CaMK4 controls follicular helper T cell expansion and function during normal and autoimmune T-dependent B cell responses

Scherlinger, Marc; Li, Hao; Pan, Wenliang; Li, Wei; Karino, Kohei; Vichos, Theodoros; Boulougoura, Afroditi; Yoshida, Nobuya; Tsokos, Maria G.; Tsokos, George C.

doi:10.1038/s41467-024-45080-x

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Published: 29 January 2024

CaMK4 controls follicular helper T cell expansion and function during normal and autoimmune T-dependent B cell responses

Nature Communications volume 15, Article number: 840 (2024) Cite this article

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Abstract

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by dysregulated B cell compartment responsible for the production of autoantibodies. Here, we show that T cell-specific expression of calcium/calmodulin-dependent protein kinase IV (CaMK4) leads to T follicular helper (T_fh) cells expansion in models of T-dependent immunization and autoimmunity. Mechanistically, CaMK4 controls the T_fh-specific transcription factor B cell lymphoma 6 (Bcl6) at the transcriptional level through the cAMP responsive element modulator α (CREMα). In the absence of CaMK4 in T cells, germinal center formation and humoral immunity is impaired in immunized mice, resulting in reduced anti-dsDNA titres, as well as IgG and complement kidney deposition in the lupus-prone B6.lpr mouse. In human T_fh cells, CaMK4 inhibition reduced BCL6 expression and IL-21 secretion ex vivo, resulting in impaired plasmablast formation and IgG production. In patients with SLE, CAMK4 mRNA levels in T_fh cells correlated with those of BCL6. In conclusion, we identify CaMK4/CREMα as a driver of T cell-dependent B cell dysregulation in autoimmunity.

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Introduction

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by a dysregulated B cell compartment which leads to the emergence of pathogenic autoantibodies and subsequent tissue damage¹. The standard of care relies on non-targeted immunosuppressive drugs such as corticosteroids and cytotoxic drugs, which increase the risk of infection that nowadays represents the first cause of death in SLE^2,3. Although recent advances in our understanding of SLE pathogenesis have fostered the development of promising targeted treatments⁴, many have failed in phase-3 trials. There is, therefore, a major need to identify new therapeutic targets to treat patients with SLE.

T-cell dysregulation has been consistently associated with lupus pathogenesis. We previously reported that SLE T cells are characterized by an aberrant T cell receptor complex (TCR) signaling, whereby spleen tyrosine kinase (Syk) is recruited in the place of Zap70 and induces aberrant signaling including enhanced and earlier intracellular calcium concentrations following the engagement of the CD3/TCR^5,6. This increased intracytoplasmic calcium is responsible for the activation of protein kinase C and other calcium-controlled kinases, including the serine/threonine calcium-calmodulin dependent kinase IV (CaMK4)⁶. In patients with SLE, especially in those with active disease, increased T cell calcium activates CaMK4, which skews T cells toward the proinflammatory Th17 compartment and promotes T regulatory (Treg) cell dysfunction^7,8. Interestingly, lupus-prone mice with a Camk4 genetic deletion are characterized by a normalized T cell subset distribution together with decreased levels of autoantibodies⁹, suggesting the importance of CaMK4 in the humoral (auto-) immune response.

Since B cells express low levels of CaMK4¹⁰, we hypothesized that CaMK4 might control humoral response through distinct T cell subsets. T follicular helper (T_fh) cells are a subset of CD4⁺ T cells which express the Bcl6 transcription factor and the CXCR5 chemokine receptor, which allows T_fh migration into the secondary lymphoid structures through a gradient of CXCL13¹¹. T_fh cells accumulate in the T zone of the germinal center and drive B cell affinity maturation and isotype switching, notably through the expression of CD40L and the production of interleukin-21 (IL-21). In murine and human SLE, T_fh cells have been shown to be abnormally expanded and activated and to drive autoantibody production and tissue damage^12,13. The mechanisms that mediate T_fh expansion are likely multifactorial and include increased T_fh differentiation through the OX40/OX40L axis and platelet activation^14,15, transdifferentiation from regulatory T cells (T_reg)¹⁶, and impaired T follicular regulatory (T_fr) cell function^17,18. In the current study, we investigated the importance of the expression of CaMK4 in T cells in the activation and functional expansion of T_fh cells in the setting of immunization with nominal antigens and autoantigens.

