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Abstract
Objectives The tuning effects of JAK/TYK2 inhibitors on the imbalance between T follicular helper (Tfh) and T regulatory (Treg) cells, related to systemic lupus erythematosus (SLE) pathogenesis, were investigated using human peripheral blood samples.
Methods Peripheral blood mononuclear cells from untreated patients with SLE and healthy controls were analysed. Tfh1 cells were identified in nephritis tissue, and the effect of Tfh1 cells on B-cell differentiation was examined by coculturing naïve B cells with Tfh1 cells.
Results Tfh1 cell numbers were increased in the peripheral blood of patients, and activated Treg cell counts were decreased relative to Tfh1 cell counts. This imbalance in the Tfh to Treg ratio was remarkably pronounced in cases of lupus nephritis, especially in types III and IV active nephritis. Immunohistochemistry revealed Tfh1 cell infiltration in lupus nephritis tissues. Co-culture of Tfh1 cells (isolated from healthy individuals) with naïve B cells elicited greater induction of T-bet+ B cells than controls. In JAK/TYK2-dependent STAT phosphorylation assays using memory CD4+ T cells, IL-12-induced STAT1/4 phosphorylation and Tfh1 cell differentiation were inhibited by both JAK and TYK2 inhibitors. However, phosphorylation of STAT5 by IL-2 and induction of Treg cell differentiation by IL-2+TGFβ were inhibited by JAK inhibitors but not by TYK2 inhibitors, suggesting that TYK2 does not mediate the IL-2 signalling pathway.
Conclusions Tfh1 cells can induce T-bet+ B cell production and may contribute to SLE pathogenesis-associated processes. TYK2 inhibitor may fine-tune the immune imbalance by suppressing Tfh1 differentiation and maintaining Treg cell differentiation, thereby preserving IL-2 signalling, unlike other JAK inhibitors.
- systemic lupus erythematosus
- cytokines
- T-lymphocytes
Data availability statement
Data are available on reasonable request.
This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/.
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WHAT IS ALREADY KNOWN ON THIS TOPIC
IL-12-induced increase in T follicular helper (Tfh1) cell numbers is observed in some systemic lupus erythematosus (SLE) cases. TYK2 inhibitor has demonstrated efficacy in phase 2 trials for SLE, and phase 3 trials are currently underway.
WHAT THIS STUDY ADDS
Patients with SLE often have an immune imbalance with increased Tfh1 and decreased activated T regulatory (Treg) cell numbers; Tfh1 cells induce the differentiation of T-bet+ B cells, which may be involved in the pathogenesis of lupus nephritis. TYK2 inhibitor hinder Tfh1 cell differentiation; however, unlike other JAK inhibitors, they can preserve Treg cell differentiation without inhibiting the IL-2-JAK1/3-STAT5 signalling pathway.
HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY
TYK2 inhibitor, unlike other JAK inhibitors, preserves Treg cell differentiation and fine-tune T cell subsets. They may have potential in the treatment of SLE and allow effective stratified therapy for populations with increased Tfh1 cell counts and reduced Treg cells.
Introduction
Systemic lupus erythematosus (SLE) is an autoimmune disease characterised by multiple organ involvement and the production of diverse autoantibodies. It is caused by a complex immune abnormality consisting of both acquired and innate immunity, involving a breakdown of immune tolerance based on genetic predisposition and environmental factors. In simpler terms, SLE is caused by abnormalities in innate immunity, such as the excessive production of type I interferons (IFNs) by plasmacytoid dendritic cells that recognise immune complexes comprising auto-nucleic acids and autoantibodies. In addition, abnormalities in acquired immunity contribute to SLE pathogenesis, such as the excessive activation and differentiation of autoreactive T cells and increased differentiation of B cells into antibody-producing cells.1
T follicular helper (Tfh) cells are involved in the formation of germinal centres (GCs) and the selection of high-affinity B cells in GCs.2 Similar to Th1, Th2, Th17 and T regulatory (Treg) cells, there are Tfh1, Tfh2, Tfh17 and T follicular regulatory (Tfr) cells. These subpopulations have differential effects in regulating B cell responses.3–6 Considering their functional significance, Tfh cells may contribute substantially to the pathogenesis of SLE. We previously reported that the number of Tfh1 cells, which exhibit the characteristics and functions of both Tfh and Th1 cells, increased in the peripheral blood of patients with SLE and were induced by IL-12 stimulation.7 Although previous studies have demonstrated the significance of Tfh cells and IL-12 signalling in SLE,8–12 reports on Treg cells remain controversial. Various viewpoints suggest that Treg and Tfr cells are either reduced in number or exhibit impaired function in patients with SLE compared with healthy individuals.13–17 However, it has been argued that the balance between Tfh cells and Treg cells may be disrupted in SLE.15 17 Consequently, in this study, we hypothesised that Tfh1 cells also play a role in the pathogenesis of SLE.
