Journal: Journal of Human Immunity

Article Title: Refractory infantile IPEX with Treg-restricted FOXP3null expression caused by a novel variant in FOXP3

doi: 10.70962/jhi.20250249

Figure Lengend Snippet: Patient CD4 + Tregs demonstrate no meaningful expression of FOXP3. (A) Flow cytometry analysis of patient PBMC and healthy control show CD4 + and CD8 + T cells within the CD3 + compartment (first row), Treg phenotype (CD25 + CD127 − ) (second row), expression of FOXP3 (third row), Helios (fourth row), and TIGIT (fifth row) within the Tregs. (B) FOXP3 expression in the CD4 + Teffs and Tregs, represented as histograms. (C) Quantification of TSDR-demethylated cells in CD3 + T cells (left) and CD4 + T cells (right) from patient samples and male healthy controls (adults, n = 33 and pediatric controls, n = 11) as well as the patient’s newborn screening dried blood spots (DBSs). (D) Kinetics of the percentage of TSDR/CD4 demethylated cells in patient blood since birth. ( E ) Detection of TSDR-demethylated cells within different CD4 + T effector compartments. GD-253 (IPEX patient [Pt]), GD-260 (patient’s mother), and HD-2417 and HD-2645 (healthy donors/controls). Gating strategies for are in .

Article Snippet: CD4 + T cells were isolated from freshly isolated PBMC using human CD4 + T cells Isolation kit (130-096-533; Miltenyi) according to the manufacturer’s instructions.

Techniques: Expressing, Flow Cytometry, Control

Journal: Journal of Human Immunity

Article Title: Refractory infantile IPEX with Treg-restricted FOXP3null expression caused by a novel variant in FOXP3

doi: 10.70962/jhi.20250249

Figure Lengend Snippet: Activated patient CD4 + T cells express FOXP3. (A) Expression of FOXP3 in baseline and activated CD4 + T cells (first row) showing total FOXP3 (clone: 259D) and CTLA4 (second row) as well as total FOXP3 (clone: 259D) and full-length FOXP3 (clone: 150D) (third row) expression by flow cytometry in patient’s mother, healthy control, and patient (Pt) CD4 + T cells. (B) Western blot analysis of FOXP3 expression in the activated CD4 + T cells, showing the full-length and delta2 isoforms of the FOXP3 protein along with the quantification of the protein levels. Source data are available for this figure: .

Article Snippet: CD4 + T cells were isolated from freshly isolated PBMC using human CD4 + T cells Isolation kit (130-096-533; Miltenyi) according to the manufacturer’s instructions.

Techniques: Expressing, Flow Cytometry, Control, Western Blot

Journal: Signal Transduction and Targeted Therapy

Article Title: Targeting GPR34 in damage-associated macrophages enhances anti-tumor immunity and the efficacy of Surufatinib in pancreatic cancer