Using mice with a T cell-specific genetic deletion of Camk4, we showed that CaMK4 is responsible for the expansion of murine T_fh cells after immunization and is necessary for the development of a full-blown humoral response. Mechanistically, CaMK4 promotes the expression of Bcl6 through the activation of the cAMP response element modulator α (CREMα) transcription factor. In the B6.lpr lupus-prone mouse, the deletion of Camk4 in T cells leads to the reduction of circulating and tissue T_fh cells, decreased levels of multiple pathogenic B cell subsets, and improvement of biological and clinical hallmarks of the disease. At a translational level, CaMK4 inhibition in primary human T_fh cells led to the downregulation of BCL6 expression, IL-21 production, and B cell-stimulating properties of T_fh. Overall, this study identifies CaMK4 as a modulator of T-dependent humoral immunity in the normal and autoimmune response.

Results

CaMK4 drives T_fh expansion through the transcriptional control of Bcl6 expression

We previously found that murine Treg cells express high levels of CaMK4 compared to B cells⁸. To evaluate CaMK4 expression in various immune compartments, we used fluorescent-activated cell sorting to isolate B (B220⁺CD3⁻) cells, T_fh (CD3⁺CD4⁺CD25⁻CD44⁺CXCR5⁺PD1⁺) cells and T_reg (CD3⁺CD4⁺CD25⁺) cells from the splenocytes of C57Bl/6 mice. Using reverse-transcription quantitative qPCR, we confirmed that Treg cells express high levels of Camk4 while B cells express minimal amounts (Fig. 1a). Interestingly, T_fh cells also express high levels of Camk4 (Fig. 1a), suggesting its role in T_fh biology. To evaluate the importance of Camk4 expression for T_fh cell development, we immunized wild-type C57Bl/6 (designed as WT, Camk4^fl/fl) or T cell-specific Camk4-deficient (Camk4^fl/fl.dlck-Cre) mice with sheep red blood cells (SRBC). On day 7 after immunization, WT mice had an expansion of CXCR5⁺ PD1⁺ T_fh cells within splenocytes (Fig. 1b; Fig. S1A, p < 0.001), while those with T cell-specific CaMK4 deficiency did not (p = ns). Conversely, CXCR5⁻PD1⁺ T peripheral helper (T_ph) cells were not affected by T cell-specific Camk4 deletion (Fig. S1B, C). Impaired expansion of Tfh cells upon SRBC immunization was confirmed using intracellular staining for BCL6 (Fig. 1c and Fig. S1D). Mice with global Camk4 deletion had a similar impaired expansion of CXCR5⁺ PD1⁺ T_fh after immunization (Fig. S1E, F). Since we previously showed that CaMK4 expression negatively affects Treg cell differentiation and function in the settings of autoimmunity⁸, we asked whether this was also true in our model of T cell-dependent immunization. Neither T_reg, nor T_fr (CD3⁺CD4⁺CD25⁺FoxP3⁺CXCR5⁺) cells were affected by CaMK4 expression at day 7 after SRBC immunization (Fig. S1G, H).

**Fig. 1: CaMK4 drives T_fh expansion during T cell-dependent immunization.**

To investigate the molecular mechanism underlying these differences, we differentiated in vitro CD62L⁺ CD4⁺ T cells to inducible T_fh (iT_fh) cells (see methods), leading to the expression of Bcl6 and PD1 (Fig. 2a; gating Fig. S2A). CaMK4 deficiency led to a significant downregulation of Bcl6 (p < 0.001) but not PD1 on iT_fh (Fig. 2b, c). Conversely, transfection of iTfh cells with a CaMK4-overexpressing vector led to increased Bcl6 expression (Fig. S2B). We and others have previously shown that CaMK4 positively or negatively controls the expression of several genes through the expression of cAMP-responsive element modulation α (CREMα)^7,9. Indeed, activated CaMK4 binds and phosphorylates (i.e., activates) CREMα, which leads to CREMα relocalization to the nucleus and the modulation of gene transcription. By analyzing the promoter for the murine Bcl6 gene, we found 2 CRE-binding sites, one of which was highly conserved among mammals (human, mouse, rabbit). We, therefore, hypothesized that CaMK4 may promote Bcl6 gene expression through CREMα. To address this, we cloned the Bcl6 gene promoter on a pGL3 Renilla luciferase reporter vector and conducted a site-directed mutagenesis to delete the highly conserved CRE-binding site (Mut vector, Fig. 2d). The Renilla vector was transfected, along with a control firefly luciferase vector, in cells two days after the initiation of iTfh differentiation and luciferase activity assessed at day 3. The normalized luciferase activity was significantly decreased in the Mutated (Mut) vector compared to the WT vector (p < 0.01, Fig. 2e). Furthermore when the reporter vectors were transfected in Camk4⁻^/⁻ or in Icer/Crem⁻^/⁻ T cells, the luciferase activity was similarly decreased (Fig. 2e). To confirm these results, we conducted chromatin immunoprecipitation (CHIP−) PCR on iTfh cells (see Methods). DNA pulldown with a CREMα-specific antibody led to significant enrichment of the Bcl6 gene promoter region (Fig. 2f). Conversely, in Camk4⁻^/⁻ iTfh, the enrichment was significantly lower compared to WT iTfh (Fig. 2f), suggesting that CaMK4 expression is instrumental in CREMα binding to the Bcl6 gene promoter region. Overall, our results suggest that CaMK4 is responsible for the expansion of T_fh cells during T cell-dependent immunization through the promotion of the Bcl6 gene expression by CREMα.

