues were then pooled and cut into small pieces using surgical blade in 5 ml of digestion medium (collagenase I and Ia (Sigma-Aldrich, catalog nos. C0130 and C9891, respectively), and 20 μg/ml DNAse I (Roche, catalog no. 10104159001) and digested at 37°C for 30 min. Enzymatic digestion was stopped by adding EDTA to a final concentration of 5 mM. To homogenize the tissue, the suspension was repeatedly sucked up and ejected using a 5-ml syringe with an 18G needle until a uniform milky homogenate was formed, avoiding excessive foaming. The homogenate was filtered through a 70-μm nylon cell strainer into a 50-ml Falcon tube, which was then filled with media (RPMI 1640 with 1% FCS) and centrifuged at 1300 rpm for 8 min at 4°C to pellet the cells and myelin. The supernatant was discarded by pouring it off gently, and the pellet was resuspended in a final volume of 5 ml of 30% Percoll and mixed. The 30% Percoll-containing CNS mixture was poured gently into a 15-ml tube containing 5 ml of 70% Percoll and then centrifuged at 1800 rpm for 30 min at 4°C, without brakes. After centrifugation, a PBS–Percoll gradient formed, with each layer containing a specific fraction of the homogenate. The myelin was carefully sucked off using a suction pump or pipette. The large middle layer containing the leukocytes was transferred without the bottom layer of RBCs into a Falcon tube. The supernatant was discarded and the cells were resuspended in media (RPMI 1640 with 1% FCS).

Single-cell suspensions were stained with a fixable viability dye (LIVE/DEAD fixable blue stain), then pretreated with unlabeled anti-CD16/CD32 (Fc Block, BD Pharmingen). Cells were then stained in FACS buffer (PBS containing 2.5% FCS and 0.1% sodium azide) with mixtures of directly conjugated Abs. The following Abs were used: CD8a (53-6.7), CD3 (17A2), CD3 (145-2C11), CD4 (GK1.5 and RM4-5), CD44 (IM7), B220 (RA3-6B2), TCRβ (H57-597), TNF-α (MP6-XT22), IFN-γ (XMG1.2), GM-CSF (MP1-22E9), γH2AX (2F3), p-ATM (10H11.E12), CD45 (30-F11), CD11b (M1/70), Ly6G (1A8), and F4/80 (BM8) from BioLegend; CD62L (RM4317) and Alexa Fluor 647 streptavidin conjugate from Invitrogen; CD3 (17A2), IL-17A (TC11-18H10), and Siglec-F (E50-2440) from BD Biosciences; and Foxp3-biotin (FJK-16s) from eBioscience.

For intracellular cytokine staining, a single-cell suspension was incubated with PMA (30 ng/ml; Calbiochem) and ionomycin (1 μM; Abcam) in the presence of GolgiPlug (1 μg/ml; BD Biosciences, catalog no. 555029) and GolgiStop (1 μg/ml; BD Biosciences, catalog no. 554724) for 4 h at 37°C and stained using the Cytofix/Cytoperm kit (BD Biosciences). For Foxp3 staining, a Foxp3 staining kit (eBioscience) was used according to the manufacturer’s instruction.

For the T cell proliferation assay, naive CD4⁺ T cells isolated from control mice were treated with DMSO or KH-4-43 for the last 20 h of 3-d cultures. Briefly, CD4⁺ T cells (100,000/well) were incubated with CellTrace Violet (CTV) (Thermo Fisher Scientific) at a final concentration of 5 μM for 10 min at 37°C. Cells were washed three times with ice-cold complete RPMI 1640 and stimulated with anti-CD3 (145-2C11; BioLegend)/CD28 (37.51; BioLegend) Abs (5 μg/ml). After day 2, cells were treated with DMSO or KH-4-43 (in concentrations as indicated). On day 3, cells were harvested and the change in CTV intensity was measured. Samples were analyzed using an LSRFortessa flow cytometer (BD Biosciences) at the CHOP Flow Cytometry Core Facility. Data were analyzed using FlowJo software v10 (Tree Star, Ashland, OR). Results are expressed as the percentage of positive cells or median fluorescence intensity (MFI).

