PMC7314954_01
Pedigree.
fimmu-11-00884-g0001.jpg
PMC7314954_02_fimmu-11-00884-g0001.jpg
F1
P2 is the 9-year-old brother of P1 (Figure 1), with history of transient diarrhea (but reportedly normal GI endoscopies), asthma, and GH deficiency (16kg/Z -5, 106 cm/Z -5). The father has insulin-dependent diabetes mellitus diagnosed at 13 years, and the mother is healthy (Figure 1). CARMIL2 variant and one DOCK8 variant (Figure 1). CARMIL2 p.Gln455_Leu464del results in the loss of ten amino acids from the leucine-rich repeat (LRR) domain, which is highly-conserved between species, maintains protein stability, and mediates protein-protein interactions, thus favoring the pathogenicity of this variant. FOXP3+Tregs was decreased (Figure 2E, Table 1), similar to previous reports described in other CARMIL2-deficient patients, and supported by the critical role of CD28 in Treg lymphopoiesis. Furthermore, expression of CD25 on FOXP3+Tregs was substantially diminished (Figure 2E), which likely compromises Treg suppressor function and contributes to P1's immune dysregulatory phenotype with IBD.
(A) Flow cytometric evaluation of T lymphocyte proliferation. T cell proliferation after stimulation with soluble anti-CD3, soluble anti-CD3 and anti-CD28, and soluble anti-CD3 with recombinant interleukin-2 (IL-2) was measured using an Edu-based  flow assay with CD45 and CD3 as markers for total lymphocytes and CD3+ T cells, respectively. Data is expressed as either % CD45+ lymphocytes relative to total CD45+ lymphocytes, and % CD3+ T cells relative to total CD3+ T cells. Data is shown with an experimental healthy control. This was performed as a clinical test validated in 101 healthy controls, and therefore multiple replicates were deemed unnecessary as clinical assays are stringently analytically validated for regulatory oversight. Further, the volume of blood required to repeat this clinical testing was contraindicated in the patient. (B) CD28 expression on CD4+ and CD8+T cells by flow cytometry. (C) CARMIL2 protein expression in CD4+ and CD8+T cells by flow cytometry. (D) DOCK8 protein expression in peripheral blood mononuclear cell subsets by flow cytometry. (E) Regulatory T-cell (Treg) phenotyping. Flow cytometric assays shown in (B-E) were performed on the proband (P1), his younger affected sibling (P2), their parents (carriers), and HCs. Assays were performed at least twice on each sample yielding consistent results. Samples from P1 and P2 were inadequate for further replicates. The frequency of CD28+ T cells is shown in the first graph, and the median fluorescence intensity (MFI) is shown in the second graph (right). The parents also show decreased frequency of CD28+ CD8+ T cells, though the MFI shows overlap between the two patients and a couple of healthy controls. CARMIL2 protein expression was assessed in both CD4+ and CD8+ T cells, as well as CD3-negative lymphocytes (B and NK cells). For DOCK8 protein expression, while the frequency of DOCK8 is comparable between P1 and P2, parents (carriers), and healthy controls, the MFI is decreased in all lymphocyte subsets and monocytes in P1. The expression (MFI) of CD25 in FOXP3+ Tregs is substantially reduced in both P1 and P2.
fimmu-11-00884-g0002.jpg
PMC7314954_02_fimmu-11-00884-g0002.jpg
F2
IL-2 (Figure 2A) was significantly blunted compared to healthy controls (HC), especially for soluble anti-CD3 and anti-CD3+anti-CD28. CD28 protein expression (median fluorescence intensity, MFI) on P1 and P2's CD4+ T cells was reduced compared to HCs and parents, but the expression of CD28 on CD8+ T cells was normal in both P1 and P2 and parents, comparable to the HCs (Figure 2B). On the other hand, the frequency of CD28+CD8+ T cells was decreased in P1 and both parents, but normal in P2 (Figure 2B). CARMIL2 protein expression in CD4+ and CD8+T cells is shown in Figure 2C. More importantly, there were no CD8+T cells or CD3-negative lymphocytes (B and NK cells) expressing CARMIL2 (Figure 2C). T cells compared to HCs, but not in CD3-negative lymphocytes. (Figure 2C), but they had no relevant clinical phenotype. There were no significant differences in CD28 (Figure 2B) and CARMIL2 expression (MFI) (Figure 2C) observed between the proband and his younger sibling despite the differences in clinical phenotype, which may reflect age-related progression of disease, and potentially other epigenetic factors. DOCK8 protein expression was also analyzed (Figure 2D) in P1, as he had compound heterozygous variants of uncertain significance. P1 demonstrated reduced expression of DOCK8 within monocytes, T, B, and NK cells compared to his kindred (P2 and parents) and HCs, although the frequency of DOCK8-expressing cells appeared normal (Figure 2D). FOXP3+Tregs was decreased (Figure 2E, Table 1), similar to previous reports described in other CARMIL2-deficient patients, and supported by the critical role of CD28 in Treg lymphopoiesis. Furthermore, expression of CD25 on FOXP3+Tregs was substantially diminished (Figure 2E), which likely compromises Treg suppressor function and contributes to P1's immune dysregulatory phenotype with IBD.
