PMC6473200_01
A novel Y524S variant in p110delta causes APDS. (A) Pedigree showing a de novo variant in p110delta. (B) Molecular model showing the location of the Y524S variant in relation to p85alpha. Note the loss of the hydrogen bond and buried surface area when Tyr 524 is mutated to Ser. (C) Levels of phospho-Akt (Ser473) and beta-Actin in CD4 cells purified from control or patient PBMCs were assayed by Western blotting. Cells were unstimulated (-) or stimulated with anti-CD3 and anti-CD28 for 5 min (+). Results are representative of three experiments. (D) Flow cytometry of control or patient CD4+ PHA blasts. Cells were assayed for phospho-Akt (Ser473) and phospho-S6 (Ser240/244) with or without stimulation for 10 min with anti-CD3 and anti-CD28. Results are representative of two experiments. (E) Western blotting for phosphotyrosine in freshly purified control or patient CD4+ T cells, either unstimulated or stimulated for the indicated times with anti-CD3.
fimmu-10-00753-g0001.jpg
PMC6473200_01_fimmu-10-00753-g0001.jpg
F1
Whole exome sequencing revealed a novel, de novo variant in exon 13 of p110delta, NM_005026.4:c.1571A>C (g.9780849 (chr1, hg19)) (Figure 1A). The helical domain interacts with the nSH2 domain of the inhibitory subunit p85alpha, and Y524 lies on the surface of p110delta directly adjacent to another APDS-causing variant (E525K) (Figure 1B). APDS-causing variants, including E525K, have been shown to increase Akt phosphorylation both basally and after TCR stimulation. Akt phosphorylation was enhanced in freshly purified CD4+ cells from the patient upon stimulation, however, basal phospho-Akt levels were not different than controls (Figure 1C). Enhanced phosphorylation of Akt and S6 was apparent, regardless of activation (Figure 1D). T cells with anti-CD3epsilon mAb and assaying phosphotyrosine levels by Western blot. The patient's T cells responded similarly to controls (Figure 1E). These results show that the Y524S variant increases PI3K activity in a similar fashion to other APDS variants. The interaction between iOPN and p85alpha was increased in the presence of the Y524S p110delta variant (Figures 4B, S1A). We saw enhanced co-immunoprecipitation of iOPN when p85alpha was pulled down (Figures 4C, S1B). WT or Y524S p110delta and examined nuclear and cytoplasmic fractions for the presence of iOPN. We found increased translocation of iOPN in the presence of the Y524S mutant p110delta (Figures 4D, S1C).
Peripheral and lymph node CD4+ T cells have a TFH phenotype in the Y524S APDS patient. (A) Serial sections of a lymph node biopsy from the patient stained for H&E, CD10, CD4, Bcl-6, CXCR5, ICOS, and PD-1. Arrowheads indicate areas of CD4+ cells that are also positive for CXCR5, ICOS, and PD-1. (B) FACS analysis of CXCR5 and PD-1 on CD4+ T cells from fresh control and patient PBMCs. (C) CXCR5 and PD-1 expression on activated T cells. Freshly isolated CD4+ T cells from control and Patient I were stimulated for 4 days with the indicated concentrations of plate-bound anti-CD3, with or without 2 ug/mL ICOS-L-Fc. Anti-CD28 was included at 1 ug/mL in all cultures. Results are representative of two experiments.
fimmu-10-00753-g0002.jpg
PMC6473200_01_fimmu-10-00753-g0002.jpg
F2
ICOS, and PD-1 revealed intense staining in CD4+ T cells surrounding CD10+Bcl-6+ germinal centers (Figure 2A). In comparison, a reactive lymph node from a subject without primary immunodeficiency has scattered PD-1+ T cells stained in the germinal center but not significantly in the band of lymphocytes that surround the germinal center (Figures S2A,B). In agreement with recent results from APDS patients bearing variants at E525 or E1021, peripheral CD4+ T cells also had a circulating TFH phenotype. More than 30% of peripheral CD4+ T cells were CXCR5+PD-1+, compared to approximately 5% in a healthy control (Figure 2B). We found that both patients with the pathogenic E81K variant had increased peripheral TFH cells, albeit to a lesser degree than Patient I (Figure 2B). TFH phenotype when offered sub-optimal levels of stimulation and roughly doubled the percentage of CXCR5+PD-1+ cells after 4 days of stimulation with a high concentration of anti-CD3 in the absence of differentiation factors (Figure 2C). This increase likely represents increased receptor expression among previously differentiated cells upon stimulation. In the presence of ICOS-L, control and patient cells differentiated to a similar extent upon high stimulation, while maximal differentiation occurred at 10-fold lower levels of stimulation in the patient's cells (Figure 2C).