Naive CD4⁺ T cells were isolated by magnetic separation using the Miltenyi Biotec naive CD4⁺ T cell isolation kit. Briefly, cells were isolated from spleen and lymph nodes. Single-cell suspensions were resuspended in MACS buffer and stained with Ab-conjugated beads. For naive CD4⁺ T cell isolation, a mixture of biotin-conjugated mAbs against CD8, CD11b, CD11c, CD19, CD25, CD45R (B220), CD49b (DX5), CD105, anti–MHC class II, Ter-119, and TCRγ/δ was used. Microbeads conjugated to monoclonal anti-biotin Abs and CD44 microbeads were added. After staining, cell suspensions were loaded onto a column and cells that flowed through the column (unlabeled cells) were collected and used as naive CD4⁺ T cells.

Naive CD4⁺ T cells were stimulated in vitro in complete RPMI (RPMI 1640 supplemented with 10% FBS [Atlanta Biologicals], HEPES [Thermo Fisher Scientific], nonessential amino acids, sodium pyruvate [Thermo Fisher Scientific], 2 mM glutamine, antibiotics, and 2-ME) with plate-bound anti-CD3 (clone 17A2, BioLegend) and anti-CD28 (clone 37.51, BioLegend) Abs. The tissue culture plates were coated with the Abs overnight at 4°C (5 μg/ml). Cells were cultured at 37°C with 10% CO₂. For Th1 differentiation, cells were stimulated with plate-bound anti-CD3 and anti-CD28 (5 μg/ml each) as well as IL-2 (10 U/ml), IL-12 (20 ng/ml), and anti–IL-4 (5 μg/ml). For Th17 differentiation, cells were cultured in IMDM media and stimulated with plate-bound anti-CD3 (5 μg/ml) and anti-CD28 (2 μg/ml) together with TGF-β (0.5 ng/ml), IL-1β (20 ng/ml), IL-6 (20 ng/ml), IL-23 (50 ng/ml), and anti–IL-2 (2 μg/ml). For induced Treg (iTreg) differentiation, naive CD4⁺ T cells were stimulated with plate-bound anti-CD3 and anti-CD28 (5 μg/ml each) together with TGF-β (5 ng/ml) and IL-2 (50 U/ml).

Annexin staining was performed to determine cell viability under various in vitro conditions. In brief, naive CD4⁺ T cells were stimulated with anti-CD3/CD28 (5 μg/ml) for 48 h to generate an asynchronous population. Cells were then treated with DMSO (vehicle) or KH-4-43 (in concentration as indicated) for 20 h. Cells were then harvested, resuspended in annexin binding buffer (10 mM HEPES [pH 7.4], 150 mM NaCl, 0.25 mM CaCl₂), and incubated with FITC-conjugated annexin V (Thermo Fisher Scientific) in the dark for 15 min at room temperature. After incubation, 400 μl of annexin binding buffer was added and cells were analyzed.

Brain was gently removed from the skull using curved forceps, after which 1- to 2-mm transverse sections of the brain were taken and fixed in 10% neutral buffered formalin. Tissues were then paraffin embedded and sectioned to 5-μm thickness and stained with H&E. Images were obtained using a Leica DM4000 B upright microscope paired with a Spot RT/SE slider camera with a ×10 objective lens.