PMC7314954_02
Pedigree.
fimmu-11-00884-g0001.jpg
PMC7314954_02_fimmu-11-00884-g0001.jpg
F1
P2 is the 9-year-old brother of P1 (Figure 1), with history of transient diarrhea (but reportedly normal GI endoscopies), asthma, and GH deficiency (16kg/Z -5, 106 cm/Z -5). The father has insulin-dependent diabetes mellitus diagnosed at 13 years, and the mother is healthy (Figure 1). CARMIL2 variant and one DOCK8 variant (Figure 1). CARMIL2 p.Gln455_Leu464del results in the loss of ten amino acids from the leucine-rich repeat (LRR) domain, which is highly-conserved between species, maintains protein stability, and mediates protein-protein interactions, thus favoring the pathogenicity of this variant. FOXP3+Tregs was decreased (Figure 2E, Table 1), similar to previous reports described in other CARMIL2-deficient patients, and supported by the critical role of CD28 in Treg lymphopoiesis. Furthermore, expression of CD25 on FOXP3+Tregs was substantially diminished (Figure 2E), which likely compromises Treg suppressor function and contributes to P1's immune dysregulatory phenotype with IBD.
(A) Flow cytometric evaluation of T lymphocyte proliferation. T cell proliferation after stimulation with soluble anti-CD3, soluble anti-CD3 and anti-CD28, and soluble anti-CD3 with recombinant interleukin-2 (IL-2) was measured using an Edu-based  flow assay with CD45 and CD3 as markers for total lymphocytes and CD3+ T cells, respectively. Data is expressed as either % CD45+ lymphocytes relative to total CD45+ lymphocytes, and % CD3+ T cells relative to total CD3+ T cells. Data is shown with an experimental healthy control. This was performed as a clinical test validated in 101 healthy controls, and therefore multiple replicates were deemed unnecessary as clinical assays are stringently analytically validated for regulatory oversight. Further, the volume of blood required to repeat this clinical testing was contraindicated in the patient. (B) CD28 expression on CD4+ and CD8+T cells by flow cytometry. (C) CARMIL2 protein expression in CD4+ and CD8+T cells by flow cytometry. (D) DOCK8 protein expression in peripheral blood mononuclear cell subsets by flow cytometry. (E) Regulatory T-cell (Treg) phenotyping. Flow cytometric assays shown in (B-E) were performed on the proband (P1), his younger affected sibling (P2), their parents (carriers), and HCs. Assays were performed at least twice on each sample yielding consistent results. Samples from P1 and P2 were inadequate for further replicates. The frequency of CD28+ T cells is shown in the first graph, and the median fluorescence intensity (MFI) is shown in the second graph (right). The parents also show decreased frequency of CD28+ CD8+ T cells, though the MFI shows overlap between the two patients and a couple of healthy controls. CARMIL2 protein expression was assessed in both CD4+ and CD8+ T cells, as well as CD3-negative lymphocytes (B and NK cells). For DOCK8 protein expression, while the frequency of DOCK8 is comparable between P1 and P2, parents (carriers), and healthy controls, the MFI is decreased in all lymphocyte subsets and monocytes in P1. The expression (MFI) of CD25 in FOXP3+ Tregs is substantially reduced in both P1 and P2.
fimmu-11-00884-g0002.jpg
PMC7314954_02_fimmu-11-00884-g0002.jpg
F2
IL-2 (Figure 2A) was significantly blunted compared to healthy controls (HC), especially for soluble anti-CD3 and anti-CD3+anti-CD28. CD28 protein expression (median fluorescence intensity, MFI) on P1 and P2's CD4+ T cells was reduced compared to HCs and parents, but the expression of CD28 on CD8+ T cells was normal in both P1 and P2 and parents, comparable to the HCs (Figure 2B). On the other hand, the frequency of CD28+CD8+ T cells was decreased in P1 and both parents, but normal in P2 (Figure 2B). CARMIL2 protein expression in CD4+ and CD8+T cells is shown in Figure 2C. More importantly, there were no CD8+T cells or CD3-negative lymphocytes (B and NK cells) expressing CARMIL2 (Figure 2C). T cells compared to HCs, but not in CD3-negative lymphocytes. (Figure 2C), but they had no relevant clinical phenotype. There were no significant differences in CD28 (Figure 2B) and CARMIL2 expression (MFI) (Figure 2C) observed between the proband and his younger sibling despite the differences in clinical phenotype, which may reflect age-related progression of disease, and potentially other epigenetic factors. DOCK8 protein expression was also analyzed (Figure 2D) in P1, as he had compound heterozygous variants of uncertain significance. P1 demonstrated reduced expression of DOCK8 within monocytes, T, B, and NK cells compared to his kindred (P2 and parents) and HCs, although the frequency of DOCK8-expressing cells appeared normal (Figure 2D). FOXP3+Tregs was decreased (Figure 2E, Table 1), similar to previous reports described in other CARMIL2-deficient patients, and supported by the critical role of CD28 in Treg lymphopoiesis. Furthermore, expression of CD25 on FOXP3+Tregs was substantially diminished (Figure 2E), which likely compromises Treg suppressor function and contributes to P1's immune dysregulatory phenotype with IBD.