doi: 10.1038/s41392-026-02641-4

Figure Lengend Snippet: Cohort and in vitro studies reveal the role of GPR34 in macrophages. a Volcano plot showing differentially expressed genes between responder group and non-responder group from the macrophage subclusters of scRNA sequencing data. The horizontal dashed line represents the P -value cutoff ( P < 10⁻⁵⁰), and the vertical dashed line represents the log 2 FC cutoff (-1 or 1). FC fold change, sig significance, R Responder, NR Non-responder. b , c UMAP plot showing the expression of GPR34 in all cells ( b ) and macrophages ( c ). Mac: Macrophage, cl cluster. d Representative mIF staining images (100x) of surgical specimens from responders and non-responders in the clinical trial ( n = 26). Navy: GPR34, Magenta: CD68, Red: CD8, Green: Tim-3, Yellow: CK19, Blue: DAPI. White arrowheads: Tim-3 + CD8 + exhausted T cells (Tex); White arrows: GPR34 + CD68 + macrophages. White scale bar = 100 μm. e Bar plot comparing the proportion of CD68 + macrophages (top) and the proportion of GPR34 + cells among CD68 + macrophages (bottom) between responders and non-responders. Two-tailed unpaired t-test. Data are presented by mean ± SD. * P < 0.05. f Bar plot comparing the proportion of CD8 + T cells (top) and the proportion of Tim-3 + cells among CD8 + T cells (bottom) between responders and non-responders. Two-tailed unpaired t-test. Data are presented by mean ± SD. g Representative mIF staining images (200x) of surgical specimens from clinical trial patients ( n = 26). Navy: GPR34, Magenta: CD68, Red: MPO, Green: CD3, Dark yellow: CD20, Yellow: CK19, Blue: DAPI. White scale bar = 50 μm. h Representative mIF staining images (200x) of clinical trial patient surgical specimens ( n = 26). Navy: GPR34, Magenta: CD68, Red: α-SMA, Green: CD31, Dark yellow: CD117, Yellow: CD56, Blue: DAPI. White scale bar = 50 μm. i Bar plots showing the ratio of GPR34 + cells in different cell types ( n = 26). One-way ANOVA with Dunnett’s test was used to compare other cell types versus macrophages. Data are presented by mean ± SD. j Bar plots showing the ratio of GPR34 + cells in different cell types ( n = 26) examined by flow cytometry. One-way ANOVA with Dunnett’s test was used to compare other cell types versus macrophages. Data are presented by mean ± SD. k , l Kaplan-Meier curves for overall survival (OS) and Recurrence-free survival (RFS) in the prospective cohort ( k ) and retrospective cohort ( l ), comparing prognosis between groups with high versus low infiltration of GPR34 + macrophages. The cutoff for the proportion of GPR34 + cells among CD68 + cells were dichotomized using a 20% . Log-rank test was used for comparison. m Flow cytometry analysis of GPR34 + cells in tumor tissue versus adjacent non-tumorous tissue from prospective cohort patients ( n = 42). Scatter plot shows the paired infiltration proportion of GPR34 + cells in tumor and normal tissue from the same patient . Two-tailed paired t -test was used. n Flow cytometry analysis of GPR34 + cells in tumor tissue from prospective cohort patients ( n = 42). Bar plots show the infiltration proportions of CD45 + cells, CD8 + T cells, Tim-3 + PD-1 + T cells, MRC1 + macrophages, and MHC-I + macrophages in the low GPR34 ( ≤ 20%) versus high GPR34 ( > 20%) groups. Two-tailed unpaired t-test. Data are presented by mean ± SD. o BMDMs from C57BL/6 mice were cultured until day 5, stimulated with KPC cell TCM for 12 h, followed by treatment with Surufatinib (4 nM) or CSF-1R inhibitor (PLX3397, 20 nM) for 24 h, then analyzed by flow cytometry ( n = 3). Bar plot compares the gMFI of GPR34 among groups. One-way ANOVA with post-hoc Tukey’s test was used. Data are presented by mean ± SD. NS no significance, * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: CD8+ T cells were isolated from spleens of OT-1 or C57BL/6 mice using the human CD8 + T Cell Isolation Kit (Negative Isolation) (Miltenyi, Cat. No. 130-104-075).

Techniques: In Vitro, Sequencing, Expressing, Staining, Two Tailed Test, Flow Cytometry, Comparison, Cell Culture

Journal: Signal Transduction and Targeted Therapy

Article Title: Targeting GPR34 in damage-associated macrophages enhances anti-tumor immunity and the efficacy of Surufatinib in pancreatic cancer