In recent years, treatment options for SLE have increased; the efficacy of anti-IFN receptor antagonist antibodies and anti-B-cell activating factor (BAFF) antibodies has been demonstrated, and JAK or TYK2 inhibitors are also attracting interest.18 19 There are three types of JAKs, JAK1, JAK2, and JAK3, and TYK2, each of which transduce different cytokine signals. Among the cytokines involved in the breakdown of immune tolerance in SLE, those that bridge the innate and adaptive immune systems, such as type I IFNs, interleukin (IL)-12, and IL-23, and those that activate T cell–B cell interactions, such as IL-21, IL-6 and IL-4, all have JAK-STAT pathway-dependent activity; therefore, JAK inhibitors have potential applications in the treatment of SLE.20 However, a phase 3 trial of SLE using baricitinib, a JAK1 and JAK2 selective inhibitor, failed to demonstrate clear efficacy.21 One plausible reason is that the multitargeted action of JAK inhibitors may not always promote a therapeutic effect, as several JAK inhibitors, including baricitinib, are involved in triggering not only inflammation-inducing cytokines but also inflammation-regulating cytokines. JAK inhibitors with an ATP-binding competitive inhibitory mechanism (such as tofacitinib, baricitinib, upadacitinib and filgotinib), which are currently used to treat rheumatoid arthritis (RA), are known to inhibit JAK1 phosphorylation. However, recently developed TYK2-selective inhibitor can inhibit TYK2 without inhibiting JAK122; this may distinguish them from other inhibitors. We have previously investigated the effects of JAK inhibitors on human lymphocyte differentiation and found that baricitinib inhibits the differentiation of some T cells, including Th1 and Th17 cells.23 However, the impact of TYK2 inhibitor on human lymphocyte differentiation remains unexplored. Further, JAK and TYK2 inhibitors vary significantly in terms of cytokine selectivity, potentially leading to differential inhibitory effects and subsequent alterations in the distribution of lymphocyte subsets, including T-cell subsets. Of note, these differences may ultimately impact the pathogenesis of various immune disorders, including SLE.
Therefore, the objective of this study was to evaluate the immune dysregulation between individual Tfh subsets and Treg cells in untreated patients with SLE and establish a scientific basis for the effective implementation of JAK or TYK2 inhibitors in modulating the immune imbalance using human lymphocytes.
Materials and method
Study design and patients
This study was conducted as part of the FLOW registry, which involved several facilities led by the University of Occupational and Environmental Health, Japan. Participating institutions included Wakamatsu Hospital, Tobata General Hospital, Kitakyushu General Hospital and Shimonoseki Saiseikai Hospital. The study analysed a total of 82 patients diagnosed with newly diagnosed or relapsed SLE between 2014 and 2021, whether as inpatients or outpatients. All patients met the 2012 Systemic Lupus International Collaborating Clinics SLE classification criteria or the 2019 European Alliance of Associations for Rheumatology/American College of Rheumatology classification criteria and were referred to the Hospital of the University of Occupational and Environmental Health, Japan for diagnosis, evaluation of organ damage and peripheral blood lymphocyte analysis.24 25 Patients taking immunomodulators, immunosuppressants or glucocorticoids at the time of analysis were excluded. Furthermore, 66 healthy subjects matched for age and sex, with no autoimmune or infectious complications, were included as healthy controls. Blood samples were obtained from patients and healthy subjects, respectively. SLE Disease Activity Index (SLEDAI) was assessed, and a score of 5 or higher was defined as active SLE. The British Isles Lupus Assessment Group (BILAG) index 200426 27 was used to evaluate organ damage. The clinical background of the patients included is shown in table 1.
Demographic and clinical characteristics of study subjects
Test compounds
Tofacitinib, baricitinib and PF-06700841 were synthesised at Advanced ChemBlock (USA). Upadacitinib was synthesised at Abbvie (USA). Peficitinib was synthesised at Astellas Pharma (Japan). Filgotinib was synthesised at Gilead Sciences (USA). TYK2 inhibitor (BMS-986202) was synthesised at Bristol–Myers Squibb (USA). The compounds were dissolved in DMSO and then used in stepwise dilutions. A solution containing DMSO (maintaining the DMSO concentration below 0.1% to prevent cytotoxic effects) without inhibitors served as a control.
Cell separation
Peripheral blood mononuclear cells were isolated from blood samples using a lymphocyte separation medium (Cedarlane, ON, Canada) and suspended in RPMI 1640 medium (Wako, Tokyo, Japan) supplemented with 10% fetal bovine serum and 1% (v/v) penicillin-streptomycin (PS). CD4+ T cells and CD19+ B cells were purified using a CD4+ T Cell Isolation Kit and CD19+ B-Cell Isolation Kit (MiltenyiBiotec, Bergisch Gladbach, Germany, Biolegend, California, USA), respectively. For coculture experiments, Memory CD4+ T cells were purified once from healthy peripheral blood and stained with the antibodies listed in online supplemental table 1 for 30 min. CD4+ CD45RA− CXCR5+ CXCR3− CCR6− Tfh cells, CD4+ CD45RA− CXCR5+ CXCR3− CCR6+ Tfh17 cells, CD4+ CD45RA− CXCR5+ CXCR3+ Tfh1 cells and CD4+ CD45RA− CXCR5− CXCR3+ Th1 cells were then isolated by FACS Aria II. Cell purity was consistently >90%.