Prior to harvesting, cell cultures were supplemented with DMSO or bortezomib (50 nM) for 2–4 h. Subcellular fractionation was carried out as described (31) with some modifications. CD4⁺ T cells were stimulated with anti-CD3/CD28 mAb for 40 h and were then treated with either DMSO or KH-4-43 for 6 h. Cells were harvested and washed twice with PBS (Ca²⁺ and Mg²⁺-free) and resuspended in solution A (10 mM HEPES [pH 7.4], 10 mM KCl, 1.5 mM MgCl₂, 0.34 M sucrose, 10% glycerol, 1 mM DTT, 10 mM NaF, 1 mM Na₂VO₃, EDTA-free complete protease inhibitor mixture [Roche], deubiquitylase inhibitors PR-619 [LifeSensors], and Zn²⁺-chelator ortho-phenanthroline [LifeSensors]). Triton X-100 was added to a final concentration of 0.1%, and the cells were incubated for 30 min on ice. The soluble fraction was separated from the chromatin-bound fraction by centrifugation (4 min, 1800 × g). The chromatin-bound fraction was washed once in solution A and the final chromatin pellet was resuspended in SDS sample buffer and sonicated to release chromatin-bound proteins. Samples were resolved on 4–12% Novex Tris-glycine gels (Thermo Fisher Scientific) and then transferred onto a polyvinylidene difluoride membrane (Amersham Pharmacia Biotech, Piscataway, NJ). The membrane was probed with the primary Abs cyclin E2 (Cell Signaling Technology) and p21 (Santa Cruz Biotechnology, 1:200). GAPDH (Millipore, 1:5000) and lamin B1 (Cell Signaling Technology, 1:1000) were used as loading controls. Immunostaining was performed using appropriate secondary Abs at a dilution of 1:5000 and developed with the LI-COR Odyssey imaging system.

KH-4-43 was supplied by DaVita Labs and Pan Laboratories and was prepared according to reported procedures (29). C57BL/6 mice at 8–10 wk of age with body weight not <20 g at the study starting point were injected with KH-4-43. KH-4-43 (40 mg/kg) was formulated in 100% DMSO and the injection volume was kept at <50 µl/mouse. The first dose was given at day 9 and the last dose on day 14. KH-4-43 was administered i.p. daily from day 9 to 14. For vehicle control, 50 μl/mouse of 100% DMSO was administered i.p. daily.

Results are expressed the mean ± SEM. Statistical analysis was performed using Prism software 7 and 9. The p values were calculated using a Student two-tailed t test. Two-sided p values <0.05 were considered statistically significant.

Our prior studies showed that Cul4b deletion in T cells (Cul4b^fl/flCD4Cre or Cul4b^cKO) did not impact overall CD4⁺ and CD8⁺ T cell frequencies or numbers in the thymus, spleen, or lymph nodes of mice. However, reductions were seen among CD44⁺ (Ag-experienced) T cells, particularly when Cul4b-deficient T cells were raised in a competitive setting with control/WT T cells (i.e., mixed chimera). Consistent with this, when CD4⁺ or CD8⁺ Cul4b-deficient T cells were cocultured with WT T cells and activated using anti-CD3/CD28 mAbs, the Cul4b-deficient T cells quickly became outnumbered by the WT T cells. Interestingly, when Cul4b^fl/flCD4Cre mice were infected with lymphocytic choriomeningitis virus, few lymphocytic choriomeningitis virus–specific CD4⁺ or CD8⁺ T cells could be identified by day 7 postinfection, supporting an important role for Cul4b in T cell expansion (13). However, it was not known whether Cul4b might be required for T cell expansion or whether this protein could be a good therapeutic target to treat T cell–mediated autoimmune disease. To test this, we employed the EAE model, as it is a well-characterized T cell–mediated and induced (not genetic) autoimmune disease. We induced EAE in control and Cul4b^cKO mice using MOG_35–55 emulsified in CFA at day 0 and pertussis toxin was provided at day 0 and day 2. Mice were then monitored for clinical scores for 14 d. Between days 7 and 10 following EAE induction, both control and Cul4b^cKO mice showed an initial increase in clinical score. However, between days 11 and 14 the control mice showed a rapid progression of clinical score whereas the Cul4b^cKO mice did not (Fig. 1A). Instead, Cul4b^cKO mice maintained a very low clinical score for the duration of the analysis, with a mean disease score of 0.77. This was significantly lower than control mice, which showed a mean disease score of 2.3 (Fig. 1B). The increased clinical score and onset of neurologic symptoms after EAE induction in control mice coincided with a reduction in their body weight (Fig. 1C), which was also not observed in Cul4b^cKO mice. At day 14 we performed histopathological analysis of the CNS from control and Cul4b^cKO mice. Inflammatory infiltration in brain parenchyma was assessed using H&E staining, and an influx of immune cells observed in control mice was higher than that observed in Cul4b^cKO mice (Fig. 1D, 1E). Collectively, these results strongly suggested that when T cells lack Cul4b, they are much less likely to induce clinical disease following EAE induction.