**Fig. 2: CaMK4 modulates *Bcl6* gene expression through CREMα.**

T cell CaMK4 controls germinal center formation and B cell response during immunization

Next, we evaluated T_fh differentiation and function in vivo using the murine SRBC immunization model. Splenic germinal center B (GCB; B220⁺CD93⁻CD95⁺GL7⁺) cells were significantly decreased at 7 and 14 days after immunization in mice with T cell-specific Camk4 knockdown compared to controls (Fig. 3a, b). Before and after immunization, other B cell subpopulations were unremarkable (Fig. S3A–C). Consistently, we observed impaired formation of organized splenic germinal centers in Camk4^fl/fl.dlck^Cre mice (Fig. 3c, d). Since germinal center formation is important for positive selection and affinity maturation of IgG-producing B cells¹⁹, we evaluated the humoral response with another model of T cell-dependent immunization, the 4-Hydroxy-3-nitrophenylacetyl chicken γ-globulin (NP-CGG) model (Fig. 4a). After the immunization, the levels of total IgG were similar in Camk4^fl/fl.dlck-Cre and Camk4^fl/fl mice (Fig. 4b). However, NP-specific GC B cells and plasma cells were significantly decreased in Camk4^fl/fl.dlck-Cre mice compared to their Camk4-sufficient counterparts (Fig. 4c, d). This was associated with decreased levels of high affinity (NP-7) IgG, but not mixed affinity IgG (Fig. 4e), with a decrease of the anti-NP7/NP44 IgG ratio suggesting an impaired affinity maturation (Fig. 4f). The study of IgG subclasses identified that IgG1 and IgG3 were mainly affected by T cell-specific Camk4 deletion (Fig S4A–C). Importantly, mice with a global Camk4 deletion also developed a similarly impaired antibody response to NP-CGG (Fig. S4D). Overall, these results demonstrate that The T-cell expression of CaMK4 positively controls the germinal center formation and the high-affinity humoral response in the setting of T-dependent immunization.

**Fig. 3: T cell CaMK4 drives germinal center formation during immunization.**

**Fig. 4: T cell CaMK4 drives antigen-specific IgG response to immunization.**

T cell CaMK4 controls pathogenic B cell development and autoantibody production in autoimmunity

To evaluate the relevance of our findings in autoimmunity, we crossed the B6.lpr with the Camk4^fl/fl.dlck-Cre mouse to generate a T cell-specific, Camk4-deficient lupus-prone mouse. B6.lpr.Camk4^fl/fl and B6.lpr.Camk4^fl/fl.dlck-Cre littermates were sacrificed at 34 weeks of age. B6.lpr, which did not express CaMK4 in T cells, was characterized by decreased levels of PD1⁺ T_fh cells in the spleen, cervical lymph nodes (cLNs) and the blood (gating Fig. S5A; Fig. 5a and Fig. S5B) while the levels of Tfr cells were similar (Fig. S5C). Furthermore, the deletion of Camk4 in T cells led to a change in splenic B cell populations, with a decrease in the pathogenic age-associated B cells (B220⁺CD93⁻CD23⁻CD21/35⁻; ABC) and normalization of follicular B cells (B220⁺CD93⁻CD23⁺CD21/35⁺; FOB; Fig. 5b; Fig. S5D, E). Similar to our findings in the immunization model, GCB cells were significantly decreased in the spleen and cLNs from B6.lpr.Camk4^fl/fl.dlck-Cre mice (Fig. 5c and Fig. S5F). Importantly, antibody-producing CD138⁺ plasma cells were also decreased in T cell-specific Camk4-deficient mice (Fig. 5d and Fig. S5G). These differences resulted in decreased total and subclass-specific anti-dsDNA antibodies (Fig. 5e, f). Hypergammaglobulinemia, a hallmark of B cell hyperactivation in autoimmunity, was absent in B6.lpr.Camk4^fl/fl.dlck-Cre mice (Fig. 5g). At the tissue level, we had previously shown that the B6.lpr.Camk4^fl/fl.dlck-Cre were characterized by improved kidney pathology⁸. These mice also showed a significant decrease in the glomerular IgG and C3 deposition as evaluated by immunofluorescence (Fig. 5h, i). Overall, the deletion of the Camk4 gene in T cells leads to decreased T_fh cells, normalization of B cell subsets, decreased auto-antibody production, and organ damage in systemic autoimmunity.

