Journal: bioRxiv

Article Title: Binding of inhibitory checkpoints to CD18 in cis hinders anti-cancer immune responses

doi: 10.1101/2025.09.10.675342

Figure Lengend Snippet: ( A ) Existing model by which SIRPα suppresses phagocytosis by interacting in trans with CD47 on target cells. See text for details. The 3 Ig-like domains of SIRPα (1 IgV and 2 IgCs) and the single Ig-V domain of CD47 are shown as ellipses. Mβs, macrophages. ( B ) Depiction of SIRPα variants and their functional characteristics. SIRPα FFFF contained substitution of tyrosine (Y)-to-phenylalanine (F) substitution at Y436, 460, 477, and 501; SIRPα ΔIC lacked most of the cytoplasmic domain of SIRPα, ending with arginine 401; SIRPα T96V carried a threonine (T)-to-valine (V) mutation at position 96 (shown by lavender star), which abolishes CD47-binding; SIRPα T96V,FFFF had the T96V and FFFF mutations; SIRPα T96V,ΔIC had the T96V and the ΔIC mutations. KO, knock-out. ITIM, immunoreceptor tyrosine-based inhibitory motif. ( C to G ) SIRPα variants or empty vector were expressed in SIRPα KO BMDMs and tested. Wild-type (WT) BMDMs were used as control. ( C ) Schematic representation of assays performed. Fc, fragment crystallizable. ( D ) Flow cytometry analyses of SIRPα expression and CD47-binding. APC, allophycocyanin. AF647, Alexa fluor 647. ( E and F ) Representative ( E ) and compiled data ( F ) of pHrodo-based phagocytosis assays using L1210 derivatives expressing Tac and opsonized with Tac monoclonal antibody (mAb) 7G7, as targets. Positive cells with percentages are boxed. G , Efficiency of phagocytosis inhibition in SIRPα KO BMDMs expressing or not the indicated SIRPα variants was calculated using the values in ( F ). SIRPα KO expressing WT SIRPα or empty vector displayed 100% and 0% inhibition efficiency, respectively. All data are means ± s.e.m., **** p < 0.0001. Results in ( D and E ) are representative of 6 independent experiments, except for SIRPα T96V , SIRPα T96V, FFFF and SIRPα T96V, ΔIC that are representative of 3 experiments. Results in ( F and G ) are pooled from a total of 6 mice studied in 6 independent experiments, except for SIRPα T96V , SIRPα T96V, FFFF and SIRPα T96V, ΔIC that involved 3 mice in 3 experiments. Each symbol in ( F ) represents one mouse.

Article Snippet: For opsonization of target cells with IgG, Fc-intact human CD25 mAb 7G7 (mouse IgG2a, BioXCell, Lebanon, NH) and Fc-intact human CD20 mAb rituximab biosimilar (human IgG1, BioXCell) were used.

Techniques: Functional Assay, Mutagenesis, Binding Assay, Knock-Out, Plasmid Preparation, Control, Flow Cytometry, Expressing, Inhibition

Journal: bioRxiv

Article Title: Binding of inhibitory checkpoints to CD18 in cis hinders anti-cancer immune responses

doi: 10.1101/2025.09.10.675342

Figure Lengend Snippet: ( A ) FRET assays of donor-labeled mouse SIRPα with acceptor-labeled mouse CD18 and unlabeled mouse CD11b, in the presence of Fc-silent mouse SIRPα mAbs, as was done for , D to F. ( B ) Binding of a soluble CD47-Fc fusion protein to EL-4 cells, expressing or not expressing mouse SIRPα, was studied by flow cytometry. ( C to K ) Generation and impact of bispecific antibody (BsAb) against mouse SIRPα. ( C ) Schematic representation of Fc-silent BsAb combining one arm of mAb #17 with one arm of mAb #27, using the “knob-into-hole” technology. Phagocytosis of IgG-opsonized L1210 cells ( D ) and EL-4 cells ( E ) by WT BMDMs, in the presence of mAbs, was assessed by a microscopy assays. ( F to K ) Schematic depictions of the assays are shown in (F and I). RAG-1 KO mice injected subcutaneously with Tac + L1210 cells ( G and H ), or C57BL/6J mice injected subcutaneously with Tac + EL-4 cells ( J and K ), were treated by intraperitoneal injection of Fc-silent mAbs, alongside Tac mAb 7G7 for opsonization. Tumor volume was measured using a caliper ( G and J ) and survival was recorded ( H and K ). ( L ) FRET assays of donor-labeled human SIRPα V1 or V2 with acceptor-labeled human CD18 and unlabeled human CD11b in the presence of Fc-silent Ctrl IgG and human SIRPα mAbs KWAR23, 40A, 50A, or 18D5, as was done for , D to F. The mAbs were rendered Fc-silent by the LALAPG mutation. ( M ) Phagocytosis of IgG-opsonized Raji cells by human macrophages in the presence of Fc-silent Ctrl IgG and SIRPα mAbs KWAR23, 40A, 50A, or 18D5, was assayed as for . ( N ) FRET assays of donor-labeled human 2B4 (SLAMF4), PD-1 or LILRB1 with acceptor-labeled human CD18, in the presence of Ctrl IgG or human CD18 mAb were done as for , D to F. All data are means ± s.e.m. ns, not significant; * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001. Results are pooled from a total of two ( H and K ), three ( A , D , E , G , J , L and N ) or five ( B and M ) independent experiments. Each symbol in ( A , D , E and L to N ) represents one healthy donor, cell or mouse.