Altered FOXP3 expression and cytokine production in T cells from APDS patients. (A) FACS analysis of CD25+FOXP3+ Treg cells in freshly isolated PBMCs and in PHA blasts with or without restimulation with anti-CD3/28 beads and IL-2. Results are representative of three experiments. (B) Intracellular staining of CD4+ T cell blasts stimulated with PMA and ionomycin. (C,D) FACS analysis of chemokine receptors on CD4+ cells from freshly isolated PBMCs. CXCR5- non-Tfh cells (C) and CXCR5+PD-1+ Tfh cells (D) were assayed for CXCR3 and CCR6 expression.
fimmu-10-00753-g0003.jpg
PMC6473200_01_fimmu-10-00753-g0003.jpg
F3
Treg cells and whether FoxP3 was normally upregulated in CD4+ T cells upon TCR mediated activation. The percentage of peripheral CD4+CD25+FoxP3+ Tregs was normal in the patient, but PHA blasts from Patient I were impaired in their ability to express FoxP3 upon stimulation (Figure 3A). These results suggest that lymphoproliferation and autoimmunity in these patients is due to impaired cell intrinsic regulation of proliferation rather than to lack of Treg cells. While IL-21 production was not significantly enhanced compared to control, there was a striking reduction of IFN-gamma-producing cells Patient II.A and a moderate reduction in Patient II.B (Figure 3B). Furthermore, the percentage of cells expressing CXCR3, the chemokine receptor associated with Th1 cells, was not significantly enhanced in either CXCR5+PD-1+ TFH cells or CXCR5- non-TFH cells (Figures 3C,D).
APDS variants enhance p85alpha-iOPN interactions. (A) Immunoprecipitation of WT or Y524S p110delta with p85alpha. 293T cells were co-transfected with HA-p110delta and Myc-p85alpha, and p85alpha was immunoprecipitated with an anti-Myc affinity gel. Results are representative of two experiments. (B) Co-immunoprecipitation of p85alpha with iOPN in the presence of WT or Y524S p110delta. 293T cells were co-transfected with WT or variant HA-p110delta, iOPN-FLAG and the indicated amount of Myc-p85alpha. iOPN was immunoprecipitated with an anti-FLAG affinity gel. Results are representative of two experiments. (C) Co-immunoprecipitation of iOPN with p85alpha from control and patient CD4+ T cell blasts. Results are representative of two experiments. (D) Intracellular OPN expression in nuclear and cytoplasmic fractions of transduced (GFP+) cells. CD4+ T cells from a healthy donor ere transduced with WT or Y524S p110delta. Blotting for GAPDH shows proper fractionation. Non-specific low molecular weight bands from the OPN blot show equal protein loading in the nuclear fractions. Results are representative of two experiments. (E) Model showing that enhanced interaction of iOPN with p85alpha in the presence of the variant p110delta drives TFH differentiation.
fimmu-10-00753-g0004.jpg
PMC6473200_01_fimmu-10-00753-g0004.jpg
F4
As previously shown for the pathogenic variants E525K and E1021K, immunoprecipitation experiments demonstrated that there was no difference in p110delta-p85alpha interactions for the Y524S variant (Figure 4A). The interaction between iOPN and p85alpha was increased in the presence of the Y524S p110delta variant (Figures 4B, S1A). We saw enhanced co-immunoprecipitation of iOPN when p85alpha was pulled down (Figures 4C, S1B). WT or Y524S p110delta and examined nuclear and cytoplasmic fractions for the presence of iOPN. We found increased translocation of iOPN in the presence of the Y524S mutant p110delta (Figures 4D, S1C).