Supplemental material
Cell stimulation and culture
Naïve CD4+ T cells were cultured in 96-well plates with a flat bottom at a density of 1×105 cells per well. The plates were precoated with anti-CD3 antibody and soluble anti-CD28 antibody in RPMI 1640 medium (Wako, Tokyo, Japan) supplemented with 10% fetal bovine serum and 5% PS for 3 days. After the initial stimulation, the cells were transferred to new 96-well plates to halt TCR stimulation. Fresh medium was added, followed by cytokine stimulation and the addition of JAK/TYK2 inhibitors. Cells were then cultured for an additional 2 days and collected on day 5. IL-12 stimulation was performed for the induction of Tfh1 cell differentiation, and TGFβ and IL-2 stimulation were performed for Treg cell differentiation. For the coculture experiments, isolated T cells and naïve B cells were cultured with anti-CD3 antibody and soluble anti-CD28 antibody for 4 days. In Phosflow, after the induction of and Treg cell subset differentiation by the method described above, the cell culture medium was replaced with fresh RPMI 1640 medium supplemented with 10% FCS and 5% PS on day 5; the cells were then cultured for 1 day to terminate cytokine stimulation. On day 6, the culture medium was replaced with fresh RPMI 1640 medium supplemented with 10% FCS and 5% PS; the cells were then incubated at 37°C for 10 min with the specified concentrations of test compounds. The cells were then treated with recombinant human cytokine for 15 min, fixed in Fix buffer I (BD Biosciences, New Jersey, USA) at 37°C for 10 min and incubated in Perm buffer III (BD Biosciences) for 30 min on ice. After fixation, the cells were washed and stained with anti-pSTAT antibody for 90 min at 4°C in the dark. Finally, the cells were washed, resuspended in a wash buffer and stored on ice until flow cytometric analysis. The stimulants used are shown in online supplemental table 2.
Evaluation of IC50 values by STAT phosphorylation assay
To determine the IC50 values, CD45RA− CD4+ T cells were preincubated with seven concentrations of the test compounds (ranging from 1 nM to 1000 nM) for 10 min at 37°C, followed by 15 min of incubation with recombinant human cytokines. To assess IL-12-stimulated Tfh1 cell-induced STAT phosphorylation, naïve CD4+ T cells were subjected to TCR stimulation for 3 days to induce IL-12 receptor expression on the cell surface and then subjected to treatment with JAK inhibitor and IL-12 stimulation. For cell fixation, treatment with BD Phosflow Lyse/Fix buffer I (BD Biosciences) was performed at 37°C for 10 min, followed by treatment with Perm buffer III (BD Biosciences) for 30 min at 4°C. After washing, cells were stained with anti-phospho STAT antibody at 4°C in the dark. After staining, cells were washed and analysed by flow cytometry. For cell fixation, treatment with BD Phosflow Lyse/Fix buffer I (BD Biosciences) was performed at 37°C for 10 min, followed by treatment with Perm buffer III (BD Biosciences) for 30 min at 4°C. After washing, cells were stained with anti-phospho STAT antibody at 4°C in the dark. After staining, cells were washed and analysed by flow cytometry.
Flow cytometry
The phenotype of immune cell subsets was determined based on the Human Immunology Project protocol of comprehensive 8-colour flow cytometric analysis proposed by the National Institutes of Health/Federation of Clinical Immunology Societies.28 The identification of Tfh cells was based on the original criteria, with ICOS being utilised as an indicator of activation markers (online supplemental figure 1). For cell surface staining, the antibodies in online supplemental table 1 are used. For intracytoplasmic staining, cells were fixed and permeabilised with Transcription Factor Buffer Set (BD Biosciences) for 30 min at 4°C and washed with Perm/Wash Buffer (BD Biosciences). For intracytoplasmic cytokine staining, cells were stimulated with phorbol 12-myriSTATe 13-acetate (50 ng/mL), ionomycin (1 µg/mL) and brefeldin A (2.5 µg/mL) for 5 hours before cell fixation. Flow cytometric analysis was performed using viable cells that did not express Fixable Viability Dye (eBioscience, California, USA). Isotype-matched control antibodies were used as background controls. Flow cytometric analysis was performed using a BD FACS Lyric flow cytometer and analysed using FlowJo software (FlowJo, Ashland, Oregon, USA).
Immunofluorescence
Histopathological evaluation of lupus nephritis was conducted on cases categorised as type III according to the 2018 revision of the International Society of Nephrology/Renal Pathology Society (ISN/RPS) classification of lupus nephritis.29 Negative controls consisted of cases diagnosed as type II using the same classification criteria. Multiplex immunofluorescence was performed using the Opal four-colour fluorescence immunohistochemistry kit (Akoya Biosciences, Marlborough, Massachusetts, USA) following the manufacturer’s protocol. The antibodies employed and the detailed staining procedures are shown in online supplemental table 3.
Statistical analysis
Differences between the two groups were analysed by Student’s unpaired t-test or Mann-Whitney U test, and more than two groups were analysed by analysis of variance; in the evaluation of the relationship between the two variables, Spearman’s correlation coefficient analysis was used; in in vitro experiments. Data were analysed as the mean±SEM of three or more independent experiments with different donors. Values of p<0.05 were considered significant. Analyses were performed with GraphPad Prism software V.9.
