Human peripheral blood mononuclear cells (PBMC) were isolated through standard gradient centrifugation using Ficoll-lymphoprep (Axis-Shield) from buffy coats obtained from healthy blood donors (Sanquin Blood Supply). Donors gave informed consent approved by the local institutional review board and the Medical Ethics Committee of Sanquin Blood Supply. CD4⁺ T cells were purified from PBMCs using anti-CD4 Dynabeads and DETACHaBEAD (Invitrogen). Untouched naive CD4⁺ T cells (CD4⁺CD45RO^-) were isolated with high purity (>98%) using CD45RO-PE antibodies and anti-PE beads (MACS; Miltenyi Biotec). Cells were cryopreserved in liquid nitrogen.

Naive CD4⁺ T cells labeled with Cell Trace CFSE according to manufacturer’s instructions (Invitrogen) were cultured in 96-round bottom well plates at a density of 2.5 x 10³/well in a total volume of 200 µl complete RPMI 1640 medium (Invitrogen), supplemented with 5% FCS (Bodinco), 100 U/ml penicillin (Invitrogen), 100 µg/ml streptomycin (Invitrogen), 2 mM L-glutamine (Invitrogen), 50 µM 2-ME (Sigma) and 20 µg/ml human apotransferrin (Sigma; depleted for human IgG with protein G Sepharose (Amersham Biosciences)). Cells were activated with varying doses of anti-CD3 (clone 1XE; Sanquin) and 1 μg/mL anti-CD28 (clone 15E8; Sanquin) with or without 50 ng/mL IL-21 (Invitrogen), 50 ng/mL IL-4 (Cellgro), and 50 ng/mL IFN-γ (Peprotech).

Surface markers and intracellular cytokines were detected by the following FACS anti-human antibodies: CD4 (SK3; BD Biosciences); IL-21 (3A3-N2; eBiosciences); IFN-γ (B27; BD Biosciences); and IL-4 (3010.211; BD Biosciences). Before staining, cells were stimulated in a complete medium with 0.1 µg/ml PMA (Sigma), 1 µg/ml ionomycin (Sigma), and 10 µg/ml brefeldin A (Sigma) for 5 hours. Samples were washed twice with PBS and stained with a Fixable Near-IR Dead Cell Stain Kit (Invitrogen). Cells were fixed with 4% PFA for 15 minutes, permeabilized with 0.5% saponin in PBS containing 1% BSA, and incubated with fluorescent antibodies for 30 minutes at room temperature. All cells from the samples were collected on an LSRII flow cytometer (BD Bioscience) and analyzed with FACSDiva software (BD) and FlowJo version 10 (Treestar).

RT-PCR was performed as previously described (32). RNA was reverse transcribed to cDNA using random hexamers, Superscript II, and an RNase H-reverse transcriptase kit. Primers for 18S rRNA, IL-21, CXCR5 and BCL6 were designed to prevent genomic DNA amplification ( Supplementary Table 1 ). Gene expression levels were measured in triplicate using SYBR green method in StepOnePlus (Applied Biosystems), normalized to 18S rRNA as the internal control.

Statistical analyses were performed using Prism 9 (Graphpad). The statistical tests used are indicated in the figure descriptions. Data show the mean of multiple donors, and error bars represent mean ± SEM.

We examined the impact of T cell receptor (TCR) stimulation strength on naive CD4⁺ T cell differentiation into IL-21-secreting Tfh-like cells. It is noteworthy that under basal conditions, IL-21 expression remains undetectable in human naïve CD4+ T cells ( Figure 1A ). These cells, subjected to varying doses of CD3 and optimal CD28 stimulation (1 μg/mL), exhibited increased proliferation and live cell numbers, particularly evident at later time points ( Figures 1B, C ). Analysis of IL-21 production at distinct time intervals revealed an anti-CD3 dose-dependent induction of IL-21 expression, peaking shortly (3 days) after TCR-CD3 stimulation ( Figures 1D–F ). Accordingly, IL-21 production occurred early during cell division, indicating a prompt response to TCR-CD3 stimulation ( Figure 1G ).

To ascertain whether IL-21-expressing cells exhibited a Tfh-like phenotype, we evaluated CXCR5 and PD1 expression ( Figure 1H ). Stimulation with PMA/Ionomycin, supplemented with Brefeldin A, notably reduced CXCR5 expression, consistent with previous findings (33) (data not shown). To evaluate IL-21 expression and other Tfh characteristics, we isolated CXCR5^-PD1^-and CXCR5⁺PD1⁺populations during the peak of IL-21 production (3 days) induced by robust TCR-CD3 stimulation. Subsequent analysis of CXCR5 and IL-21 mRNA expression in both populations revealed a higher expression in CXCR5⁺PD1⁺ compared to CXCR5^-PD1^- CD4⁺ T cells, whereas the expression of BCL6 was similar, confirming the Tfh-like phenotype of IL-21-secreting cells ( Figure 1H ).

