PMC9459137_01
Timeline of clinical events, therapeutic interventions, and diagnostic procedures. (A) Summary of major clinical manifestations and therapeutic interventions. IVIG: intravenous immunoglobulins; SCIG: subcutaneous immunoglobulins; RTX: Rituximab. At each follow-up, an extensive immune cell phenotyping was conducted. (B) Timeline of fasting blood glucose (FBG), 2-h blood glucose (BG2h), fasting C-peptide (CPEP), and 2-h C-peptide (CPEP2h), HOMA index, fasting insulin (FI) and 2-h insulin (I2h), glycohemoglobin (HbA1c), and platelet count (PLT) from April 2016 to August 2021.
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Similar to the index patient, the father expressed reduced levels of BAFFR on the surface of his circulating B cells ( Figure S1 ). FU ( Table 1 ). Further analyses on cTfh cell subset distribution and activation status identified a remarkable shift toward Tfh1 (CXCR3+CCR6-) cells at the expense of the Tfh2 and Tfh17 subsets (CXCR3+CCR6- and CXCR3-CCR6+, respectively) ( Figure 2D ) that was maintained throughout the 5-year FU ( Figure S2B ,  Table 1 ). HC (median, IQR = 21.30, 16.40-25.30), whereas ICOS+ cTfh cell frequency remained higher since disease onset (~4 times higher than the control, 6.15% vs. HC median, IQR = 1.45%, 0.91-2.32) ( Figure 2E ,  Table 1 ). IQR = 2.42%, 1.30-4.58) and increased over time (3-FU = 18.30%; 4-FU = 38.00%; 5-FU = 30.50%; 6-FU = 26.30%) ( Figure 3B ,  Table 1 ). In contrast to the index patient, circulating B cell frequency and subset distribution in the father were normal ( Table 1 ).
Genetic and immunological characteristics of a patient with CVID and stage 2 T1D. (A) Genetic testing identified low T1D GRS (0.22817, HLA: X/X), and H159Y mutation in BAFFR inherited in a patrilineal fashion. The father was diagnosed with autoimmune thyroiditis, and the brother was positive for anti-TPO autoantibody production. (B) Representative gating strategy to evaluate BAFFR distribution on B cells, Tfh, and Treg. White, dark, and light gray slopes for control, HC, and CVID013, respectively. (C) FoxP3 and CXCR5 staining on CD3+CD4+ lymphocytes identifies Tfh (CXCR5+FoxP3-), Tfr (CXCR5+FoxP3+), and Treg (CXCR5-FoxP3+) cells, and (D) CXCR3 and CCR6 staining on CD4+CXCR5+CD45RA-. The following subsets were identified: cTfh1 (CXCR3+CCR6-), cTfh2 (CXCR3-CCR6-), and cTfh17 (CXCR3-CCR6+). (E) PD-1+ and ICOS+ expressing cells among cTfh are increased over time compared to the HC group (PD-1+ median, IQR = 21.3%, 16.4-25.3, n = 65; ICOS+ median, IQR = 1.45, 0.91-2.32, n = 65). Solid dots and squares represent the index patient and the proband father, respectively. (F) CXCL13 was evaluated in plasma by ELISA assay. Stable higher levels of this chemoattractant were detected over time in CVID patient when compared with the HC (median, IQR = 47.68 pg/ml, 29.52-68.24; n = 65), represented by the continuous line within the light gray area. (G, H) Treg gating strategy based on CD25 and CD127 expression, and FoxP3 expressing cells among CD25+CD127- Treg. (I) CD25+CD127- Treg were reduced in the index patient at the first follow-up (HC mean +- SD = 3.42% +- 0.51) and expressed lower levels of FoxP3 (mean HC FoxP3 +- SD = 88.7% +- 3.3; mean HC FoxP3 MFI +- SD = 1080 +- 254.2).