Results
Elevated Tfh1 cells and imbalance in Tfh1/Activated Treg cell ratio in SLE
Although previous studies have analysed Tfh subsets in patients with SLE, numerous patients take therapeutic drugs, and the influence of drug modification cannot be ruled out, especially because glucocorticoids significantly impact lymphocyte differentiation. To detect abnormal lymphocyte differentiation more accurately, CD4+ T cell and CD19+ B-cell subsets were detected in blood samples from 82 recently diagnosed or relapsed untreated patients with SLE and 66 age-matched and sex-matched healthy subjects with no autoimmune or infectious disease complications (table 1).
The results showed a significant increase in Tfh1 cell counts in patients with SLE while Treg cell counts showed a slight increase compared with HD cell counts (figure 1A). When comparing Tfh1 and Treg cells, a predominant increase in Tfh1 cell counts was observed, especially when compared with activated Treg cells with a regulatory function, revealing a more significant difference (figure 1B). This suggests an imbalance between the autoimmune immune-inducible subset and the immune-regulatory subset. Additionally, in cases of active SLE, the number of activated Treg cells decreased as the number of Tfh1 cells increased, indicating a negative relationship between Tfh1 cells and activated Treg cells (figure 1C). Conversely, the number of Tfh17 cells exhibited a positive relationship with the number of activated Treg cells, and the total number of Tfh cells showed no association with the number of activated Treg cells.
Comparison of CD4+ T cell and CD19+B cell subsets between patients with SLE healthy subjects. Flow cytometry analysis of CD4+ T cells and CD19+ B cells in peripheral blood mononuclear cells (PBMCs) isolated from peripheral blood of patients with SLE (n=82) and healthy subjects (n=66). (A) Comparison of CD4+ T-cell subsets and CD19+ B-cell subsets in peripheral blood of 82 patients with SLE and 66 healthy subjects. (B) Comparisons between the ratio of Tfh1 cells to activated Treg cells in 82 patients with SLE. Data are shown in logarithmic scale. (C) Correlation between Tfh subsets and activated Treg cells in patients with high disease activity of SLE was assessed. The criterion for high disease activityof SLE was a SLEDAI score of 5 or higher; 64 patients with active SLE were enrolled. Spearman’s coefficient of correlation was used for assessment. (D) Comparison of the ratio of Tfh1 cells and activated Treg cells in cases of nephritis based on the BILAG index and SLEDAI score and the ratio of Tfh1 cells and activated Treg cells according to the pathological type of nephritis based on the International Society of Nephrology/Society of Renal Pathology (ISN/RPS) criteria. Comparisons between two groups were assessed using Mann-Whitney U test. Symbols represent individual subjects; bars represent mean±SD. Anti-ds-DNA antibody, anti-double-stranded DNA antibody; Anti-SM antibody, anti-Smith antibody; BILAG, British Isles Lupus Assessment Group Index; CH50, 50% haemolytic complement activity; HD, healthy donor; SLEDAI, Systemic Lupus Erythematosus Disease Activity Index.
Next, we explored the potential contribution of the imbalance in the Tfh1 and Treg cell ratio to organ involvement in SLE. We compared the percentage of Tfh1 cells to that of Treg cells based on the presence or absence of organ involvement. Our comparative analysis, based on organ damage assessment using the BILAG index and SLEDAI score, revealed that Tfh1 cell counts were increased in cases of active lupus nephritis while Treg cell counts were decreased (figure 1D). Further examination of the Tfh1 to Treg cell ratio, classified by lupus nephritis class according to the ISN/RPS classification, demonstrated that the imbalance between these cells was most pronounced in active lupus nephritis, particularly types III and IV. Furthermore, when analysing the association with SLEDAI based on the percentage of Tfh1 cells, we found that Tfh1 cell counts were elevated in patients with proteinuria, haematuria and hypocomplementaemia; however, no correlation was found with other clinical findings (online supplemental figure 2). In the BILAG assessment, Tfh1 cell counts were significantly increased in patients with active nephritis (BILAG A or B). Conversely, no notable increases in other T cell subset counts were observed in cases of active nephritis (online supplemental figure 3).
These findings suggest that Tfh1 cells are a subset of cells associated with disease activity, particularly in the context of lupus nephritis, and that the imbalanced relationship between Tfh1 cells and Treg cells may be a key factor in the pathogenesis of SLE.
Tfh1 cells induce T-bet+ B cells and may play a role in the pathogenesis of lupus nephritis
Next, to assess the significance of Tfh cells in lupus nephritis, we examined the localisation of Tfh1 cells in renal tissue using specimens obtained from ten patients diagnosed with type III lupus nephritis, as per the 2018 revised classification of the ISN/RPS. While some type III lupus nephritis specimens used for staining exhibited zero Tfh1 cell detection in cases with minimal lymphocytic infiltration, Tfh1 cells were ultimately identified in five cases. Figure 2A portrays representative findings, where T-bet+ Bcl6+ CD4+T cells coexpressing T-bet and Bcl6, the main transcription factors of Tfh1 cells, were observed infiltrating the nephritis tissue in type III lupus nephritis cases. In contrast, no such cells were observed in control lupus nephritis type II cases. Additionally, the infiltration of CXCR3+ CXCR5+ CD4+T cells, which coexpress CXCR3 and CXCR5 (the surface antigens of Tfh1 cells), was observed in type III lupus nephritis cases while these cells were absent in a control type II lupus nephritis case (online supplemental figure 4). Overall, these findings suggest that Tfh1 cells may play a role in the pathogenesis of lupus nephritis.