**Fig. 5: Deletion of CaMK4 in T cells corrects B cell dysregulation and improves autoimmunity.**

CaMK4 controls BCL6 expression and T_fh cell function in healthy individuals and patients with SLE

To provide translational value of our findings from mice, we next sorted T_fh (CD4⁺CXCR5⁺CD25⁻CD127⁺), B memory (CD19⁺IgD⁻CD27⁺), and B naïve (CD19⁺IgD⁺CD27⁻) cells from healthy donors using FACS (Fig. S2A, B). Using reverse transcriptase qPCR, we found that human T_fh cells expressed significantly more CAMK4 compared to B cells (Fig. 6a), similar to our findings in mice. Primary human T_fh cells cultured for 48 h with a CaMK4 inhibitor (KN93) significantly downregulated the expression of BCL6 and IL21 (p < 0.05, Fig. 6b), while T_fh activation (as assessed by PDCD1 expression) was not affected (Fig. 6b). We confirmed at the protein level that IL21 production was significantly downregulated upon CaMK4 inhibition (Fig. 6c). To confirm that the impact on BCL6 expression was mediated by CAMK4 inhibition, we transfected human primary Tfh cells with CAMK4 target siRNA which led to BCL6 downregulation (Fig. S6). To investigate whether the T_fh cell function was affected by CaMK4 inhibition, we cocultured freshly sorted human T_fh cells and B memory cells together with staphylococcus enterotoxin B (SEB). After 7 days of coculture, the percentage of CD38⁺CD27⁺ plasmablasts was significantly decreased in the presence of the CaMK4 inhibitor KN93 (Fig. 6d). Furthermore, the IgG production as measured in the coculture supernatant was significantly decreased in the presence of KN93 (Fig. 6e). Finally, we sought to evaluate the relevance of these findings in human SLE. We sorted T_fh, Treg, and B cells from frozen PBMCs of 13 patients with SLE (Table S1). CAMK4 expression was significantly higher in the T_fh cells compared with the other subsets (Fig. 7a). There was no difference in Tfh cells CAMK4 expression between healthy donors and SLE patients (Fig. S7A). As previously shown²⁰, CAMK4 expression is not affected by SLE disease activity (Fig. S7B), whereas its activation and nuclear localization (which mediates its genetic effect) is²⁰. In SLE T_fh cells, there was a significant correlation between CAMK4 and BCL6 expression (r² = 0.626, p = 0.002; Fig. 7b), suggesting that CaMK4 may control BCL6 expression in human SLE in the same way that we described in mice.

**Fig. 6: CaMK4 modulates human T_fh cell function.**

**Fig. 7: CaMK4 expression in SLE T_fh cells is increased compared with T_reg cells and correlates with T_fh *BCL6* expression.**

Discussion

In the current study, we have demonstrated that T cell-specific CaMK4 expression drives T_fh cell expansion and, ultimately, humoral immune response during immunization and nominal antigens and autoimmunity. A T cell-specific deletion of Camk4 led to impaired T_fh cell expansion, decreased germinal center formation, and overall antibody responses after immunization with T cell-dependent antigens. Mechanistically, we found that CaMK4 controls the transcription of the Bcl6 lineage T_fh-determining transcription factor through the activation of CREMα. Using human primary T_fh cells, we confirmed that CaMK4 was necessary for the B cell helper function of T_fh.

CaMK4 is expressed in multiple subsets of CD4⁺ T cells, and its activation is also involved with the differentiation and function of Th₁₇^7,21 and Treg cells^8,9. In the immunization model, we used a mouse with a genetic deletion of Camk4 in all T cells, which opens the possibility that other subsets of T cells were affected by this deletion. However, we did not find any changes in the levels of T_reg nor T_fr cells (Fig. 1d), supporting the fact that the differences observed in terms of B cell responses are linked to the role of CaMK4 on the T_fh cell compartment.