EAE is characterized by the invasion of autoreactive T cells into the CNS, leading to the recruitment of innate immune cells such as neutrophils, inflammation, and demyelination (1). Immune cells in the brain and spinal cord on day 14 of MOG_35–55-induced EAE were analyzed using flow cytometry. Cul4b^cKO mice had decreased percentages and numbers of CD4⁺ T cells in the CNS (Fig. 2A–C). Although CD4⁺ T cells are the primary drivers of EAE, CD8⁺ T cells have also been shown to promote EAE pathogenesis (32). In keeping with this, we found decreased percentages and numbers of CD8⁺ T cells in the CNS of Cul4b^cKO mice compared with controls (Fig. 2A–C). Day 14 after immunization, the percentages of CD4⁺ and CD8⁺ T cells in the spleen of Cul4b^cKO showed either no change or a decrease, respectively, compared with control mice (Supplemental Fig. 1A). However, the absolute number of CD4⁺ T cells was higher in Cul4b^cKO compared with control mice whereas numbers of CD8⁺ T cells remained unchanged between the two groups (Supplemental Fig. 1B). These variations may be due to changes that occur in CD4⁺ and CD8⁺ T cells numbers as EAE progresses in the WT mice (33, 34).

Considering the importance of cytokine-producing CD4⁺ effector T cells in triggering inflammatory disease, we sought to measure proinflammatory cytokine production in CD4⁺ T cells. We found that Cul4b-deficient CD4⁺ T cells showed little production of IFN-γ, IL-17A, TNF-α, and GM-CSF production on day 14 (Fig. 2D–H). This was evident in both frequencies and numbers of cytokine-producing T cells compared with control mice (Fig. 2I–L). Given the reduced clinical score in Cul4b^cKO mice, we also assessed Treg numbers to determine whether these might be increased in frequency (among total CD4⁺ T cells) or number. We found that Foxp3⁺ Treg numbers in the CNS of Cul4b^fl/flcKO mice were reduced (Fig. 2M–O), although the frequencies of these cells were similar to those observed in controls, as this reduction mirrored the reduction in conventional T cell numbers. These data support that the lack of clinical progression in the Cul4b^cKO mice is likely due to decreased numbers of autoreactive effector T cells rather than an imbalance in the ratio of Tregs to T effector cells.

Given that T cells can instruct myeloid cells to drive inflammation and trigger pathogenic changes during experimental EAE, we assessed the composition of different myeloid cell populations in the CNS of Cul4b^cKO and control mice following EAE induction. Consistent with reduced T effector cell numbers, we found fewer neutrophils and macrophages in the Cul4b^cKO mice (Supplemental Fig. 1C–H). Thus, Cul4b expression in T cells is required for autoreactive T cells to drive CNS inflammation and clinical progression of EAE.