Identification of Tfh1 cells in lupus nephritis tissue and induction of T-bet+B-cell differentiation by Tfh1 cells. (A) Immunostaining was performed on tissue specimens from patients with lupus nephritis type III, using lupus nephritis type II as control. The infiltration of Tfh1 cells, characterised by T-bet+ Bcl6+ CD4+ T cells in the renal parenchyma was examined. (B) For coculture experiments, Non-Tfh1 and Tfh17 (CXCR3− CXCR5+ CCR6− CD45RA− CD4+ T cells), Tfh17 cells (CXCR3− CXCR5+ CCR6+ CD45RA− CD4+ T cells), Tfh1 cells (CXCR3+ CXCR5+ CCR6− CD45RA− CD4+ T cells) and naïve CD19+ B cells (CD19+ IgD+ CD27− B cells) were isolated from peripheral blood of healthy subjects. Isolated T cells (2×104 cells) and naïve B cells (4×104 cells) were cultured in U-bottom 96-well plates coated with anti-CD3 antibody (1.0 µg/mL), soluble anti-CD28 antibody (0.5 µg/mL) for 4 days to examine the induction of T-bet+ B cells. Samples with a purity of over 96% were qualified to be used. In the flow cytometry analysis, the total number of acquired events was set to 50 000 cells. (C) T-bet+ B-cell expression was compared by mean fluorescence intensity (MFI). Data are presented as the mean (SEM) of four independent experiments using samples from three donors. Symbols represent individual subjects; **p<0.01.
In SLE, T-bet+ double-negative B cells contribute to lupus nephritis.30–32 Therefore, we first examined the association between Tfh1 and B cells in the peripheral blood of patients with SLE. When examining the relationship between T cell subsets and B cells, we noted that the proportion of Tfh1 cells positively related to the increased double-negative B cell counts in SLE samples. However, no similar association was observed with other T cell subsets (online supplemental figure 5).
To evaluate whether Tfh1 cells contribute to the induction of T-bet+ B-cell differentiation, non-Tfh1-Tfh17-Th1, Tfh17, Th1 and Tfh1 cells were isolated from the peripheral blood of healthy subjects and cocultured with naïve B cells in vitro. The results revealed that Tfh1 cells induced a marked differentiation of naïve B cells into T-bet+ B cells, compared with non-Tfh1-Tfh17 and Tfh17 cells (figure 2B,C).
These results suggest that Tfh1 cells may be involved in the pathogenesis of lupus nephritis, as they may induce the differentiation of naïve B cells into T-bet+ B cells.
TYK2 inhibitors inhibit IL-12 but not IL-2 signalling
Previous studies have suggested that an imbalance between Tfh1 and active Treg cells may contribute to the pathogenesis of SLE. The impact of JAK inhibitors on cell differentiation is considered, given the key role that cytokines play in lymphocyte differentiation. The IC50 values of each compound varies due to differences in the dependency of cytokines on JAK isoforms and the kinase selectivity of JAK inhibitors. Thus, detecting such differences may provide insights into the role of JAK inhibitors during lymphocyte differentiation. Therefore, we first evaluated the IC50 values of various JAK inhibitors on stimulating human CD45RA− CD4+ T cells with various cytokines. We also detected pSTAT inhibition on IL-12 and IFNγ stimulation in CD45RA− CD4+ T cells, a phenomenon that has not been reported previously.
We then examined the IC50 values of tofacitinib (JAK1/3 selective), baricitinib (JAK1/2 selective), filgotinib (JAK1 selective), peficitinib (Pan JAK), upadacitinib (JAK1 selective), BMS-986202 (TYK2 selective) and PF-06700841 (JAK1/TYK2 selective) on the stimulation of CD4+CD45RA-T cells with IL-2, IL-4, IL-6, IL-12, IFNα and IFNγ. IL23 is also a TYK2-mediated signalling cytokine but was not included in this study because IL-23 receptor expression in circulating human CD45RA- CD4+ T cells is low and cannot be assayed.33 The average mean fluorescence intensity (MFI) values for the inhibition of pSTAT expression following stimulation with various cytokines are presented in online supplemental table 4. Additionally, the fold change in JAK selectivity between cytokine signals, based on the lowest IC50 value of each JAK/TYK2 inhibitor, is depicted in table 2, using mean MFI values. All JAK inhibitors and the TYK2 inhibitor blocked the JAK1-mediated and TYK2-mediated phosphorylation of STAT1 and STAT3 under IFNα stimulation, as well as IL-12-stimulated STAT1 phosphorylation with low IC50 values. Upadacitinib, a selective JAK1 inhibitor, also inhibited JAK2/TYK2-mediated IL-12-stimulated STAT1 phosphorylation with a low IC50. This observation suggests the cross-reactivity of upadacitinib with JAK2. In contrast, the IC50 values were the lowest for upadacitinib, tofacitinib and baricitinib on IL-2-stimulated JAK1/3-mediated STAT5 phosphorylation, IL-4-stimulated JAK1/3-mediated STAT6 phosphorylation, IL-6-stimulated JAK1/JAK2/TYK2-mediated STAT1/STAT3 phosphorylation and IFNγ-stimulated JAK1/JAK2-mediated phosphorylation of STAT1, but characteristically high for TYK2 inhibitors. Regarding IL-6-induced expression of pSTAT1 and pSTAT3, STAT1 expression was more JAK 1-dependent, and pSTAT1 expression was suppressed in the presence of JAK inhibitors as previously reported. This suggests that, unlike other JAK inhibitors, TYK2 inhibitors demonstrate minimal and highly selective involvement in these signalling pathways.