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T1D GRS was calculated by typing 30 common HLA and non-HLA genetic variants associated with T1D, as previously described. The index patient did not have a T1D-risk HLA (X/X for DR3/DR4-DQ8) and his T1D GRS score was 0.22817 ( Figure 2A ). BAFFR was associated with reduced gMFI BAFFR expression on the B cell, Tfh, and T regulatory cell (Treg) surface as compared with HC. BAFFR decrease was more pronounced in B cells (MFI reduction 82.1%) than in T cells (reduction 15.4%, 18.9%, and 18.5% in Tfh, Treg, and Tfr, respectively) ( Figure 2B ). Table 1 ). Treg cells (CD25+CD127-/loFoxP3+) and FoxP3 levels (gammaMFI) in the proband were reduced at disease ( Figures 2G-I ). FU ( Table 1 ). Further analyses on cTfh cell subset distribution and activation status identified a remarkable shift toward Tfh1 (CXCR3+CCR6-) cells at the expense of the Tfh2 and Tfh17 subsets (CXCR3+CCR6- and CXCR3-CCR6+, respectively) ( Figure 2D ) that was maintained throughout the 5-year FU ( Figure S2B ,  Table 1 ). HC (median, IQR = 21.30, 16.40-25.30), whereas ICOS+ cTfh cell frequency remained higher since disease onset (~4 times higher than the control, 6.15% vs. HC median, IQR = 1.45%, 0.91-2.32) ( Figure 2E ,  Table 1 ). Table 1 ,  Figure 2F ). The father had normal frequencies and subset distribution of follicular T cells ( Table 1 ).
Functional analysis of B- and T-cell subsets. (A) Representative gating strategy for CD38lowCD21low autoreactive B cells, gated on CD19+ cells and (B) their frequency over time. CD38lowCD21low cell percentage was higher compared with the HC median, IQR (2.42%, 1.3-4.58; n = 85), increasing from 11.10% at the first follow-up up to 38.00% in 2020, and decreasing to 30.50% in the last monitoring. (C, D) Functional analysis of IgM and IgG production. Sorted B memory or B naive cells were co-cultured with Tfh cells (1:1 ratio) in autologous (solid dot) or heterologous settings (CVID B cells with HC Tfh, solid square, or HC B cells with CVID Tfh, clear square), and the percentage of CD38+CD20- was analyzed within CD19+CD4- cells after 1 week. The black continuous line is representative for the mean HC percentage value +- SD (66.32% +- 12.46, n = 16) represented by the light gray area within the two dashed lines. The production of IgM and IgG was evaluated in the supernatant (E,

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). The white dots and squares are representative for the 1-FU and 2-FU, respectively, whereas the black dots represent the HC. (F, G) Evaluation of IFN-gamma, IL-17, and IL-21 production in CD4+CXCR5+ cells after 2-h stimulation with PMA/Ionomycin. The HC and patient slopes are identified with the light and dark gray, respectively, whereas the unstimulated control is represented by the dashed line. IFN-gamma and IL-17 production was lower compared with the HC (IFN-gamma mean +- SD = 16.66% +- 6.84; IL-17 mean +- SD = 8.35% +- 6.63; n = 65), whereas IL-21 production was lower than HC mean and comprised within the SD (IL-21 mean +- SD = 8.74% +- 4.30; n = 65). (H) IFN-gamma, IL-17, and IL-21 production in CD4+CXCR5- cells after 2-h stimulation with PMA/Ionomycin. IFN-gamma and IL-21 production was higher compared with the HC (IFN-gamma mean +- SD = 3.69% +- 2.86; IL-21 mean +- SD = 3.32% +- 2.06; n = 65), whereas IL-17 production was comparable with HC (IL-17 mean +- SD = 1.14% +- 0.37; n = 65). .
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IQR = 2.42%, 1.30-4.58) and increased over time (3-FU = 18.30%; 4-FU = 38.00%; 5-FU = 30.50%; 6-FU = 26.30%) ( Figure 3B ,  Table 1 ). In contrast to the index patient, circulating B cell frequency and subset distribution in the father were normal ( Table 1 ). FACS-sorted memory and naive B cells were co-cultured with cTfh cells in autologous (CVID B cells with CVID Tfh cells) and heterologous settings (CVID B cells with HC Tfh cells or vice versa) and the percentage of CD20-CD38+ plasmablasts together with IgM and IgG levels were evaluated after a week ( Figures 3C-E ). The percentage of plasmablast differentiating from memory B cells in the presence of autologous Tfh cells was lower with respect to HC at diagnosis but improved in 2-FU ( Figure 3D , left panel). Tfh cells co-cultured either with autologous or heterologous B naive cells were unable to induce class switching and IgG production in vitro ( Figures 3D, E ). Total PBMCs were activated with PMA/Ionomycin and the expression of interferon-gamma (IFN-gamma), interleukin-17 (IL-17), and IL-21 was evaluated by flow cytometry (FC) ( Figures 3F, G ). On the contrary, higher frequencies of IFN-gamma and IL-21 producing cells were observed within the CXCR5- compartment ( Table 1 ,  Figure 3H ).