Journal: Nature Communications

Article Title: CD8 + T cell loss induces autoinflammation in inborn errors of cell death

doi: 10.1038/s41467-026-70317-2

Figure Lengend Snippet: a , b Schematic diagram showing serum cytokine microarray analysis of continuous P2’s serum samples before (PS1 and PS2) or after (PS3, flare; PS4 and PS5, non-flare) the onset of recurrent fever ( a ). PCA analysis of sample variance determined by semi-quantitative 174 cytokines antibody array on serum samples of C1-C3 from three unaffected controls and PS1-PS5 from P2 ( b ). c–e The integrated relative MFI plots of P2’s serum cytokine levels. 26 cytokines peaked in PS3 were enrich in group 1 (left), 24 cytokines higher than control were enriched in group 2 (right) ( c ). The relative MFI was calculated by normalizing the average MFI of the unaffected controls ( n = 3) to 0, and setting the largest MFI to 1 or the smallest MFI to −1 across 5 samples from P2. Boxplots exhibiting expression scores of cytokines of group 1 ( d ) and group 2 ( e ) in indicated cell with Seurat AddModuleScore function. The cell number of each subtype (listed in the “Methods” section), was employed for p value calculations. Boxes show the median (center line), 25th and 75th percentiles (upper and lower hinges), and whiskers extend to 1.5× the interquartile range, representing the maximum and minimum values. f Average expression values (arcsinh transformed) of cl. Casp-3 across identified peripheral immune cell lineages in PBMCs freshly isolated from four unaffected controls (C) and two patients (P, 1 sample from P1 and 2 samples from P2) by mass cytometry by time-of-flight (CyTOF) analysis. g–j Representative contour plots of cl. Casp-3 ( g , h ) and histograms of total RIPK1, pRIPK1(S166) levels ( i , j ) in CD4 + T and CD8 + T cells in freshly isolated PBMCs from unaffected controls (for cl. Casp-3, n = 12; for pRIPK1(S166), n = 10) and patients ( n = 4 for P1 and P2) (left). Summary histograms of cl. Casp-3 and pRIPK1(S166) (right) were shown. k Representative histogram and statistical analysis of surface CD69 expression on CD3 + T cells from unaffected controls ( n = 7) and two patients ( n = 3 for P1 and P2). l Serum IFNγ, sCD25, sCD95 and CD95L of unaffected controls (for IFNγ, n = 9; for sCD25, n = 15; for sCD95 and CD95L, n = 10), two patients (for IFNγ, P1, n = 5, P2 = 6; for sCD25, P1, n = 5, P2, n = 4; for sCD95, P1 and P2, n = 7; for CD95L, P1 = 7; P2 = 8) and their parents (for IFNγ and sCD25, n = 7; for sCD95 and CD95L, n = 6) as well as two CRIA syndrome patients ( n = 6) determined by ELISA. T denotes 10 ng ml -1 TNF; S denotes 250 nM SM164; Z denotes 25 μM z-VAD-fmk. All histogram graphs show mean ± SD. P values were determined by two-tailed unpaired t-test. Data in g–k were pooled from two independent experiments.

Article Snippet: Human or mouse serum cytokines were measured by enzyme-linked immunosorbent assay (ELISA) (Multi Sciences: 70-EK1280, 70-EK194, 70-EK182HS, 70-EK106HS, 70-EK191, 70-EK180HS, 70-EK1F01, 70-EK1F02, EK206HS, EK282HS/2) according to the manufacturer’s instructions.

Techniques: Microarray, Ab Array, Control, Expressing, Transformation Assay, Isolation, Mass Cytometry, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Nature Communications

Article Title: CD8 + T cell loss induces autoinflammation in inborn errors of cell death