Distinct Janus kinase (JAK) inhibitor and tyrosine kinase 2 (TYK2) inhibitor selectivity for JAK heterodimeric cytokine signalling
JAK and TYK2 inhibitors inhibit Tfh1 cell differentiation
As IL-12 induces Tfh1 cells to differentiate via pSTAT1 and pSTAT4,7 we investigated the differences in the inhibition of Tfh1 cell differentiation using JAK inhibitors with different selectivity.
First, stimulation of CD4+ T cells with IL-12 resulted in the efficient induction of IFNγ+ IL-21+ producing cells (majorly Tfh1 cells). figure 3A presents comparative data obtained using upadacitinib as a representative JAK inhibitor; reference data for TYK2 inhibitor are also presented. Of note, both inhibitors suppressed the differentiation of cytokine-producing cells. Other JAK inhibitors also inhibited differentiation in a concentration-dependent manner; however, filgotinib showed no inhibitory effect (figure 3B). IL-12 stimulation then efficiently induced the phosphorylation of STAT1 and STAT4. All JAK and TYK inhibitors, except Filgotinib, inhibited phosphorylation in a concentration-dependent manner (figure 3C). Next, IL-12 stimulation induced the differentiation into Bcl6+ T-bet+ cells expressing key transcription factors of Tfh1 cells. This process was inhibited in a concentration-dependent manner by all JAK and TYK inhibitors, except Filgotinib (figure 3D).
IL-12-induced Tfh1 cell differentiation suppressed by JAK and TYK2 Inhibitors. Naïve CD4+ T cells were isolated from healthy donor peripheral blood, and after 3 days of TCR stimulation, Tfh1 cells were induced to differentiate by IL-12 stimulation in the presence of JAK and TYK2 inhibitors. The expression ratio of Tfh1 cells was compared for each inhibitor concentration (0 nM, 30 nM, 100 nM, 300 nM, 1000 nM) with TCR stimulation as the reference. (A) As a comparison of representative data obtained by flow cytometry, the results of suppressing IL-21+ IFNγ+ CD45RA− CD4+ cells with Upadacitinib and TYK2 inhibitors are shown. (B) Expression ratios of IL-21+ IFNγ+ CD45RA− CD4+ cells were compared. (C) Expression ratios of pSTAT1+ pSTAT4+ CD45RA− CD4+ cells were compared. (D) Expression ratios of T-bet+ Bcl6+ CD45RA- CD4+ cells were compared. Data are presented as the mean (SEM), and each dot represents an independent experiment using samples obtained from three different donors; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
These results suggest that JAK and TYK2 inhibitors, except filgotinib, inhibit Tfh1 cell differentiation.
TYK2 inhibitor preserve Treg differentiation unlike other JAK inhibitors
There was marked difference in the differentiation of CD4+ T cells into IL-10+ Foxp3high Treg cells induced by IL-2 and TGFβ stimulation on treatment with inhibitors targeting JAK and TYK2. A representative comparison between upadacitinib, which had the lowest IC50 values for IL-2 signalling inhibition, and TYK2 inhibitor is shown in figure 4A. The disparity in Treg cell differentiation inhibition was significant; TYK2 inhibitors failed to inhibit IL-2 signalling but preserved Treg cell differentiation, whereas upadacitinib dose-dependently inhibited Treg cell differentiation. Moreover, Treg cell differentiation was also inhibited by other JAK inhibitors in a concentration-dependent manner (figure 4B). STAT5 phosphorylation was dose-dependently inhibited by all JAK inhibitors except filgotinib. In contrast, the TYK2 inhibitor did not affect phosphorylation (figure 4C). Furthermore, the IL-2-TGFβ stimulation-mediated generation of CD25+ Foxp3high Treg cells was inhibited in a concentration-dependent manner by all JAK inhibitors (except Filgotinib) but not by TYK2 inhibitors (figure 4D).
TYK2 inhibitors preserve Treg cell differentiation Unlike JAK inhibitors. Naïve CD4+ T cells were isolated from HD peripheral blood, and after 3 days of TCR stimulation, Treg cells were induced by IL-2+TGFβ stimulation in the presence of JAK and TYK2 inhibitors. Treg cell expression ratios were compared for each inhibitor concentration (0 nM, 30 nM, 100 nM, 300 nM, 1000 nM) with TCR stimulation as the reference. (A) As a comparison of representative data obtained by flow cytometry, the results of suppressing IL-10+ Foxp3high CD45RA− CD4+ cells with Upadacitinib and TYK2 inhibitors are shown. (B) Expression ratios of IL-10+ Foxp3high CD45RA− CD4+ cells were compared. (C) Expression ratios of pSTAT5+ CD45RA− CD4+ cells were compared. (D) Expression ratios of CD25+ Foxp3high CD45RA− CD4+ cells were compared. Data are presented as the mean (SEM), and each dot represents an independent experiment using samples obtained from three different donors; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
These results suggest that the TYK2 inhibitor, unlike other JAK inhibitors, does not inhibit IL-2 and TGFβ-induced Treg cell differentiation.