Interestingly, our results are different from those observed by Nakaya and coauthors²². They observed that the day 3 expression of CAMK4 in PBMCs from individuals immunized with the inactivated influenza vaccine negatively correlated with the vaccinal antibody response at day 28²². However, a recent transcriptomic atlas encompassing more than 3000 individuals immunized with 13 different vaccines failed to identify an association between CAMK4 expression (or any other gene) and antibody response²³. Furthermore, Haralambevia and colleagues found that T-cell CAMK4 expression was (non-significantly) negatively associated with the Fluad vaccine antibody response and (non-significantly) positively associated with the Fluzone vaccine antibody response²⁴. Nakaya and colleagues²² also showed that mice with a Camk4 germline deletion developed enhanced IgG response to flu vaccines, which is in contrast to our results with NP-CGG immunization (Fig. S4D). The differences might be explained by the different routes of immunization (intraperitoneal vs. intramuscular injections)²⁵ or the presence of adjuvant (Alum in our study vs. no adjuvant). Indeed, different vaccination routes result in different diffusion of the antigen to secondary lymphoid organs that might impact the T/B cell response to the antigen.

CREMα belongs to a superfamily of transcription factors, which also includes the inducible cAMP early repressor (ICER) and the cAMP-responsive element binding proteins—1 and 2 (CREB-1/2)²⁶. All these transcription factors recognize the CRE-binding site and result in either the upregulation or the downregulation of the gene. For example, CaMK4 activation of CREMα is responsible for the upregulation of the Il17a gene, which promotes the expansion of the Th₁₇ subset in autoimmunity^7,27. Conversely, CaMK4 activation of CREMα leads to the downregulation of the Il2 gene, which results in the impairment of Treg cell expansion and function and may worsen autoimmunity^9,20. Importantly, CAMK4 gene expression is similar between healthy donors and SLE patients (Fig. S6A), but CaMK4 protein phosphorylation (i.e., activation) and nuclear localization are increased in SLE^8,20. In the current study, we found that the presence of the CRE-binding site is necessary for the transcription control of Bcl6 in murine T_fh cells and confirmed using Icer/Cremα⁻^/⁻ mice that this effect is mediated by ICER/CREMα and not through CREB.

Although SLE is viewed as an auto-antibody-mediated disease, strategies aiming at the containment of the B cells compartment, notably with rituximab or other B cell-depleting antibodies, have been disappointing^28,29. Advances in our understanding of the molecular mechanisms underlying T cells and B cell dysregulation in autoimmunity have led to the development of new targeted treatments. These include targeted synthetic disease-modifying anti-rheumatic drugs (tsDMARD) such as Janus kinase (JAK), non-receptor tyrosine kinase 2 (Tyk2) or Bruton’s tyrosine kinase (BTK) inhibitors⁴. This study, along with previous studies from our team, suggests that the inhibition of CaMK4 signaling represents a valuable strategy in the treatment of patients with SLE. Indeed, CaMK4 is involved in the dysregulation of several subsets of T cells, including T_fh, T_reg^8,9, and Th₁₇ cells^7,21, but also in the promotion of kidney damage through its expression in podocytes^30,31. The main issue with tsDMARD are the off-target effects. For example, these off-target effects may be responsible for elevated risk of thrombosis and cancer of the JAK inhibitor Tofacitinib³². CaMK4 is also expressed in the hippocampus³³, underlying the importance of developing compounds or drug delivery strategies that do not cross the blood-brain barrier, such as CD4-tagged nanolipogels loaded with a CaMK4 inhibitor³⁴.

The strengths of our study include the confirmation of our findings in mice using primary human T_fh cells and the study of a group of SLE patients, with nearly half of them having high disease activity at the time of the sampling (Table S1). The main limitation of our study is the use of mice with deletion of Camk4 in all T cells and not only in T_fh cells. For example, restored IL-2 production in CaMK4-deficient mice may participate in Tfh cell dysfunction and impaired follicular center formation³⁵. However, we believe that CaMK4 activation is an early event during naïve T cell activation and differentiation. Therefore, the deletion of CaMK4 in fully differentiated T cells by using Camk4^fl/fl.Bcl6-Cre mice may not recapitulate the events that happen during the generation of T_fh cells while a normal or autoimmune response evolves.

In conclusion, this study identifies CaMK4 activation in T cells as a major factor responsible for the expansion of T follicular helper cells and a full-blown T-dependent humoral response in the setting of immunization and autoimmunity. These findings reinforce the rationale for CaMK4 inhibition as a multitarget therapy in autoimmune diseases such as SLE.

Methods

Human samples

Healthy donors and patients with a diagnosis of SLE fulfilling the ACR/EULAR 2019 classification criteria of SLE were recruited in the study. All participating volunteers provided written informed consent. The Beth Israel Deaconess Medical Center Institutional Review Board approved this study protocol. Peripheral blood was drawn in heparin-lithium tubes and processed either fresh or after thawing of previously frozen PBMCs. Blood collars from healthy blood donors were obtained from the Boston Children’s Hospital donor center.