Article Snippet: For opsonization of target cells with IgG, Fc-intact human CD25 mAb 7G7 (mouse IgG2a, BioXCell, Lebanon, NH) and Fc-intact human CD20 mAb rituximab biosimilar (human IgG1, BioXCell) were used.

Techniques: Labeling, Binding Assay, Expressing, Flow Cytometry, Microscopy, Injection, Mutagenesis

Journal:

Article Title: T cell activation up-regulates cyclic nucleotide phosphodiesterases 8A1 and 7A3

doi: 10.1073/pnas.101131098

Figure Lengend Snippet: Up-regulation of PDE7A1, PDE7A3, and PDE8A1 mRNA in CD4+ T cells. CD4+ T cells were stimulated with CD3 and CD28 antibodies and harvested at the indicated time points (hours). RT-PCR analysis was performed as described in Materials and Methods. (A) Comparison of methods of preparation of CD4+ T cells. Cells were prepared as described in Materials and Methods by using either the CD4+ T cell isolation kit in combination with the CD69 microbeads (a) or a mixture of mAbs and goat anti-mouse magnetic beads (b). (B) Time course of the induction of PDE7A1, PDE7A3, and PDE8A1 mRNA compared with a GAPDH control. The bands were scanned and quantified by using nih image. Within each image, the maximum intensity of the band was set to a value of 1 and other bands were calculated as a fraction of the maximum. The values were normalized to the GAPDH signal. The results of the densitometry are graphed. (C) Time course of induction of PDE7A1, PDE7A3, and PDE8A1 by using serially diluted cDNA as described in Materials and Methods. (D) RT-PCR was performed for PDE8A1 from cells harvested at 1 and 16 h after stimulation by using either CD3, CD28, or a combination of the antibodies (Left). Cells were harvested 16 h after stimulation and analyzed by Western blotting by using a PDE8A1 polyclonal antibody (PIL9) (Right).

Article Snippet: The anti-CD8 (G10–1), CD16 (FC2), CD20 (2H7), CD25 (7G7), and HLADR (HB10a) mouse mAbs were provided by E. A. Clark ( 19 ), and the anti-CD3 and CD28 mAbs were purchased from PharMingen.

Techniques: Reverse Transcription Polymerase Chain Reaction, Cell Isolation, Magnetic Beads, Western Blot

Journal:

Article Title: T cell activation up-regulates cyclic nucleotide phosphodiesterases 8A1 and 7A3

doi: 10.1073/pnas.101131098

Figure Lengend Snippet: Up-regulation of PDE7A1, PDE7A3, and PDE8A1 mRNA in CD4+ T cells. CD4+ T cells were stimulated with CD3 and CD28 antibodies and harvested at the indicated time points (hours). RT-PCR analysis was performed as described in Materials and Methods. (A) Comparison of methods of preparation of CD4+ T cells. Cells were prepared as described in Materials and Methods by using either the CD4+ T cell isolation kit in combination with the CD69 microbeads (a) or a mixture of mAbs and goat anti-mouse magnetic beads (b). (B) Time course of the induction of PDE7A1, PDE7A3, and PDE8A1 mRNA compared with a GAPDH control. The bands were scanned and quantified by using nih image. Within each image, the maximum intensity of the band was set to a value of 1 and other bands were calculated as a fraction of the maximum. The values were normalized to the GAPDH signal. The results of the densitometry are graphed. (C) Time course of induction of PDE7A1, PDE7A3, and PDE8A1 by using serially diluted cDNA as described in Materials and Methods. (D) RT-PCR was performed for PDE8A1 from cells harvested at 1 and 16 h after stimulation by using either CD3, CD28, or a combination of the antibodies (Left). Cells were harvested 16 h after stimulation and analyzed by Western blotting by using a PDE8A1 polyclonal antibody (PIL9) (Right).

Article Snippet: The anti-CD8 (G10–1), CD16 (FC2), CD20 (2H7), CD25 (7G7), and HLADR (HB10a) mouse mAbs were provided by E. A. Clark ( 19 ), and the anti-CD3 and CD28 mAbs were purchased from PharMingen.