doi: 10.1038/s41392-026-02641-4

Figure Lengend Snippet: Macrophage-specific GPR34 knockout improves chemotherapy outcomes in mice. a Orthotopic pancreatic injection of KPC-GFP-LUC cells into Gpr34 flox/flox and Gpr34 Δ Lyz2 mice. After tumor formation, chemotherapy was administered to simulate tumor killing. Tumor bioluminescence intensity was dynamically monitored by bioluminescence imaging. Representative bioluminescence images show tumor growth in each group ( n = 5). b Time-course curve of bioluminescence intensity in orthotopic tumor-bearing mice (n = 5). Two-way ANOVA with post-hoc Tukey’s test was used. Data are presented by mean ± SEM. c Bar plot of tumor weight on day 21 post-implantation in orthotopic tumor-bearing mice ( n = 5). One-way ANOVA with post-hoc Tukey’s test was used. Data are presented by mean ± SD. NS: no significance, *: P < 0.05. d Flow cytometry analysis of the proportions of CTL, Tex, M1 macrophages, and M2 macrophages in tumor tissues of orthotopic tumor-bearing mice (n = 5). Bar plots show the infiltration differences of immune cells among groups. One-way ANOVA with post-hoc Tukey’s test was used. Data are presented by mean ± SD. e KPC mice were irradiated with a total dose of 10 Gy, followed by tail vein injection of bone marrow cells from Gpr34 +/+ or Gpr34 −/− mice. After successful transplantation confirmed by flow cytometry. Tumor growth was monitored by B-ultrasound during chemotherapy simulating injury signals ( n = 8–10). Representative B-ultrasound images compare tumor size among groups. Black dashed lines outline tumor boundaries. Tumor volume was calculated as 0.5 * long diameter * (short diameter) ². S: spleen; K: kidney. Black arrows point to the tumor. f Time-course plot of tumor volume monitored by B-ultrasound. Each solid line represents an individual mouse. Two-way ANOVA with post-hoc Tukey’s test was used. Data are presented by mean ± SD. g Kaplan-Meier curve of KPC spontaneous tumor-bearing mice ( n = 8–10). Log-rank test was used. h mIF staining of paraffin sections from orthotopic tumors in the bone marrow transplantation KPC mice ( n = 6). Representative fluorescence images (top 100x, bottom 400x) show exhausted T cell infiltration among groups. Red: CD8α, Green: Tim-3, White: CK19, Blue: DAPI. White scale bar = 50 μm. i Bar plot showing the proportion of Tim-3 + CD8 + T cells from mIF staining in KPC spontaneous tumor-bearing mice ( n = 6). One-way ANOVA with post-hoc Tukey’s test was used. Data are presented by mean ± SD. j Fluorescence imaging of macrophages sorted by flow cytometry from tumors of KPC-GFP-LUC orthotopic injection mice model. Representative images show GFP phagocytosis by macrophages in each group ( n = 6). Green: GFP, Blue: DAPI. White scale bar = 20 μm. k Bar plot showing the gMFI of GFP phagocytosis by flow-sorted macrophages from orthotopic tumors ( n = 5). One-way ANOVA with post-hoc Tukey’s test was used. Data are presented by mean ± SD. l Bar plot of the gMFI of GFP in macrophages from KPC cell orthotopic tumor-bearing mice ( n = 6). One-way ANOVA with post-hoc Tukey’s test was used. Data are presented by mean ± SD. m Bar plot showing the proportion of MerTK + or AXL + macrophage from flow cytometry analysis in KPC mice ( n = 6). One-way ANOVA with post-hoc Tukey’s test was used . Data are presented by mean ± SD. n BMDMs from Gpr34 +/+ and Gpr34 −/− mice were cultured until day 5, cultured with TCM for 12 h, followed by co-culture with CD8 + T cells for 24 h. Flow cytometry detected the proportion of MerTK + or AXL + macrophage. Bar plots show differences between Gpr34 +/+ and Gpr34 −/− groups ( n = 6). Two-tailed unpaired t-test was used. Data are presented by mean ± SD. NS no significance, * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: CD8+ T cells were isolated from spleens of OT-1 or C57BL/6 mice using the human CD8 + T Cell Isolation Kit (Negative Isolation) (Miltenyi, Cat. No. 130-104-075).

Techniques: Knock-Out, Injection, Imaging, Flow Cytometry, Irradiation, Transplantation Assay, Staining, Fluorescence, Cell Culture, Co-Culture Assay, Two Tailed Test

Journal: Signal Transduction and Targeted Therapy

Article Title: Targeting GPR34 in damage-associated macrophages enhances anti-tumor immunity and the efficacy of Surufatinib in pancreatic cancer