The inability of Cul4b-deficient T cells to produce IFN-γ and IL-17A in vivo could be due to a defect in the differentiation of naive CD4⁺ T cells into Th1 and Th17 effector cells. To evaluate this possibility, we differentiated naive CD4⁺ T cells isolated from WT and Cul4b^cKO mice under Th1 and Th17 conditions. Naive CD4⁺ T cells from the spleen and lymph nodes were isolated using a Miltenyi Biotec naive CD4⁺ T cell isolation kit, following the procedure detailed in Materials and Methods. Specifically, we cocultured naive CD4⁺ T cells isolated from control (CD45.1⁺) and Cul4b^cKO (CD45.2⁺) mice in the presence of Th1 and Th17 differentiating conditions and assessed cells by flow cytometry after 5 d in culture. The relative proportions of Cul4b-deficient CD4⁺ T cells, following 5 d of culture, were lower than those of control cells (Fig. 3A). Furthermore, cells from WT mice showed a robust induction of IL-17A following restimulation with PMA/ionomycin. In contrast, fewer Cul4b-deficient cells produced IL-17A⁺ following restimulation (Fig. 3B, 3C). In addition, the MFI of IL-17A, a reflection of the amount of cytokine produced per cell, was significantly higher in control cells compared with Cul4b-deficient cells (Fig. 3D). A similar defect in IFN-γ production was observed in Cul4b-deficient T cells following Th1 differentiation. Cul4b-deficient T cells were found at lower frequencies and were less likely to produce IFN-γ than were control T cells (Fig. 3E–H). Taken together, these data support that Th1 and Th17 differentiation is compromised in the absence of Cul4b. We next analyzed the ability of naive CD4⁺ T cells to differentiate into iTregs. Naive CD4⁺ T cells from WT mice showed robust induction of Foxp3, signifying conversion into Tregs, whereas T cells from Cul4b^cKO mice were less likely to show Foxp3 expression (Fig. 3I–K), and the MFI of Foxp3 was somewhat lower in Cul4b-deficient cells (Fig. 3L).

Given that Cul4b-deficient T cells proliferated less and were much more likely to undergo apoptosis following anti-CD3/CD28 stimulation in vitro, we posited that CRL4 inhibition in WT T cells would drive apoptosis of recently activated lymphocytes. KH-4-43, a CRL4 inhibitor that was recently described, was able to induce cytotoxicity in a panel of 36 tumor cell lines by compromising the DNA damage response (DDR) (29). Thus, we tested this CRL4 inhibitor in activated CD4⁺ T cells. To evaluate whether KH-4-43 might inhibit CRL4 function in T cells, we used flow cytometry with annexin V staining to detect cells undergoing apoptosis. We found that activated CD4⁺ T cells were much more likely to undergo apoptosis following treatment with KH-4-43 in a dose-dependent manner (Fig. 4A, 4B). Treatment of cells with 10 μM KH-4-43 for 20 h induced 2-fold more apoptosis than with DMSO-treated cells. Furthermore, CRL4 inhibition increased γH2AX and p-ATM levels (Fig. 4C–F), in a manner reminiscent of that shown in Cul4b-deficient T cells (13). KH-4-43 treatment of WT cells also reduced proliferation in T cells following mitogenic stimulation (Fig. 4G, 4H). These data support that KH-4-43 inhibits CRL4 in T cells and KH-4-43 treatment of WT T cells shows substantial overlap with features of Cul4b-deficient T cells (12).

