**2. Materials and Methods**

Twenty-three pediatric patients (12–36 months) with an initial diagnosis of unPAD, attending the Pediatric Immunopathology and Allergology Unit/ Regional Referral Center for PIDs at Policlinico Tor Vergata in Rome, the Department of Pediatrics of University of Pisa, and the Pediatric Immunology Center of Federico II University in Naples were enrolled in the study. All patients matched ESID diagnostic criteria for unPAD (https://esid.org/Working-Parties/Registry-Working-Party/Diagnosis-criteria; accessed on 15 April 2023). A local ethical committee approved the study, and written informed consent was obtained from all participants or legal guardians.

According to general practice, immunological work-up of these patients included serum Ig levels by nephelometry, serum IgG subclass values by radial immunodiffusion, extended T and B cell immunophenotype by fluorescence-activated cell sorting (FACS), and specific IgG antibody response to tetanus and pneumococcal vaccines by ELISA (VaccZyme TM Tetanus toxoid IgG kit and VaccZyme TM Anti PCP- IgG, Binding Site, Birmingham, England). In patients with allergy, skin prick tests (SPT) and serum IgE (sIgE) were performed. Serum IgE was tested by ImmunoCAP FEIA, whereas airborne and/or food allergen extracts were used for SPT, according to clinical phenotype. In a limited number of patients, genetic analysis (next-generation sequencing—NGS) for the main PAD-associated genes was also performed. The NGS panel included the following genes: ICOS, TNFRSF13B, TNFRSF13C, TNFRSF12, CD19, CD81, CR2, CD20, CD27, IL21, IL21R, LRBA, CTLA4, PRKCD, PLCG2, NFKB2, NFKB1, PIK3CD, PIK3R1, PTEN, VAV1, RAC2, BLK, IKZF1, IRF2BP2, BTK, CD40L, SYK, LYK, FYK, MYD88, IRAK4, TNFSF13B, TNFSF17, TNFRSF17, RELB, REL, IKBE, IKBA, IKBB, IKK-alpha, IKK-beta, MAP3K14, RELA, STK4, AKT, LAT, IL12RB1, IL12B, IFNGR1, IFNGR2, ISG15, EVER1, EVER2, AICDA, CD40, UNG, CD79A, CD79B, PAX5, TCF3, BLNK. Sanger sequencing was used to confirm genetic variants detected by NGS as previously reported [13]. Clinical and immunological data were prospectively collected at enrollment and during follow-up (FU) at the following times: age 4 and then every 6–12 months according to common clinical practice. The

last evaluation was performed on April 2023. The mean FU period was 14 years (range 3–32 years, median 16 years).