doi: 10.1038/s41392-026-02641-4

Figure Lengend Snippet: Validation of GPR34 function in macrophage and CD8 + T cell co-culture system. a Gpr34 flox/flox and Gpr34 Δ Lyz2 mice were treated with anti-CD8α or IgG, followed by orthotopic pancreatic injection of KPC-LUC cells. After tumor formation, chemotherapy was administered to simulate an injury signal. Tumor bioluminescence was dynamically monitored. Representative bioluminescence images show tumor growth in different groups ( n = 6). b Time-course curve of bioluminescence imaging for the KPC-LUC orthotopic model ( n = 6). Two-way ANOVA with post-hoc Tukey’s test was used. Data are presented by mean ± SD. NS no significance, *** P < 0.001. c Bar plot showing tumor weight on day 21 in the KPC-LUC orthotopic model ( n = 6). One-way ANOVA with post-hoc Tukey’s test was used. Data are presented by mean ± SD. NS no significance. d , e BMDMs from Gpr34 +/+ and Gpr34 −/− mice were cultured until day 5, then co-cultured with TCM and KPC-GFP cells for 12 h. BMDMs were then isolated and co-cultured with CD8 + T cells for 24 hours . Flow cytometry analyzed the expression of functional molecules in BMDMs ( d ) and CD8 + T cells ( e ). Bar plots show levels in Gpr34 +/+ vs Gpr34 −/− groups ( n = 3). Two-tailed unpaired Student’s t test was used. Data are presented by mean ± SD. f , g BMDMs from Gpr34 +/+ and Gpr34 −/− mice were cultured until day 5, pre-stimulated with SIINFEKL, then cultured with TCM for 12 h, followed by co-culture with CD8 + T cells from OT-1 mice for 24 h. Flow cytometry detected T cell-specific killing function ( f ) and BMDM antigen presentation function ( g ). Bar plots show differences between Gpr34 +/+ and Gpr34 −/− groups ( n = 3). Two-tailed unpaired t-test was used. Data are presented by mean ± SD. h Violin-box plots of cytokine transcript expression in macrophage clusters from scRNA sequencing data. White dot and solid lines in boxes represent medians and quartiles. Two-tailed Wilcoxon test. i BMDMs from Gpr34 +/+ and Gpr34 −/− mice were cultured until day 5, then stimulated with TCM and chemotherapy-induced apoptotic KPC-GFP cells for 12 h. qPCR detected Cxcl16 transcript levels. Bar plot compares Cxcl16 transcripts between groups ( n = 3). Two-tailed unpaired t-test was used. Data are presented by mean ± SD. j ELISA detection of cytokine secretion in supernatant from BMDMs stimulated with apoptotic KPC-GFP cells. Bar plot shows CXCL16 protein secretion levels from Gpr34 +/+ and Gpr34 −/− BMDMs ( n = 10). Two-tailed unpaired t-test was used. Data are presented by mean ± SD. k , l BMDMs from C57BL/6 mice were cultured until day 5, transiently transfected with siRNA, then co-cultured with TCM, LysoPS and chemotherapy-induced apoptotic KPC-GFP cells for 12 h. BMDMs were then isolated and co-cultured with CD8 + T cells. Flow cytometry detected T cell exhaustion ( k ) and cytotoxicity levels ( l ) ( n = 3). One-way ANOVA with Dunnett’s test compared siRNA groups versus control. Data are presented by mean ± SD. NS no significance, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: CD8+ T cells were isolated from spleens of OT-1 or C57BL/6 mice using the human CD8 + T Cell Isolation Kit (Negative Isolation) (Miltenyi, Cat. No. 130-104-075).

Techniques: Biomarker Discovery, Co-Culture Assay, Injection, Imaging, Cell Culture, Isolation, Flow Cytometry, Expressing, Functional Assay, Two Tailed Test, Immunopeptidomics, Sequencing, Enzyme-linked Immunosorbent Assay, Transfection, Control

Journal: Signal Transduction and Targeted Therapy

Article Title: Targeting GPR34 in damage-associated macrophages enhances anti-tumor immunity and the efficacy of Surufatinib in pancreatic cancer

