Differentiation of human T follicular helper cells in vitro requires co-operation between STAT3 and SMAD signalling cytokines that is restrained by IL-2

Keywords

• T follicular helper cells (Tfh)

• human Tfh differentiation in vitro

• STAT3 and SMAD signalling cytokines (IL-2)

Main Findings

Naive CD4+ T cells can differentiate into different T helper subsets depending on the cytokine environment present during their activation and priming phase. T follicular helper cells (Tfh) that are responsible for initiating germinal center reaction and production of high-affinity antibody by interacting with the B cells are the focus of this preprint. The cytokines IL-6, IL-12, TGF-β, Activin A, and anti-IFN-𝛾 are known to mediate Tfh differentiation. Phenotypically, Tfh cells express the transcription factors BCL6 and MAF, as well as the surface markers PD-1 and CXCR5 while BLIMP-1 is downregulated and acts as a negative regulator of BCL6. This study addresses how human naïve CD4+ T cells differentiate into Tfh in vitro in a way that resembles germinal centre Tfh in vivo. The aim is to establish a reproducible in vitro system by investigating which cytokine combinations and signalling sequences are sufficient to drive human Tfh differentiation in vitro.

Using human naïve CD4+ T cells from peripheral blood, the authors added an established cocktail of cytokines known to drive Tfh differentiation (IL-6, IL-12, IL-1ß, TGF-β, Activin A after stimulation with ICOSL and anti-CD3/anti-CD28) to show that Tfh-like cells can be generated from naïve human CD4+ T cells in vitro. Their results highlight IL-12 and TGF-βas playing dominant and distinct roles in Tfh differentiation in vitro. Accordingly, STAT and SMAD signalling cytokines promote specific Tfh phenotypes. STAT3 signalling was essential for the survival of CD4+ T cells in conditions favouring Tfh differentiation. When compared to in vivo tonsil Tfh and T follicular regulatory (Tfr), in vitro generated Tfh cells upregulated core in vivo TFH transcriptomic signatures and did not express FOXP3 or other regulatory transcripts. Further, Tfh cells produced IL-2 during differentiation, which sets up an autocrine negative feedback loop by activating STAT5 signalling that antagonizes Bcl6 expression and thereby limits excessive Tfh differentiation. This demonstrates that IL-2 neutralization unmasks a more germinal centre (GC)-like Tfh phenotype.

Together, the results in this preprint show that human Tfh differentiation in vitro requires coordinated STAT3- and SMAD-mediated cytokine signalling, while IL-2 acts as an intrinsic negative regulator that restrains the development of a germinal centre-like Tfh program.

Limitations & Suggestions

• The study primarily relies on inhibitors to investigate loss-of-function mechanisms. However, inhibitors can have off-target effects that may influence the interpretation of the results. To strengthen the conclusions, genetic approaches such as CRISPR–Cas9 knockout or siRNA knockdown of key regulators of Tfh differentiation (e.g., STAT3 or BCL6) could be used to confirm whether the observed effects are specifically mediated by these pathways.

• It is also crucial to assess B-cell functionality in more detail, such as IgG/IgA class switching, plasmacytosis, and antibody titers or affinity, to show that in vitro generated Tfh cells are functional and can support B-cell maturation.

• The conclusion regarding the role of IL-2 signalling in the differentiation system was somewhat unclear. In particular, it was not clearly stated whether anti-IL-2 antibodies were added to the culture conditions. Since IL-2 is known to inhibit Tfh differentiation, clarifying this point would help strengthen the interpretation of the results.

• Some figures were difficult to interpret due to limited description and labeling. Improving figure legends, labeling of experimental conditions, and overall layout would make the data easier to follow. Additionally, some results were discussed without corresponding data being shown; including supplementary figures would improve clarity and transparency

Significance/Novelty

This study addresses an important gap in the field by investigating how human naïve CD4⁺ T cells can be differentiated into TFH cells in vitro in a way that resembles germinal centre TFH cells in vivo. The authors aim to establish a reproducible in vitro system and systematically test which cytokine combinations and signalling sequences are sufficient to drive TFH differentiation. A key finding of the study is that autocrine IL-2 signalling acts as an intrinsic “brake” on TFH differentiation, separating T cell proliferation from the acquisition of a full TFH phenotype. Neutralizing IL-2 allows the cells to acquire a more germinal centre–like TFH phenotype, highlighting the importance of tightly controlled IL-2 signalling during TFH development.

Developing a reliable human in vitro TFH differentiation system is valuable because it provides a controlled platform to study CD4⁺ T cell activation, TFH differentiation, and T–B cell interactions in human cells. This can help immunologists investigate mechanisms of germinal centre responses, antibody production, and vaccine responses, which are otherwise difficult to study directly in humans.

Credit

Reviewed by Antra Gaur and Katherine Dickinson as part of a cross-institutional journal club between the Max-Delbrück Center Berlin, the Ragon Institute Boston (Mass General, MIT, Harvard), the University of Virginia, the Medical University of Vienna and other life science institutes in Vienna.

The author declares no conflict of interests in relation to their involvement in the review.