Journal: eLife

Article Title: Biallelic mutations in calcium release activated channel regulator 2A (CRACR2A) cause a primary immunodeficiency disorder

doi: 10.7554/elife.72559

Figure Lengend Snippet: Figure 4. Patient T cell responses to T cell receptor stimulation. (A) Representative flow plots showing expression of IFN-γ in human PBMCs from a healthy donor and the patient. PBMCs were stimulated with anti-CD3 and anti-CD28 antibodies for 48 hr and cultured for further 4 days in the presence of IL-2 before re-stimulation with PMA plus ionomycin for 5 hr for cytokine analysis. Cells were gated for CD4+ T cells. (B) Quantitative mRNA expression analysis (± s.d.m.) of indicated cytokines from human PBMCs (cultured as mentioned above) with or without stimulation with PMA plus ionomycin for 5 hr. (C) Levels of IL-2 and TNF in human PBMCs from culture supernatants of cells stimulated as described above (B) were determined by ELISA. (D) Representative traces showing averaged (± SEM) SOCE responses from healthy control and patient PBMCs (cultured as indicated in A), after transient stimulation with anti-CD3 antibody cross-linking, or ionomycin (0.5 µM) in the presence of external solution containing 2 mM Ca2+ (left) as indicated. Bar graphs show baseline subtracted ratio values for anti-CD3 antibody cross-linking or ionomycin (average± SEM) from six independent experiments (right). (E) Representative traces showing averaged (± SEM) SOCE responses from healthy control and patient PBMCs (cultured as indicated in A), after store-depletion with thapsigargin (1 µM) stimulation in Ca2+-free Ringer’s solution. SOCE was measured by addition of 2 mM Ca2+-containing Ringer’s solution as indicated (left). Bar graph shows baseline subtracted ratio values at the peak of SOCE (average± SEM) from four independent experiments (right). (F) Phosphorylated JNK levels in CD4+ T cells from healthy control and patient PBMCs (cultured as indicated in A), stimulated with anti-CD3 antibody for indicated times. Bar graphs show average± SEM from three independent experiments. ** p < 0.005, *** p < 0.0001.

Article Snippet: PBMCs from healthy control and patient were activated for 48 hr on a plate coated with 10 μg/ml of anti- CD3 antibody (OKT3, Bio X Cell) and cultured in T cell media (DMEM containing 20% fetal bovine serum and 1% Pen- Strep) supplemented with 5 μg/ml of anti- CD28 antibody (Bio X cell), and 20 U/ml IL- 2 (Peprotech) for ThN differentiation.

Techniques: Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay, Control

Journal: eLife

Article Title: Biallelic mutations in calcium release activated channel regulator 2A (CRACR2A) cause a primary immunodeficiency disorder

doi: 10.7554/elife.72559

Figure Lengend Snippet: Figure 5. Cytokine expression profiles in T cells expressing individual allelic mutations in CRACR2A. (A) Representative flow plots showing expression of IL-2 in control or CRACR2A KO Jurkat T cells stably expressing FLAG-tagged WT CRACR2A, CRACR2AE278D (E278D), or CRACR2AR144G/E300* (DM) after stimulation with PMA plus ionomycin for 16 hr (left). Bar graph shows means ± s.e.m. of pooled technical replicates from two independent experiments (right). (B) Representative flow plots showing expression of IFN-γ in primary human CD4+ T cells transduced with lentiviruses encoding CRACR2A- targeting sgRNA and those encoding cDNAs of WT or indicated mutants of CRACR2A after stimulation with anti-CD3 and anti-CD28 antibodies for 5 hr (left). Bar graph (right) shows means ± s.e.m. of pooled technical replicates from three independent experiments. (C) Representative flow plots showing expression of IFN-γ in primary human CD4+ T cells purified from healthy donors and transduced with lentiviruses encoding cDNAs for WT or indicated mutant of CRACR2A after stimulation with anti-CD3 and anti-CD28 antibodies for 5 hr (left). Bar graph (right) shows means ± s.e.m. of pooled technical replicates from three independent experiments. * p < 0.05, *** p < 0.0001.

Article Snippet: PBMCs from healthy control and patient were activated for 48 hr on a plate coated with 10 μg/ml of anti- CD3 antibody (OKT3, Bio X Cell) and cultured in T cell media (DMEM containing 20% fetal bovine serum and 1% Pen- Strep) supplemented with 5 μg/ml of anti- CD28 antibody (Bio X cell), and 20 U/ml IL- 2 (Peprotech) for ThN differentiation.

Techniques: Expressing, Control, Stable Transfection, Transduction, Purification, Mutagenesis