doi: 10.1038/s41392-026-02641-4

Figure Lengend Snippet: LysoPS-GPR34 modulates macrophage efferocytosis and inflammatory cytokine secretion. a , b BMDMs from Gpr34 +/+ and Gpr34 −/− mice were cultured until day 5, co-cultured with TCM, LysoPS and chemotherapy-induced apoptotic KPC-GFP cells for 12 h. These were then co-cultured with CD8 + T cells for 24 hours. Flow cytometry detected GFP efferocytosis ( a ) and p-AKT levels ( b ) in BMDMs. Bar plots show differences among groups. One-way ANOVA with post-hoc Tukey’s test was used. Data are presented by mean ± SD. c BMDMs from Gpr34 +/+ and Gpr34 −/− mice were treated with LysoPS and co-cultured with CD8 + T cells. Flow cytometry detected MRC1, MHC-I on macrophages, and Tim-3, GZMB on CD8 + T cells. Bar plot shows gMFI differences among groups. One-way ANOVA with post-hoc Tukey’s test was used. Data are presented by mean ± SD. d , e BMDMs from C57BL/6 mice were treated with LysoPS and AKT inhibitor, then co-cultured with chemotherapy-induced apoptotic KPC-GFP cells for 12 h, followed by co-culture with CD8 + T cells for 24 h. Flow cytometry detected GFP uptake by BMDMs ( d ) and exhaustion/cytotoxicity levels of CD8 + T cells ( e ). One-way ANOVA with post-hoc Tukey’s test was used. Data are presented by mean ± SD. f UMA P plot showing relative expression of efferocytosis-related genes from scRNA sequencing analysis. g BMDMs from Gpr34 +/+ and Gpr34 −/− mice were cultured until day 5, co-cultured with TCM and either chemotherapy-induced apoptotic KPC-GFP cells or normal KPC-GFP cells for 12 h. After removing cells, RNA was extracted for qPCR. Bar plot compares transcript levels among groups. One-way ANOVA with post-hoc Tukey’s test was used. Data are presented by mean ± SD. h BMDMs from C57BL/6 mice were cultured until day 5, co-cultured with apoptotic KPC cells of negative control for 12 hours. RNA was extracted for qPCR. Bar plot compares transcript differences among groups. One-way ANOVA with post-hoc Tukey’s test was used. Data are presented by mean ± SD. i BMDMs from C57BL/6 mice were cultured until day 5, stimulated with TCM and LysoPS for 12 h. ELISA detected CXCL16 secretion in supernatant. Bar plot shows levels in LysoPS vs. control groups. Two-tailed unpaired Student’s t test was used. Data are presented by mean ± SD. NS no significance, * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: CD8+ T cells were isolated from spleens of OT-1 or C57BL/6 mice using the human CD8 + T Cell Isolation Kit (Negative Isolation) (Miltenyi, Cat. No. 130-104-075).

Techniques: Cell Culture, Flow Cytometry, Co-Culture Assay, Expressing, Sequencing, Negative Control, Enzyme-linked Immunosorbent Assay, Control, Two Tailed Test

Journal: Signal Transduction and Targeted Therapy

Article Title: Targeting GPR34 in damage-associated macrophages enhances anti-tumor immunity and the efficacy of Surufatinib in pancreatic cancer

