Hirofumi Shibata

Th2 cells play a central role in orchestrating type 2 immune responses by producing type 2 cytokines including IL-4 and IL-13. The master transcription factor GATA3 is essential but not sufficient for type 2 cytokine production. To better define other transcription factors that regulate type 2 cytokine expression, we performed a CRISPR-Cas9 screen after preparing a retroviral library expressing sgRNAs targeting 2,394 mouse transcription factors and cofactors. The screen revealed core-binding factor beta (Cbfβ) as a specific inducer of IL-13 that simultaneously inhibited IL-4 expression. The effect of Cbfβ on IL-13 production was confirmed both in vitro and in vivo and was independent of GATA3 induction. Mechanistically, Cbfβ induced chromatin accessibility at the HS-2 site within the Il13 promoter where the Cbfβ/Runx complex bound. Hi-TrAC analysis indicated that DNA looping between HS-2 and other enhancers was reduced upon Cbfβ deletion. Overall, our results indicate that Cbfβ directly regulates IL-13 expression by binding the Il13 promoter via the Cbfβ/Runx complex and promoting enhancer–promoter interactions that facilitate Il13 transcription.

The TCR-induced tyrosine kinase cascade results in ITK-mediated phosphorylation of at least two substrates: PLC-γ1 Y783, an essential mediator of TCR signaling, and SLP-76 Y173. We previously provided evidence that SLP-76 Y173 supports proximal TCR and FcεRI signaling; but its role in thymocyte development was never explored. Here we demonstrate that SLP-76 Y173 was required for TCR/CD28-induced degradation of IκB; moreover, both ITK and SLP-76 Y173 were required for persistent TCR-induced phosphorylation of PLC-γ1, ERK and p38. To explore whether SLP-76 Y173 may mediate ITK-dependent events in vivo, we replaced SLP-76 Y173 with phenylalanine in a mouse model. Like ITK-deficient thymocytes, SLP-76 Y173F thymocytes exhibited reduced thymic cellularity, reduced CD4/CD8 ratios, and reduced surface expression of CD5. In ex vivo-stimulated thymocytes, mutation of SLP-76 Y173 or inhibition of ITK reduced the persistence of TCR-induced calcium flux; moreover, TCR-induced phosphorylation of ERK and p38 depended on SLP-76 Y173. Like ITK-deficient thymi, SLP-76 Y173F thymocytes exhibited decreased production of CD4 ⁻ iNKT ₁ thymocytes, coupled with increased production of innate-like CD4 ⁺ and CD8 ⁺ SP thymocytes. Our results provide strong evidence that the ITK substrate site, SLP-76 Y173, promotes TCR signal persistence that is required to mediate the thymic developmental effects of ITK. One Sentence Summary: An ITK substrate site, SLP-76 Y173, is a predominant mediator of the signaling and developmental effects of ITK. Key Points: An ITK substrate site, SLP-76 Y173, regulates TCR signal amplitude and persistence Persistent TCR-induced phosphorylation of p38 depends on ITK and SLP-76 Y173 The thymic developmental effects of ITK are predominantly mediated by SLP-76 Y173

Mutations in the MEFV gene, which encodes pyrin, are associated with a spectrum of inflammatory conditions called pyrin-associated autoinflammatory diseases (PAADs). Of the 400 MEFV variants listed in the Infevers database, most are classified as variants of uncertain significance. Thus, genetic diagnosis of PAADs remains challenging, and the molecular mechanisms underlying pyrin activation remain poorly understood. Here, we used a cell-based pyroptosis assay to stratify 265 missense MEFV variants and identified previously uncharacterized pathogenic variants. We then characterized the interaction between the pyrin B30.2 domain and CDC42, a key regulator of pyrin intracellular trafficking and activation. We found that classical familial Mediterranean fever (FMF)–related variants bind tightly to CDC42 to induce pyrin hyperactivation, whereas certain non-FMF variants induce pyrin hyperactivation independently of CDC42, indicating involvement of multiple pathways in pyrin activation. Our approach provides a proof of concept for a genotype-first approach, which may advance our understanding of complex human diseases.

Heterozygous carboxyl-terminal variants in the RHO guanosine triphosphatase (GTPase) CDC42 are known to cause severe autoinflammatory syndromes. Here, we identified a heterozygous amino-terminal p.T43I (Thr ⁴³ →Ile) CDC42 variant in patients with autoinflammation and uncovered a molecular link between CDC42 and the inflammasome sensor pyrin, mutated in the hereditary autoinflammatory syndrome familial Mediterranean fever. We demonstrate that the region surrounding residue T43 of CDC42 interacts with the carboxyl-terminal B30.2 domain of pyrin and regulates its localization and activation. The p.T43I substitution strengthens the CDC42-pyrin interaction through additional van der Waals interactions, leading to increased pyrin inflammasome activation, as evidenced by increased ASC (apoptosis-associated speck-like protein containing a caspase activating and recruitment domain) speck formation, enhanced pyroptosis, and excessive interleukin-1β (IL-1β) and IL-18 production. These findings identify CDC42 as a pyrin ligand and provide critical insights into the role of the pyrin B30.2 domain in inflammasome activation, suggesting dual regulation of pyrin by two RHO family GTPases, RHOA and CDC42.

Griscelli syndrome type 2 (GS2), caused by biallelic RAB27A variants, is classically associated with hypopigmentation and life-threatening hemophagocytic lymphohistiocytosis (HLH). However, a few patients exhibit normal pigmentation, termed GS2 sine albinism. We identified two unrelated Japanese patients with this presentation caused by compound heterozygous RAB27A variants, representing the first functionally characterized cases among Japanese patients. Functional studies were performed using RAB27A-null cell systems to evaluate the two allele pairs: p.Trp73Arg/p.Val143Ala (patient 1) and p.Ser106PhefsTer18/p.Ser115Arg (patient 2). The truncating allele p.Ser106PhefsTer18 and the missense allele p.Trp73Arg failed to restore peripheral melanosome distribution and lost binding to both melanophilin (MLPH) and MUNC13-4. In contrast, p.Val143Ala and p.Ser115Arg preserved MLPH interaction but showed reduced binding to MUNC13-4. These findings demonstrate that RAB27A variants can dissociate melanosome transport from immune effector function in an allele-dependent manner. Importantly, the absence of hypopigmentation does not exclude GS2. Combined genetic and functional analyses are essential for precise variant interpretation and for guiding timely curative intervention in GS2.

Mutations in the C-terminal region of the CDC42 gene cause severe neonatal-onset autoinflammation. Effectiveness of IL-1β–blocking therapy indicates that the pathology involves abnormal inflammasome activation; however, the mechanism underlying autoinflammation remains to be elucidated. Using induced-pluripotent stem cells established from patients carrying CDC42R186C, we found that patient-derived cells secreted larger amounts of IL-1β in response to pyrin-activating stimuli. Aberrant palmitoylation and localization of CDC42R186C protein to the Golgi apparatus promoted pyrin inflammasome assembly downstream of pyrin dephosphorylation. Aberrant subcellular localization was the common pathological feature shared by CDC42 C-terminal variants with inflammatory phenotypes, including CDC42*192C*24 that also localizes to the Golgi apparatus. Furthermore, the level of pyrin inflammasome overactivation paralleled that of mutant protein accumulation in the Golgi apparatus, but not that of the mutant GTPase activity. These results reveal an unexpected association between CDC42 subcellular localization and pyrin inflammasome activation that could pave the way for elucidating the mechanism of pyrin inflammasome formation.