Discussion
This exploratory study aimed to investigate the impact of JAK/TYK2 inhibitors on human lymphocyte differentiation. Specifically, we focused on the imbalance between Tfh subsets and Treg cells in patients with SLE and examined whether TYK2 inhibition could fine-tune the differentiation of Tfh1 cells and active Treg cells using lymphocytes obtained from healthy donors.
In this study, we found an equilibrium between Tfh1 cells and activated Treg cells in patients with SLE. Recent findings have revealed the presence of various Tfh cell subsets, including Tfh1, Tfh2, Tfh17 and Tfr cells, suggesting that Tfh cells exhibit plasticity and undergo cytokine-mediated modifications while expressing diverse phenotypes.2–6 It has been demonstrated that Tfh1 cells are a subset that contributes to the production of high-affinity antibodies, as evidenced by the transient elevation of these antibody levels in human peripheral blood following influenza virus vaccination.34 Despite this, the significance of Tfh1 cells in SLE remained unclear. However, our study suggests that these cells may have pathological relevance in some populations of SLE, particularly in lupus nephritis, where the unbalanced relationship between Tfh1 cells and activated Treg cells is notable. Notably, CD11c+ T-bet+ B cells have been implicated in the pathogenesis of SLE and active nephritis.30–32 Although various T cell subsets have been implicated in the generation of T-bet+ B cells in humans, our study demonstrated that Tfh1 cells induce naïve B cells to differentiate into T-bet+ B cells in vitro. Furthermore, tissue staining revealed the infiltration of Tfh1 cells into renal tissues. Therefore, it was suggested that the imbalance in the number of Tfh1 cells and activated Treg cells observed in patients with SLE may contribute to the increased production of IL-21 and IFNγ by Tfh1 cells and the induction of T-bet+ B cells in renal tissue.
As for the differentiation pathway of Tfh1 cells, previous studies have reported that the production of Tfh1 cells, characterised by the expression of the transcription factors T-bet and Bcl6, as well as cytokines such as IL-21 and IFNγ, is induced via IL-12 signalling pathways involving pSTAT1/4.7 8 Similarly, most T cell differentiation processes are mediated by JAK-dependent cytokines, and we previously reported the impact of JAK inhibitors on the differentiation of several T cell subsets.20 23 Therefore, our second research question addressed the efficacy of JAK/TYK2 inhibitors against immune dysregulation of Tfh1 cells and active Treg cells in patients with SLE. Until now, JAK inhibitors were thought to exhibit high individual selectivity, but the IC50 results from cytokine assays suggested that cross-reactivity occurs. Considering that most JAK inhibitors competitively bind to the ATP-catalysing site of the Jak homology 1 (JH1) domain and inhibit its enzymatic activity, their selectivity may be lower than previously assumed. As demonstrated in the study by Traves et al, upadacitinib, a representative JAK1 inhibitor, appeared to demonstrate cross-reactivity with JAK2 for IL-12 and GM-CSF based on IC50 assays.35 Our study found similar results. In contrast, TYK2 inhibitors exert allosteric inhibition by binding to the regulatory site within the pseudokinase domain of TYK2, altering its molecular structure through intramolecular interactions, and inhibiting enzymatic activity. Thus, we investigated the effects of seven JAK/TYK2 inhibitors on CD4+ CD45RA- T cells. We found yhat JAK1/TYK2-mediated IFNα signalling and JAK2/TYK 2-mediated IL-12 signalling was inhibited by almost all JAK and TYK2 inhibitors, whereas JAK1/JAK3-mediated IL-2 signalling was not inhibited by the TYK2 inhibitor. Correspondingly, Tfh1 cell differentiation was inhibited by almost all JAK inhibitors, whereas Treg cell differentiation was maintained only by the TYK2 inhibitor, indicating that other JAK inhibitors simultaneously inhibited Treg cell differentiation. Based on our findings, therapeutic strategies aimed at maintaining Treg differentiation while suppressing the differentiation of Tfh1 cells, particularly using TYK2 inhibitors, might be a promising approach for treating SLE, especially lupus nephritis.