doi: 10.1038/s41392-026-02641-4

Figure Lengend Snippet: Macrophage efferocytosis function influences antigen presentation ability through MHC-I. a BMDMs from C57BL/6 mice were cultured until day 5, co-cultured with TCM and chemotherapy-induced apoptotic KPC-GFP cells for 12 hours, then analyzed by flow cytometry for GFP uptake. Bar plot shows gMFI of GFP in BMDMs treated with MerTK inhibitor vs control ( n = 3). One-way ANOVA with Dunnett’s test compared MerTKi groups to control. Data are presented by mean ± SD. b BMDMs from C57BL/6 mice were cultured until day 5, co-incubated with TCM and chemotherapy-induced apoptotic KPC-OVA-GFP cells for 12 hours, treated with MerTK inhibitor, then co-cultured with CD8 + T cells from OT1 mice for 24 hours. Flow cytometry detected MHC-I, SIINFEKL loading, CD80, CD86 on BMDMs. Bar plot shows differences between MerTK inhibitor and control groups ( n = 3). One-way ANOVA with Dunnett’s test was used. Data are presented by mean ± SD. c Flow cytometry detection of Tetramer + , PD-1 + , Tim-3 + , and GZMB + cells after co-culture of BMDMs with OT1 CD8 + T cells. Bar plot shows differences between MerTK inhibitor and control groups ( n = 3). One-way ANOVA with Dunnett’s test was used. Data are presented by mean ± SD. d Apoptotic KPC cells induced by chemotherapy and labeled with Caspase3/7 green were co-cultured with BMDMs. Phagolysosome formation was detected using pHrodo red. Representative fluorescence microscopy images (1000x) show differences between MerTK inhibitor and control groups ( n = 6). Green: Caspase3/7, Red: pHrodo, Blue: DAPI. White scale bar= 20 μm. e Bar plots show total pHrodo fluorescence intensity (left) and the number of Caspase3/7 + pHrodo + vesicles per cell (right) in BMDMs after incubation with apoptotic cells ( n = 6). One-way ANOVA with Dunnett’s test was used. Data are presented by mean ± SD. f Flow cytometry analysis of pHrodo gMFI in BMDMs after incubation with apoptotic cells. Bar plot shows pHrodo gMFI levels between MerTK inhibitor and control groups ( n = 3). One-way ANOVA with Dunnett’s test was used. Data are presented by mean ± SD. g Violin-box plots of lysosome-associated gene transcript expression in macrophage subpopulations from scRNA sequencing data. Solid lines represent medians and quartiles. One-way ANOVA with Kruskal-Wallis H test compared groups (Mac_cl1 as reference). h , i BMDMs from Gpr34 +/+ and Gpr34 −/− mice were cultured until day 5, co-cultured with TCM and chemotherapy-induced apoptotic KPC cells for 12 hours. After removing apoptotic cells, RNA was extracted for qPCR. Bar plots show transcript differences between Gpr34 +/+ and Gpr34 −/− BMDMs ( h ) efferocytosis receptors, ( i ) lysosome-related/transcription factors, ( n = 3). Two-tailed unpaired t-test was used. Data are presented by mean ± SD. j BMDMs from C57BL/6 mice were cultured until day 5, transiently transfected with siRNA, then co-incubated with TCM and chemotherapy-induced apoptotic KPC cells for 12 hours and analyzed by flow cytometry. Bar plot shows differences in MHC-I protein levels between knockdown and control groups ( n = 3). One-way ANOVA with post-hoc Tukey’s test was used. Data are presented by mean ± SD. k BMDMs from C57BL/6 mice were cultured until day 5, co-incubated with TCM and chemotherapy-induced apoptotic KPC-OVA-GFP for 12 hours, treated with lysosomal inhibitor, then co-cultured with OT1 CD8 + T cells for 24 hours. Bar plot shows pHrodo gMFI in macrophages from flow cytometry, comparing lysosomal inhibitor group vs control. One-way ANOVA with Dunnett’s test was used . Data are presented by mean ± SD. l , m Flow cytometry detection of macrophage antigen presentation function ( l ) and CD8 + T cell specific killing capacity ( m ) in the BMDM-OT1 CD8 + T cell co-culture system. Bar plots show differences between lysosomal inhibitor and control groups. One-way ANOVA with Dunnett ’ s test was used. Data are presented by mean ± SD. n BMDMs from C57BL/6 mice were cultured until day 5, co-incubated with TCM, MerTK inhibitor/Lysosome inhibitor and chemotherapy-induced apoptotic KPC cells for 12 hours. Bar plot shows differences in MHC-I protein levels between different groups detected by flow cytometry ( n = 3). One-way ANOVA with post-hoc Tukey’s test was used. Data are presented by mean ± SD. o –q BMDMs from Gpr34 +/+ and Gpr34 −/− mice were cultured until day 5, transiently transfected with Cxcl16 siRNA, co-incubated with TCM, LysoPS and chemotherapy-induced apoptotic KPC-OVA-GFP for 12 hours, treated with MerTK inhibitor, then co-cultured with OT1 CD8 + T cells for 24 hours. Flow cytometry detected CD8 + T cell specific killing function (o ), cytotoxic function ( p ), and exhaustion levels ( q ). One-way ANOVA with Dunnett’s test was used. Data are presented by mean ± SD. NS no significance, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: CD8+ T cells were isolated from spleens of OT-1 or C57BL/6 mice using the human CD8 + T Cell Isolation Kit (Negative Isolation) (Miltenyi, Cat. No. 130-104-075).

Techniques: Immunopeptidomics, Cell Culture, Flow Cytometry, Control, Incubation, Co-Culture Assay, Labeling, Fluorescence, Microscopy, Expressing, Sequencing, Two Tailed Test, Transfection, Knockdown