Mice

The animals were housed in a pathogen-free Animal Facility in the Beth Israel Deaconess Medical Center (BIDMC, Center for Life Science). B6.129 × 1-Camk4tm1Tch/J (Camk4^−/−, stock #004994) and B6.MRL-Faslpr/J (B6.lpr; stock #000482) and C57Bl/6 (stock #000664) were obtained from the Jackson laboratory. B6.Camk4^fl/fl.dlck^Cre mice obtained from previous studies⁸, and were crossed with the B6.MRL-Faslpr/J to obtain B6.lpr.Camk4^fl/fl.dlck^Cre. The SV129/Bl6.Icer/Cremα⁻^/⁻ mice were obtained from Günther Schuetz (Das Deutsche Krebsforschungszentrum, Heidelberg, Germany)³⁶ and were backcrossed with C57BL/6 mice for over nine generations. Immunization of 8-week-old mice of both sexes was conducted by the i.p. injection with either 100 µL of sheep red blood cells (Thermofisher, Waltham, MA, USA), or 25 µg of Alum-conjugated 4-Hydroxy-3-nitrophenylacetyl chicken γ-globulin (NP-CGG, ratio 20-29; Biosearch technologies, Petaluma, CA, USA) diluted in sterile PBS. The recall immunization was conducted with an i.p. injection of 50 µg of NP-CGG diluted in sterile PBS. Experimental and control animals were bred together (B6.Camk4^fl/fl.dlck^Cre and B6.lpr.Camk4^fl/fl.dlck^Cre) or in separate cages (other strains). Euthanasia was conducted using CO₂ inhalation following the recommendations from the Institutional Animal Care and Use Committee. The animal study protocol was approved by the BIDMC Institutional Animal Care and Use Committee.

Cell culture and in vitro T cell differentiation

Murine CD62L⁺CD4⁺ T cells were isolated using magnetic cell sorting from mouse splenocytes according to the manufacturer’s instruction (Miltenyi Biotec, Charlestown, MA, USA). In total, 3 × 10⁵ cells were cultured in a 48-well plate coated with goat anti-hamster crosslinking antibody in RPMI 1640 medium complemented with penicillin/streptomycin, 10% fetal bovine serum, and ß-mercaptoethanol. To differentiate the cells into T_fh, the medium was supplemented with anti-CD3 (0.25 µg/ml, 145-2C11; BioLegend, San Diego, CA), anti-CD28 (0.5 µg/ml, 37.51; BioXcell, Lebanon, NH), interleukin-21 (50 ng/mL, R&D systems, Minneapolis, MN, USA) interleukin-6 (100 ng/mL, R&D systems), anti-TGFß (0.5 µg/mL, clone 1D11; R&D systems) anti-IFN-gamma (10 µg/ml; BioXcell, Lebanon, NH, USA), and anti-IL-4 (10 µg/ml; BioXcell). The cells were retrieved for analysis on day 3 of the culture.

For the coculture experiment using human T_fh and B cells, 50,000 freshly sorted autologous T_fh and B cells were cocultured at a 1:1 ratio in a 96-well plate with staphylococcal enterotoxin B (SEB, 1 ng/mL, Fisher Scientific) for 7 days at +37 °C. The CaMK4 inhibitor KN93 (Selleckchem, Houston, TX, USA) or its vehicle (dimethysulfoxide, DSMO) was used at the concentration of 10 µM.

Western blotting

Cell lysates were loaded on NuPAGE 10% Bis–Tris Gel (ThermoFisher Scientific) for electrophoresis and later transferred to a nitrocellulose membrane. The membrane was blocked with skimmed milk 6% for 1 h and incubated with the antibodies. The primary antibodies used were rabbit polyclonal anti-Bcl6 (ThermoFisher Scientific, Ref PA5-27390, dilution 1:1000) and mouse anti-ß-actin (Sigma-Aldrich, clone AC-74, dilution 1:2500). The secondary antibodies were goat anti-rabbit horseradish peroxidase- (HRP-) conjugated antibody (ThermoFisher Scientific, reference 31460) and an anti-mouse IgG HRP-conjugated antibody (Abcam, reference ab6789). The membranes were revealed using an ECL system (Cytiva, Marlborough, MA) using a ChemiDoc XRS+ (Bio-Rad, Hercules, CA USA), and the obtained picture was analyzed using the ImageJ software (NIH).