The formation of Tfh cells depends on a CD4⁺ T cell-intrinsic requirement for IL-21 (34). To determine the autocrine or paracrine effects of IL-21 during CD4⁺ T cell differentiation, exogenous IL-21 was introduced, leading to enhanced proliferation and division rates, particularly under low TCR-CD3 stimulation ( Figure 1I ). Moreover, the strength of TCR-CD3 signaling dictated the capacity of exogenous IL-21 to augment the differentiation of IL-21-producing CD4⁺ T cells. In fact, notably, the percentage of induced IL-21-producing cells through TCR-CD3 stimulation reached a plateau, beyond which supplementary IL-21 failed to further enhance differentiation ( Figure 1J ). In summary, heightened TCR-CD3 stimulation induces IL-21 in Tfh-like cells. Conversely, lower stimulation yields reduced intracellular IL-21, but exogenous IL-21 can enhance production, suggesting an intricate regulatory mechanism involving autocrine or paracrine signals.

The plasticity of Tfh cells in expressing cytokines with diverse effects on GC responses and memory B cell or antibody-secreting cell formation remains unclear. We investigated the impact of TCR-CD3 signal strength on IFN-γ and IL-4 expression as these are the main cytokines expressed by Tfh cells known to help B cell differentiation. Consistent with the Th1 and Th2 dogma (20–23), low-intensity TCR-CD3 stimulation facilitated IL-4 production at day 5, gradually decreasing thereafter ( Supplementary Figures 1A, B ). Intermediate and high stimulations initially induced fewer IL-4-producing cells, with proportions increasing later ( Supplementary Figure 1B ). Strong TCR-CD3 stimulation resulted in substantial IFN-γ production ( Supplementary Figures 1C, D ). Unlike IL-21, IL-4 and IFN-γ secretion correlated strongly with cell division, increasing progressively over divisions ( Supplementary Figures 2E, F ). Exogenous IL-4 and IFN-γ provided positive feedback, particularly for IL-4 under low TCR-CD3 stimulation ( Supplementary Figures 1E, F ).

We investigated whether IL-21-producing Tfh-like cells could coexpress IL-4 and IFN-γ through varying TCR-CD3 signaling strength ( Figures 2A–D ). Naive CD4⁺ T cell priming mainly yielded cells expressing either IL-4 or IL-21, with only a small fraction coexpressing both across TCR-CD3 stimulations ( Figure 2B ). However, variation in TCR-CD3 signaling induced more coexpression of IL-21 and IFN-γ ( Figures 2C, D ), especially under strong stimulation ( Figures 2C, D ).

To assess cytokine coexpression in more detail, IL-4 and IFN-γ production were analyzed after TCR-CD3 stimulation in IL-21-producing Tfh-like cells ( Figures 2E, F ). On day 3, most Tfh-like cells produced only IL-21, but coexpression of IFN-γ increased, especially with higher stimulation ( Figures 2E, F ). From day 5 on, the number of IL-21-producing Tfh-like cells coexpressing IL-4 increased, particularly with low TCR-CD3 stimulation ( Figures 2E, F ). In these conditions, most cells coexpressing IL-4 also expressed IFN-γ ( Figures 2E, F ). Coexpression of IL-4 alone was mainly observed with low TCR-CD3 stimulation, potentially due to positive feedforward signaling by IL-4 and limited secretion of IFN-γ and IL-21. These findings highlight the complex interplay of cytokines within the CD4⁺ T cell network, with possible positive and negative feedback loops regulating their expression. While single IL-21 expression is observed early after naive CD4⁺ T cell priming, plastic coexpression of IL-4 and IFN-γ occurs at later time points, especially with restricted IL-21 and IL-4 coexpression under low TCR-CD3 stimulation.

The impact of IL-21 and IL-4 on the plastic coexpression of IL-4 and IFN-γ in IL-21-producing Tfh cells was explored, focusing on the effective range of these exogenous cytokines: low TCR-CD3 stimulation for IL-4 and high for IL-21 ( Figure 3 , Supplementary Figures 2 , 3 ). Both IL-4 and IL-21 significantly influenced the coexpression of IL-21 and IL-4, with exogenous IL-4 inducing IL-4 production ( Figures 3A, B versus Figures 2A, B , top rows) and IL-21 inducing IL-21 without concurrent IL-4 expression ( Figures 3A, B versus Figures 2A, B , bottom rows). Interestingly, exogenous IL-21 induced moderate coexpression of IL-21 and IL-4 double-positive cells ( Figures 3A versus Figures 2A, B , bottom rows).

Effects on IL-21 and IFN-γ coexpression were observed ( Figures 3C, D ), with limited coexpression after TCR-CD3 stimulation alone ( Figures 2C, D ), significantly enhanced with exogenous IL-21 ( Figures 3C, D and Supplementary Figures 2C, D ), and mainly repressed by exogenous IL-4 ( Figures 3C, D and Supplementary Figures 3C, D ). Further analysis of IL-21-producing Tfh-like cells revealed that exogenous IL-4 markedly increased the population coexpressing IL-4 alone or in combination with IFN-γ, particularly after low stimulation ( Figures 3E, F and Supplementary Figures 3E, F versus Figures 2E, F , top rows). In contrast, exogenous IL-21 had a minimal impact on IL-4 and IFN-γ coexpression by IL-21-producing cells ( Figures 3E, F versus 2E,F, bottom rows). Therefore, substantial coexpression of IL-4 by IL-21-producing Tfh cells requires the presence of excess IL-4, potentially acting beyond the immunological synapse, as it has been shown for IL-21 (35).