doi: 10.1038/s41467-026-70317-2

Figure Lengend Snippet: a Schematic diagram of T cell-MDM co-culture system. b , c qRT-PCR analysis of TNF, IL1B and IL6 mRNA levels in GM-CSF-activated monocytes derived macrophages (MDMs) co-cultured with or without T cells for 6 h. GM-CSF-activated MDMs generated from ( b , left) unaffected control 1 (C1) or ( b , right) unaffected control 3 (C3) or P1 ( c ) were co-cultured with donor-matched T cells as well as T cells from other unaffected controls (C2 and C4) or patients as indicated. d Dot plot of IFNG, TNF in cell subtypes found in scRNA-seq of PBMCs from three unaffected controls and two patients. e Representative histograms and statistical analysis of TNF and IFNγ expression in CD8 + T cells in PBMCs isolated from unaffected controls ( n = 6) and patients ( n = 3 from P1 and P2) treated with 10 ng ml -1 TNF (T) with or without 250 nM SM-164 (S), 25 μM z-VAD-fmk (Z) for 24 h. f , T cells isolated from P2 were co-cultured with MDMs generated from unaffected control in the presence of 10 μg ml -1 adalimumab, 10 μg ml -1 emapalumab alone or in combination for 6 h. TNF, IL1B and IL6 mRNA levels of MDMs were analyzed by qRT-PCR. g Experimental timeline for generation of lineage negative bone marrow cells (Lin - BMs)-transplanted mice with conditional overexpression of human WT or mutated RIPK1 K377E or R390G in CD8 + T cell- or myeloid cell-specific lineage. h, i ELISA measurement of serum Tnf and Il6 levels in mice specifically overexpressing WT or mutated hRIPK1 in ( h ) CD8 + T cells (EV, n = 5; WT, K377E and R390G n = 7) or ( i ) myeloid cells (EV, n = 5; WT, K377E and R390G n = 6). All histogram graphs show mean ± SD. P values were determined by one-way ANOVA with Tukey’s multiple comparisons test. Data in b , c , and f show the means ± SD of technical triplicates. The results shown in b were derived from two independent experiments using cells from different donors. The experiment in ( c , h , i , f ) is representative of two independent experiments. Data in e were pooled from two independent experiments.

Article Snippet: Human or mouse serum cytokines were measured by enzyme-linked immunosorbent assay (ELISA) (Multi Sciences: 70-EK1280, 70-EK194, 70-EK182HS, 70-EK106HS, 70-EK191, 70-EK180HS, 70-EK1F01, 70-EK1F02, EK206HS, EK282HS/2) according to the manufacturer’s instructions.

Techniques: Co-Culture Assay, Quantitative RT-PCR, Derivative Assay, Cell Culture, Generated, Control, Expressing, Isolation, Over Expression, Enzyme-linked Immunosorbent Assay

Journal: Endocrinology

Article Title: Continuous PTH in Male Mice Causes Bone Loss Because It Induces Serum Amyloid A

doi: 10.1210/en.2018-00265

Figure Lengend Snippet: PTH infusion increased SAA3 expression and decreased femoral BMD in WT mice. WT and SAA3 KO male mice (3.5 mo old) were infused with vehicle (VEH) or PTH (40 µg/kg/d) for 12 d. (a) Saa3 mRNA expression in tibiae of WT mice. Both ends of each tibia were cut off to remove the growth plates and the marrow was not flushed. mRNA expression was measured by quantitative real-time PCR (qPCR) and data were reported as RQ values. (b) SAA3 protein in serum of WT and SAA3 KO mice by ELISA. Und, undetectable. (c) Change in in vivo femoral BMD calculated as: (BMD at end of infusion − BMD at start of infusion)/BMD at start of infusion. (d) Saa1, Saa2/Saa1, and (e) Ptgs2 (COX2) mRNA in WT and KO tibiae. Both ends of each tibia were cut off to remove the growth plates and the marrow was not flushed. mRNA expression was measured by qPCR and data were reported as RQ values. Bars are means ± SEM for n = 6 mice per genotype and treatment group. For (a), **P < 0.01, determined by two-tailed, unpaired t test. For (c) and (e), **P < 0.01, determined by two-way ANOVA, post hoc Bonferroni pairwise multiple comparisons.

Article Snippet: SAA1 protein accumulated in the medium of human BMMs was measured by an SAA1 human ELISA kit (KA2108; Abnova Corporation, Walnut, CA).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, In Vivo, Two Tailed Test

Journal: Endocrinology

Article Title: Continuous PTH in Male Mice Causes Bone Loss Because It Induces Serum Amyloid A

doi: 10.1210/en.2018-00265

Figure Lengend Snippet: PTH infusion increased trabecular bone formation parameters only in SAA3 KO mice but increased resorption parameters similarly in both WT and SAA3 KO mice. WT and SAA3 KO male mice were infused with vehicle (VEH) or PTH. (a) Static histomorphometry of trabecular bone in distal femurs for Ob.S/BS, Oc.S/BS, and eroded surface per bone surface (ES/BS). (b) Representative TRAP-stained (red) and counterstained hematoxylin ×200 original magnification (scale bars, 100 µm) microscopic images of trabecular bone in distal femurs. Plump cuboidal osteoblasts around trabeculae are marked with yellow arrows. (c) Dynamic histomorphometry of trabecular bone in distal femurs for MS/BS, MAR, and BFR/BS. (c) Representative calcein-labeled (green) and demeclocycline-labeled (orange-brown) ×400 original magnification (scale bars, 25 µm) microscopic images of trabeculae. (e and f) Measurement of (e) serum bone formation markers, PINP, and BGLAP (osteocalcin) and (f) serum bone resorption markers, CTX and TRAcP5b, measured by ELISA. (g and h) mRNA expression of Runx2, Bglap (osteocalcin), Tnfsf11 (RANKL), and Tnfrsf11b (OPG) in the tibiae measured by quantitative real-time PCR and reported as RQ values. Both ends of each tibia were cut off to remove the growth plates and the marrow was not flushed. Bars are means ± SEM for n = 6 mice per genotype and treatment group. *P < 0.05, **P < 0.01, determined by two-way ANOVA, post hoc Bonferroni pairwise multiple comparisons.