We next sought to test whether CRL4 inhibition would prevent clinical progression following EAE induction. To test this, KH-4-43 was administered daily from day 9 (after the start of symptoms) until day 14 (peak of disease) after EAE induction at a dose of 40 mg/kg per day (i.p.). From day 9 to 14, vehicle-treated control mice showed rapid progression to clinical disease (Fig. 5A, 5B). In contrast, KH-4-43–treated mice showed only modest progression following treatment and none of the mice advanced to full clinical disease (Fig. 5A, 5B). CRL4 inhibition by KH-4-43 also resulted in reduced immune cell infiltration in the CNS (Fig. 5C–H) following EAE induction. We found significantly fewer CD4⁺ and CD8⁺ T cells in the CNS following treatment with KH-4-43 compared with vehicle controls (Fig. 5C, 5D). Consistent with this result, we also found reduced numbers of macrophages and neutrophils in KH-4-43–treated mice compared with vehicle controls (Fig. 5E–H). Given the reduced clinical score in KH-4-43–treated mice, we also assessed Treg numbers to determine whether these mice have increased frequency (among total CD4⁺ T cells) or the number of Tregs. However, we found that Foxp3⁺ Treg numbers in the CNS of KH-4-43–treated mice were also reduced (Supplemental Fig. 2A, 2B), although the frequencies of these cells were similar to those observed in controls (Supplemental Fig. 2C). These data support that the lack of clinical progression in the KH-4-43 mice is likely due to decreased numbers of autoreactive T cells rather than an imbalance in the ratio of Tregs to T effector cells. Furthermore, at day 15 after immunization, the percentages and numbers of CD4⁺ and CD8⁺ T cells in the spleen of KH-4-43–treated mice showed no significant change compared with vehicle control mice (Supplemental Fig. 2D, 2E). We have shown previously that Cul4b-deficient T cells are unable to repair DNA damage due to a protracted accumulation of p21 and cyclin E2, known CRL4b substrates (13). Thus, we tested whether CRL4 inhibition by KH-4-43 resulted in a similar accumulation of p21 and cyclin E2. To test this, we activated CD4⁺ T cells with anti-CD3/CD28 mAbs for 40 h, after which cells were treated with KH-4-43 for 6 h and assessed for p21 and cyclin E2. We found that KH-4-43 treatment resulted in significant accumulation of both p21 and cyclin E2 (Fig. 5I, 5J). These data support that KH-4-43 inhibits CRL4b in T cells and thus promotes the accumulation of DNA damage and hence cell death. Taken together, these data support that inhibiting CRL4 function could be an attractive therapeutic option for treating T cell–mediated autoimmune diseases such as MS.

In this study we show that Cul4b deletion in T cells reduced both CD4 and CD8 numbers in the brain and spinal cord of MOG_35–55-immunized mice and prevented accumulation and pathogenic damage from myeloid cells. Supporting this, inflammation was reduced and CD4⁺ T cells were isolated on day 15 after MOG_35–55 immunization from the brain and spinal cord of Cul4b^cKO mice produced negligible amounts of IFN-γ, TNF-α, IL-17, or GM-CSF upon in vitro stimulation with MOG_35–55 peptide. Similarly, the administration of KH-4-43, a CRL4 inhibitor, ameliorated clinical signs of MOG-induced EAE. The attenuation of EAE pathogenesis by KH-4-43 was accompanied by reduced immune cell infiltration. However, it remains unclear whether CRL4b inhibits disease progression or delays its onset. Future studies will include an extended observation period to better understand the long-term impacts of CRL4 inhibition. Despite this, CRL4 inhibitors represent novel drugs that could have therapeutic potential in autoimmune diseases due to its ability to affect cytokine production and proliferation of T cells.

Th17 cells are involved in the onset and maintenance of EAE (35). IL-17⁺ T cells have been found in lesions in brain tissues from patients with MS, indicating that Th17 cells also play a crucial role in the human demyelinating disease (36). Our data showed that CRL4b promotes the expansion and differentiation of Th17. Additionally, we found that Cul4b deficiency inhibited Th1 and induced Treg expansion and differentiation in vitro. IFN-γ–secreting Th1 cells were reduced in the CNS of the MOG_35-55-immunized Cul4b^cKO mice. Thus, combined with the role of Th1 and Th17 in the development of EAE, the data indicated that CRL4b had potential regulatory functions in the activation and differentiation of CD4⁺ T subsets, which may explain why KH-4-43 treatment might be efficient in the suppression of EAE. However, Treg development was not affected after EAE induction but we did find significantly lower numbers of Tregs in the brains of Cul4b^cKO mice 15 d after MOG_35–55 immunization. This could be a reflection of overall fewer numbers of CD4⁺ T cell infiltration into the CNS of MOG_35–55-immunized Cul4b^cKO mice or could be due to the lower overall level of inflammation. Tregs are crucial in controlling various functions of effector T cells during EAE. The reduced severity in Cul4b^cKO mice thus does not appear to be due to increased Treg numbers or function. Rather, CRL4b could be working in a cell-intrinsic manner to regulate T cell proliferation and differentiation. Many factors such as strength of signal, cytokines, tissue-specific factors, and APCs can contribute to the generation of iTregs in vivo, which may not closely reflect Tregs generated in vitro. Thus, any role for CRL4b in Treg differentiation and/or function will require further study.