Various clinical trials have been conducted using JAK and IL-12/23 inhibitors for SLE, and several JAK inhibitor trials are ongoing. The phase 2 BRAVE-1 trial aimed to evaluate the efficacy of baricitinib and showed promising results, but the phase 3 BRAVE-2 trial failed to achieve its primary endpoint and had to be terminated.21 Similarly, in the phase 3 trial of ustekinumab, an IL-12/23 inhibitor, monoinhibitor therapy was deemed unsuccessful.36 However, considering the study design and withdrawal rate, the validity of the study has been debated. Peripheral blood analysis of patients with SLE involved in the BRAVE-1 trial indicated that baricitinib therapy led to the downregulation of various cytokines, suggesting a multitargeted mechanism extending beyond IFN regulation.37 Despite some successful cases, the overall efficacy of baricitinib was not demonstrated, which could be attributed to the inhibition of common-chain cytokine signalling associated with JAK1 inhibition alongside the suppression of type I IFN signatures. JAK1/3 inhibition might have hindered IL-2 signalling and diminished the number and function of Treg cells (which are known to be reduced in SLE), thereby failing to correct immune abnormalities. Moreover, solely inhibiting the IL-12/23 pathway without targeting type I IFN might also not adequately contribute to improving disease outcomes. In contrast, TYK2-selective inhibitors can selectively inhibit TYK2 without affecting JAK1, unlike JAK inhibitors, which employ an ATP competitive inhibitory mechanism. The efficacy of deucravacitinib has not yet been fully established in ongoing clinical trials, and further investigation is required, particularly regarding the lack of demonstrated dose-dependent clinical effects.38 However, there are currently no drugs that can simultaneously inhibit both IFNα and IL-12 signalling while maintaining IL-2 signalling. Based on our findings, we believe that this drug holds promise as a potential novel therapeutic agent.
This study had certain limitations. First, JAK/TYK2 inhibitors are not currently approved for the treatment of patients with SLE. Consequently, it is not possible to directly assess the intake of these inhibitors by patients and whether the observed changes in this study occur in vivo. Additionally, due to ethical considerations, the amount of blood that can be collected from patients is limited, which poses challenges in conducting in vitro differentiation inhibition experiments using patient samples. Another limitation of the in vitro experiments is that the same cell identification markers were used for both inhibition experiments and the analysis of patient peripheral blood lymphocytes. As a result, the inhibition of differentiation led to a significant decrease in cell numbers, making identification difficult. The coculture experiments were also limited to T-bet+ B cells due to low CD11c levels. Furthermore, the impact of the inhibitor on immune cells other than T cells was not investigated.
Nevertheless, despite these limitations, this study provides insights into the significance of Tfh1 cells using specimens from untreated patients with SLE and presents the initial evidence of an imbalance between Tfh1 cells and activated Treg cells, particularly in patients with nephritis. Additionally, no prior studies have investigated the effects of JAK/TYK2 inhibitors on Tfh1 and Treg cell differentiation using live human cells. To our knowledge, this study is the first attempt to explore the potential impact of TYK2 inhibitors, which are currently validated in SLE, on human lymphocyte differentiation. Furthermore, if JAK/TYK2 inhibitors are approved for use in SLE in the future, evaluating lymphocyte differentiation abnormalities in the highly heterogeneous SLE population and assessing which inhibitors align with the pathology could provide valuable insights for future treatment strategies. This could also involve exploring the potential of personalised medicine approaches.
Data availability statement
Data are available on reasonable request.
Ethics statements
Patient consent for publication
Ethics approval
This study involves human participants and was approved by Institutional Review Board of the University of Occupational and Environmental Health (UOEHCRB21-069 and UOEHCRB21-103). Informed consent was obtained from each subject. Participants gave informed consent to participate in the study before taking part.
Acknowledgments
The authors thank N Sakaguchi and C Iwasa for their excellent technical assistance.
References
Supplementary materials
Supplementary Data
This web only file has been produced by the BMJ Publishing Group from an electronic file supplied by the author(s) and has not been edited for content.
Footnotes
Correction notice This article has been corrected since it was first published online. In Figure 2A, the labels 'CXCR3' and 'CXCR5' above the image are incorrect. They have been corrected to 'CXCR3'→ 'Bcl-6' and 'CXCR5' → 'T-bet', respectively.
Contributors YS-K and SN designed the study; YS-K conducted the experiments, analysed the data and wrote the manuscript; SK, AN, RK, KY, YS-K, YF and HT helped to conduct the experiments; and YT created the research concept and supervised the research and writing of the manuscript. Manuscript guarantor: YT
Funding This work was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI (#20K08815). Provision of compounds: BMS-986202 was provided by Bristol–Myers Squibb under an agreement with Bristol–Myers Squibb. Peficitinib was provided by Astellas Pharma under an agreement with Astellas Pharma. Upadacitinib was provided by Abbvie under an agreement with Abbvie. Filgotinib was provided by Gilead under an agreement with Gilead.
Competing interests YT has received speaking fees and/or honoraria from Eli Lilly, AstraZeneca, Abbvie, Gilead, Chugai, Behringer-Ingelheim, GlaxoSmithKline, Eisai, Taisho, Bristol–Myers, Pfizer, Taiho, received research grants from Mitsubishi-Tanabe, Eisai, Chugai, Taisho. SN has received consulting fees, lecture fees, and/or honoraria from Bristol–Myers, AstraZeneca, Pfizer, GlaxoSmithKline, AbbVie, Astellas, Asahi-Kasei, Sanofi, Chugai, Eisai, Gilead Sciences, Eli Lilly, Boehringer Ingelheim. SK has received consulting fees, speaking fees, and/or honoraria from GlaxoSmithKline, Eli Lilly, and Bristol–Myers and has received research grants from Daiichi-Sankyo, Abbvie, Boehringer Ingelheim and Astellas. All other authors declare no conflict of interest. None of the material presented in our manuscript has been previously submitted or published.
Provenance and peer review Not commissioned; externally peer reviewed.
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