Article Snippet: SAA1 protein accumulated in the medium of human BMMs was measured by an SAA1 human ELISA kit (KA2108; Abnova Corporation, Walnut, CA).

Techniques: Staining, Labeling, Enzyme-linked Immunosorbent Assay, Expressing, Real-time Polymerase Chain Reaction

Journal: Endocrinology

Article Title: Continuous PTH in Male Mice Causes Bone Loss Because It Induces Serum Amyloid A

doi: 10.1210/en.2018-00265

Figure Lengend Snippet: Continuous PTH increased osteoblast differentiation in hBMSCs only when COX2 or RANKL activity was blocked. hBMSCs were cultured with vehicle (VEH) or PTH (10 nM), with or without NS398 (100 nM), a selective inhibitor of COX2, or OPG (100 ng/mL), which blocks RANKL binding to its receptor. (a) Measurement of accumulated medium PGE2 by ELISA. (b) Markers of osteoblast differentiation, ALPL (alkaline phosphatase) and BGLAP (osteocalcin), and alizarin red staining for mineralization at day 21. (c) TNFSF11 (RANKL) mRNA at day 7 and TRAP staining at day 8. Scale bar, 100 µm. (d) BGLAP mRNA and alizarin red staining at day 21. (e) RUNX2 mRNA at day 7 and IGF1 and BMP2 mRNA at day 21. (f) WNT10B and DKK1 mRNA at day 7. mRNA was measured by quantitative real-time PCR and data are reported as RQ values. Bars are means ± SEM for n = 3 independent samples. For (a), **P < 0.01, determined by one-way ANOVA, post hoc Bonferroni pairwise multiple comparisons. For (c), **P < 0.01, determined by two-tailed unpaired t test. For (d)–(f), *P < 0.05, **P < 0.01, determined by two-way ANOVA, post hoc Bonferroni pairwise multiple comparisons. Und, undetectable.

Article Snippet: SAA1 protein accumulated in the medium of human BMMs was measured by an SAA1 human ELISA kit (KA2108; Abnova Corporation, Walnut, CA).

Techniques: Activity Assay, Cell Culture, Binding Assay, Enzyme-linked Immunosorbent Assay, Staining, Real-time Polymerase Chain Reaction, Two Tailed Test

Journal: Endocrinology

Article Title: Continuous PTH in Male Mice Causes Bone Loss Because It Induces Serum Amyloid A

doi: 10.1210/en.2018-00265

Figure Lengend Snippet: NS398 blocked the RANKL induction of SAA1 and SAA2 expression in hBMMs but did not affect osteoclast-like cell numbers, and SAA1 and SAA2 inhibited PTH-stimulated osteoblastic differentiation in hBMSCs. (a–c) hBMMs were treated with M-CSF (30 ng/mL) plus vehicle (VEH) or M-CSF plus RANKL (30 ng/mL each) with/without NS398 (100 nM). (a) SAA1 and SAA2 protein measurement in the culture medium of hBMMs measured by ELISA. (b) SAA1 and SAA2 mRNA at day 3. (c) TRAP-stained microscopic images at ×100 original magnification (scale bars, 200 µm) and TRAP+ MNC counts per well. (d) ALPL (alkaline phosphatase) and BGLAP (osteocalcin) mRNA expression in hBMSCs at day 21 treated with VEH or PTH in presence of OPG (100 ng/mL) to prevent osteoclastogenesis with/without rhSAA1 (10 µg/mL) or rhSAA2 (10 to 50 ng/mL). Data are means ± SEM for n = 3 independent samples. For (a), **P < 0.01, significantly different from VEH treated at same time point, determined by two-tailed unpaired t test. For (c), **P < 0.01, significantly different from day 4, determined by one-way ANOVA, post hoc Bonferroni pairwise multiple comparisons. For (b) and (d), *P < 0.05, **P < 0.01, determined by two-way ANOVA, post hoc Bonferroni pairwise multiple comparisons.

Article Snippet: SAA1 protein accumulated in the medium of human BMMs was measured by an SAA1 human ELISA kit (KA2108; Abnova Corporation, Walnut, CA).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Staining, Two Tailed Test