Flow cytometry and cell sorting

Surface staining was conducted in phosphate-buffered saline (PBS) at room temperature for 15 min except for CXCR5 staining, which was incubated for 60 min at +4 °C. For intracellular staining, the cells were fixed with Cytofix/Cytoperm buffer (BD Bioscience, San Jose, CA, USA) according to the manufacturer’s instruction and stained overnight in Perm/wash buffer at +4 °C. For cytokine staining, the cells were cultured for 4 h in fully supplemented RMPI along with phorbol myristate acetate (500 ng/mL; Sigma-Aldrich), ionomycin (1.4 µg/mL; Sigma-Aldrich, Saint-Louis, MO, USA) and GolgiStop (BD Biosciences). A list of the antibodies used in this study is found in Table S2. The acquisition was conducted using Cytoflex (Beckman Coulter, Brea, CA, USA). Fluorescence-activated cell sorting (FACS) was conducted using a FACSAria (BD Biosciences). For human cell FACS, CD4⁺ T cells and total B cells were presorted from a blood collar using RosetteSep human CD4⁺ T cells or human B cells (Stemcell technologies, Kent, WA, USA), respectively, according to the manufacturer’s protocol. Initial gating strategies are shown in Fig. S8.

ELISA

Specific IgG was measured using an in-house enzyme-linked immunosorbent assay (ELISA)³⁷. Briefly, the target protein was coated on a MaxiSorp Nunc ELISA plate (Sigma-Aldrich) overnight (NP-7 and NP-44: 20 µg/mL; Calf DNA 50 µg/mL; Sigma-Aldrich). In the case of calf DNA, the plates were pre-coated with poly-l-lysin (50 µg/mL) for 2 h at room temperature. After blocking with PBS-BSA 1% for 2 h, the serum diluted in PBS–BSA 1% was incubated for 2 h at room temperature with shaking. The plate-bound IgG were revealed using an alkaline phosphatase-conjugated anti-mouse IgG (Jackson Immuno Research; reference 1:5000) or with an alkaline phosphatase-conjugated anti-mouse IgG1, 2c, or 3 (Jackson ImmunoResearch, West Grove, PA, USA; references 115-055-205, 115-055-208, and 115-055-209, respectively) for 1 h. The colorimetric reaction was started using diethanolamine substrate buffer (ThermoFisher Scientific) and p-Nitrophenyl phosphate phosphatase substrate, and the optical density was read at 405 nm.

Site-directed mutagenesis, vector, and siRNA transfection

A construct with the Bcl6 gene promoter the luciferase reporter (pGL3_bcl6_vector), and the CaMK4 overexpression vector was obtained from Genscript. The list of vectors used in the study is given in Table S3. Site-directed mutagenesis was conducted to induce deletion of the −62/−58 CRE binding site (CGTCA) using the Q5 site-directed mutagenesis kit (New England Biolabs, Ipswich, MA, USA) with the following primers: forward, 5′- GGAGCCCACGTGACGGCG-3′ and reverse, 5′-TCGGCGTCTTCGCTGTAGC-3′. The deletion was confirmed with Sanger sequencing (Azenta) using the RVprimer 3 primer. The cells were transfected at day 2 of differentiation with 10 µg of the vector using an Amaxa Nucleofector device (Lonza, Basel, Switzerland). The program X-001 was used with the Amaxa Mouse T cell nucleofector kit (Lonza) according to the manufacturer’s protocol. The results were assessed at day 3 (24 h after transfection).

For siRNA transfection, primary cells were transfected with 20 nM of SMARTpool CAMK4 siRNA (Horizon discovery®) using an Amaxa Nucleofector device (list of siRNA in Table S2). The program T-023 was used with the Amaxa Human T cell nucleofector kit (Lonza) according to the manufacturer’s protocol. After transfection, the cells were rested for 6 h in total RPMI and subsequently cultured on 96-well plates coated with anti-CD3 (2 µg/mL) in complete RPMI supplemented with soluble anti-CD28 (2 µg/mL) for 42 h before RNA extraction.

Luciferase assay

The firefly luciferase reporter vector with the wild-type or mutated Bcl6 promoter was transfected (10 µg of vector per transfection) along with 200 ng of a Renilla luciferase vector used as internal control. The transfection was conducted on day 2, and the luciferase activity was assessed on day 3 using the Promega Dual Luciferase Assay System (Promega, Madison, WI, USA) according to the manufacturer’s instructions.

Chromatin immunoprecipitation (CHIP-) PCR

Inducible Tfh cells were differentiated from the CD62L⁺CD4⁺ T cells from C56Bl/6 or Camk4⁻^/⁻ mice, as described previously. Cells were lysed, and immunoprecipitation was conducted using the MAGnify Chromatin Immunoprecipitation System (Thermo Fisher Scientific) with anti-CREMα (Santa-Cruz; Table S2) or control antibody according to the manufacturer’s instructions. qPCR was conducted using a primer specific for the Bcl6 gene promoter region (Table S3). Relative binding was calculated by the comparison (ΔCt) between CREMα and control IgG with the formula 2⁻^deltaCt.