Our data support that inhibiting CRL4b could be an effective method for the treatment of autoimmune disease, as deleting this protein in T cells inhibited the differentiation and proliferation of Th17 and Th1 cells. This may be advantageous over other approaches that target many immune cells or that impact the function of naive T cells. In contrast, although most of the effects of CRL4 inhibitor in our system have been attributed to T cell dysfunction, it is unclear whether this treatment affects B cells or other immune cells in which CRL4b may be active. Therefore, further studies in which Cul4b is conditionally deleted in specific cell types are warranted. In our study, we employed CRL4 inhibitor at the onset of the diseases when T cell activation has already been initiated but T cell expansion is ongoing, as we were attempting to model treatment of symptomatic MS patients. Based on our results, CRL4 inhibition may qualify as a new treatment option to prevent the progression of autoimmune diseases such as MS. However, assessing treatment options for patients at different stages of MS, as opposed to its onset, will be important. Toward this, investigating how the CRL4 inhibitor affects disease outcomes when administered at later stages of EAE in mice could further clarify the full potential of CRL4 inhibition. Furthermore, it is still unclear how CRL4 inhibition could impact the APCs required for the reactivation of T cells in the CNS. Therefore, using CRL4 inhibitors in the clinic requires further investigation to understand the potential impact on other immune cells or potential side effects.

The role of DDR signaling in the survival and function of mature T cells remains poorly understood. Multiple defects of the DDR, including mutations in ATM, ATR, DNA-PK, and other genes, are associated with immune deficiency (37). Cul4b deficiency in CD4⁺ T cells decreased their pathogenicity and also decreased the CD8⁺ T cell–mediated antiviral immune responses. Dozens of small-molecule inhibitors targeting DDR pathways, including CRL4 inhibitors, have been developed, largely for the treatment of cancer (38). However, immune effects (or toxicities) of these drugs that modulate DDR signaling have not been previously reported. Our findings suggest that such effects should be examined in future clinical tests of DDR altering small molecules. High expression of p21 has also been implicated in replicative senescence (39), wherein cells that have repeatedly entered into the cell cycle become refractory to further stimulation and are irreversibly arrested at the G₁ phase (40). The upregulation of p21 in T cells after KH-4-43 has the potential to induce replicative senescence in encephalitogenic CD4⁺ T cells that are found in abundance in EAE in mice and MS patients.

Overall, this study showed that KH-4-43 reduced inflammation in mice, supporting the notion that CRL4 inhibition may be a potential therapeutic treatment strategy for EAE. The effectiveness of specifically inhibiting activated and expanding T cells while leaving naive T cells intact would make this strategy ideal for the treatment of several inflammatory and autoimmune diseases, including graft rejection and type 1 diabetes, as well as T cell malignancies. However, more studies are needed to elucidate the mechanisms by which CRL4b works in T cells and identify the substrate receptors used by CRL4b in T cells and other immune cells. This may allow the discovery of CRL4b inhibitors that exert T cell–specific therapeutic effects. Although KH-4-43 may require further optimization to improve efficacy, our results show promise for the continued development and the clinical testing of CRL4b inhibitors for the suppression of T cell–mediated diseases.

The authors have no financial conflicts of interest.

We thank the Flow Cytometry Core and Department of Veterinary Resources at the Children’s Hospital of Philadelphia for technical support.