1. RELA haploinsufficiency in CD4 lymphoproliferative disease with autoimmune cytopenias 
William A. Comrie, PhD, Aiman J. Faruqi, Susan Price, RN, Yu Zhang, PhD, V. Koneti Rao, MD, Helen C. Su, MD, PhD, Michael J. Lenardo, MD 
Journal of Allergy and Clinical Immunology 
Volume 141, Issue 4, Pages e8 (April 2018)
DOI: /j.jaci
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2. Fig 1
RELA haploinsufficiency leads to a T-cell–mediated autoimmune lymphoproliferative disease. Patient platelet and neutrophil counts (red dashed lines, arrow, black, green, and red asterisks represent the normal range, age of onset, splenectomy, rituximab administration, and systemic immune response to pneumococcal vaccine, respectively). Black dashed and solid red lines represent MMF and eltrombopag administration, respectively (A/B). CD4+ and CD8+ cells expressing HLA-DR; red line indicates high end of normal range (C). CD4+ and CD8+ α/β T cells per microliter of patient blood; red lines indicate normal range (D). CCR7 and CD45RA expression on CD3/CD4+/+ lymphocytes (E). Representative IFN-γ and IL2 production in phorbol 12-myristate 13-acetate and ionomycin restimulated CD4+ T cells (F). IFN-γ and IL2 production in cells stimulated as in Fig 1, F, pooled from 4 separate experiments (G). Proliferation of CD4+ blasts following restimulation with 1 μg/mL anti-CD28 and the indicated dose of anti-CD3 (n = 3) (H). Family pedigree and de novo RELA variant (I). Protein expression in CD4+ T-cell blasts by Western blot (RELA/Actin ratios in red) (left), flow cytometry (center), and RELA mRNA levels determined by quantitative RT-PCR (right) (J). IkBα degradation and p65 phosphorylation following PMA/I stimulation, pooled from 3 separate experiments (K and L, respectively). Avg, Average; CFSE, carboxyfluorescein succinimidyl ester; Ctrl, control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; MFI, Mean fluorescence intensity; NC, normal control; norm, normalized; Pos, positive; Pt, patient.
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3. Fig E1
Normal patient T-cell death in response to multiple stimuli. Fas-induced apoptosis of patient and control CD4+ T-cell blasts following crosslinking of the FAS receptor for 18 hours (A). Restimulation-induced cell death of patient and control CD4+ T-cell blasts following ligation of the TCR with the indicated dose of anti-TCR antibody for 18 hours (B). IL-2 withdrawl induced cell death of patient and control cells (C). Live cells were counted as those that excluded AnnexinV and propidium iodide (PI) dyes. Results are representative of 2 different experiments. NC, Normal control; neg, negative; Pt, patient.
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4. Fig E2
Enhanced T-cell differentiation into TH1 effectors and decreased iTreg-cell formation in patient cells. Flow cytometric plots showing staining of ex vivo CD4+ T cells for the chemokine receptors CXCR3 and CCR6. Expression of these chemokine receptors denotes the indicated effector type cell (A). Flow cytometric plots showing staining of ex vivo CD4+ T cells for CD25 and CD127. The gate is drawn to show the frequency of nTreg cells in the patient compared with controls (B). FOXP3 expression on day 5 postactivation of naive CD4+ T cells grown with the indicated amounts of TGF-β, representative of 3 different experiments (C). Quantification of FOXP3 induction in cells stimulated as in Fig E2, C; % of FOXP3-positive cells was normalized to the maximum response of 8 different controls at 10 ng/mL of TGF-β for combined analysis. Pooled from 3 different experiments (D). Ctrl, Control; iTreg, inducible regulatory T cell; max, maximum; NC, normal control; norm, normal; nTreg, natural regulatory T cell; Pt, patient.
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5. Fig E3
Normal B-cell development and antibody responses in patient B cells. Flow cytometric analysis of ex vivo B-cell populations in patients compared with controls (A). Levels of IgM, IgG, and IgA measured in patient serum during repeated clinical evaluation; red dashed lines indicate the normal range (5-95th percentiles) (B, C, and D, respectively). NC, Normal control; Pt, patient.
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6. Fig E4
Patient T cells express normal levels of NF-κB subunits other than p65 and have altered p65-containing dimmers. Western blot showing levels of NF-κB subunits in NP-40 cytosolic fractions and in p65 coimmunoprecipitations, representative of 3 different experiments (A). Densitometry quantification of NF-kB subunits in input lanes from 3 different experiments (B). Densitometry quantification of NF-κB subunits in IP lanes from 3 different experiments (C). Densitometry quantification of NF-kB subunits in IP lanes, normalized to control levels of each subunit. Densitometry quantification of NF-kB subunits in IP lanes, normalized to p65 levels immunoprecipitated from the same sample (D). Ctrl, Control; IP, immunoprecipitation; NC, normal control; norm, normal; Pt, patient.
Journal of Allergy and Clinical Immunology  , e8DOI: ( /j.jaci )
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