Immunofluorescence

Mouse tissues, including both kidneys and spleens, were embedded in optimal cutting temperature (OCT) tissue media (Tissue-Tek, Torrance, CA, USA) and frozen on dry ice. Coronal 6 µm sections were cut using a cryotome and blocked using PBS-BSA 5%. Mouse kidneys were stained with anti-mouse IgG phycoerythrin (T-6390, Invitrogen, Carlsbad, CA, USA; dilution 1:100), fluorescein-conjugated anti-C3 (SKU 0855500, MP Biomedicals, Irvine, CA, USA; dilution 1:100) and mounted with ProLong Gold antifade mountant with DAPI (Thermofisher). The image acquisition was conducted using a BZ-X800 analyzer. The mean fluorescence intensity of each marker was assessed for 20 glomeruli per mouse. Spleens were stained with Biotin-conjugated anti peanut allergen (anti-PNA, Vector lab, reference 1075-5), Alexa Fluor 647 anti-mouse/human B220 (Biolegend, reference 103226), Alexa Fluor 488 anti-mouse CD4 (Biolegend, reference 100529). Secondary staining was done using Streptavidin-Alexa Fluor 405 conjugate (ThermoFisher, reference S32355). The slides were mounted with ProLong Gold antifade mountant (Thermofisher), and the image acquisition was conducted using a Zeiss LSM 780 confocal microscope.

Quantitative PCR

The RNA was isolated with the TRIzol reagent (Sigma-Aldrich), and complementary DNA was retrotranscribed from RNA using cDNA EcoDry tubes (Takara, Mountain View, CA, USA) following the manufacturer’s instructions. The qPCR was conducted using Taqman probes (see list in Table S3), Taqman reaction buffer, and cDNA according to the manufacturer’s instruction and evaluated with a LightCycler 480 thermocycler (Roche, Indianapolis, IN, USA).

Statistical analysis

The figures and statistical analysis were made using GraphPad Prism Version 9.4.0. In the figures, the points indicate an individual data point (independent experiment or mouse), and bars represent mean ± SEM. Quantitative data were compared using Student’s two-tailed t-test for 2 groups or one-way analysis of variance (ANOVA) together with Holms–Sidak’s correction unless stated otherwise. The non-parametric Spearman correlation test was used for correlation studies. To analyze the IgG response over time, we used a two-way ANOVA, taking into account sampling time and the mice (matched samples). Multiple testing was corrected using the Hold–Sidak’s correction. A p-value < 0.05 was considered statistically significant.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

Source data are provided in this paper. Uncut Western blot membranes are provided in the source data file. The raw data that support the findings of this study are available from the corresponding author upon request. Source data are provided in this paper.

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Acknowledgements

This work was funded by National Institutes of Health grant R37 AI49954 (to G.C.T.). M.S. was financially supported by the Société Française de Rhumatologie, Arthur-Sachs & Monahan fellowships, and the Philippe Foundation. M.S. current position (France) is supported by the Institut National pour la Santé et la Recherche Médicale (INSERM), the Bettencourt-Schueller Foundation, the ATIP-Avenir program and the Foundation for Research in Rheumatology (Foreum).

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Authors and Affiliations

Department of Medicine, Beth Israel Deaconess Medical Center, Boston, MA, USA
Marc Scherlinger, Hao Li, Wenliang Pan, Wei Li, Kohei Karino, Theodoros Vichos, Afroditi Boulougoura, Nobuya Yoshida, Maria G. Tsokos & George C. Tsokos
Rheumatology department, Strasbourg University Hospital of Hautepierre, Strasbourg, France
Marc Scherlinger
Laboratoire d’ImmunoRhumatologie Moléculaire, Institut National de la Santé et de la Recherche Médicale (INSERM) UMR_S 1109, Strasbourg, France
Marc Scherlinger

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Marc Scherlinger
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Hao Li
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Contributions

M.S., H.L. and G.C.T. conceptualized and designed the study. M.S., H.L., W.P., N.Y., K.K., W.L. T.V., and M.G.T. performed the experiments, data analysis, and interpretation. G.C.T. and A.B. provided the human samples. M.S. and G.C.T. prepared the paper.

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Scherlinger, M., Li, H., Pan, W. et al. CaMK4 controls follicular helper T cell expansion and function during normal and autoimmune T-dependent B cell responses. Nat Commun 15, 840 (2024). https://doi.org/10.1038/s41467-024-45080-x

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Received: 16 January 2023
Accepted: 11 January 2024
Published: 29 January 2024
DOI: https://doi.org/10.1038/s41467-024-45080-x

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