Techniques: Reverse Transcription Polymerase Chain Reaction, Cell Isolation, Magnetic Beads, Western Blot

Journal: Frontiers in Immunology

Article Title: Cysteine-specific 89 Zr-labeled anti-CD25 IgG allows immuno-PET imaging of interleukin-2 receptor-α on T cell lymphomas

doi: 10.3389/fimmu.2022.1017132

Figure Lengend Snippet: 89 Zr-CD25 IgG characterization. (A) Schema of 89 Zr-CD25 IgG (left). SDS PAGE of intact, TCEP reduced, and DFO-maleimide conjugated anti-human and anti-mouse CD25 IgG (middle). Autoradiography of column-eluted 89 Zr-CD25 IgG fractions on native PAGE (right). (B) PD10 column chromatography of anti-human and anti-mouse 89 Zr-CD25 IgG (left), and in vitro radiochemical stability in PBS and FBS over days (right; mean of duplicate samples per group).

Article Snippet: Mouse monoclonal IgG2a anti- CD25 Ab (7G7/B6) that is specific against human CD25 and does not react with mouse CD25, rat monoclonal IgG1 Ab against mouse CD25 (PC-61.5.3), and IgG2a isotype control Ab were from BioXcell (West Lebanon, NH).

Techniques: SDS Page, Autoradiography, Clear Native PAGE, Column Chromatography, In Vitro

Journal: Frontiers in Immunology

Article Title: Cysteine-specific 89 Zr-labeled anti-CD25 IgG allows immuno-PET imaging of interleukin-2 receptor-α on T cell lymphomas

doi: 10.3389/fimmu.2022.1017132

Figure Lengend Snippet: CD25 expression and 89 Zr-CD25 IgG binding to human lymphoma cells. (A) Western blots of CD25 protein and anti-human 89 Zr-CD25 IgG uptakes in H9, Jurkat, and SUDHL1 lymphoma cells (left). Complete blocking of SUDHL1 cell binding by 0.5 μM of unlabeled anti-CD25 IgG (right). (B) Lindmo binding assays for determining the immunoreactive fraction. Five concentrations of SUDHL1 cells were used, from 0.5 to 16 million cells/ml. The final concentration of 89 Zr-CD25 IgG was 50 ng/ml, and nonspecific binding was assessed with 5 μg of unlabeled anti-CD25 IgG. A conventional plot of specific and nonspecific binding over total applied radioactivity, as a function of increasing cell concentration is shown (left). A double inverse plot was then drawn using the same data as total applied radioactivity over specific binding, as a function of the inverse cell concentration (right). The immunoreactive fraction was determined through linear extrapolation to the ordinate. Data represent the mean ± S.E of values from two independent experiments [total n = 5 per group; uptake in (A) ], or the mean ± S.D of data from a single representative experiment [n = 3 per group; (B) ].

Article Snippet: Mouse monoclonal IgG2a anti- CD25 Ab (7G7/B6) that is specific against human CD25 and does not react with mouse CD25, rat monoclonal IgG1 Ab against mouse CD25 (PC-61.5.3), and IgG2a isotype control Ab were from BioXcell (West Lebanon, NH).

Techniques: Expressing, Binding Assay, Western Blot, Blocking Assay, Concentration Assay, Radioactivity

Journal: Frontiers in Immunology

Article Title: Cysteine-specific 89 Zr-labeled anti-CD25 IgG allows immuno-PET imaging of interleukin-2 receptor-α on T cell lymphomas

doi: 10.3389/fimmu.2022.1017132

Figure Lengend Snippet: Circulating free CD25 level and effect on blood 89 Zr-CD25 IgG. (A) Western blots of CD25 protein in blood obtained from the heart of 16 SUDHL1 lymphoma-bearing mice (top). Relations of tumor weight with blood 89 Zr-CD25 IgG (bottom left) and blood 89 Zr-CD25 IgG with blood human CD25 level (bottom right). (B) Relations of tumor weight with 89 Zr-CD25 IgG tumor uptake (left) and blood activity (middle) in another group of mice. Western blots and quantified band intensities of tumor CD25 protein according to tumor weight group is shown in the right. Lines were fitted by linear regression and correlation was based on nonparametric Spearman’s analysis. Bars represent the mean ± S.D of four samples per group. N.S., not significant.

Article Snippet: Mouse monoclonal IgG2a anti- CD25 Ab (7G7/B6) that is specific against human CD25 and does not react with mouse CD25, rat monoclonal IgG1 Ab against mouse CD25 (PC-61.5.3), and IgG2a isotype control Ab were from BioXcell (West Lebanon, NH).

Techniques: Western Blot, Activity Assay

Journal: Frontiers in Immunology

Article Title: Cysteine-specific 89 Zr-labeled anti-CD25 IgG allows immuno-PET imaging of interleukin-2 receptor-α on T cell lymphomas

doi: 10.3389/fimmu.2022.1017132

Figure Lengend Snippet: Pharmacokinetics, PET imaging, and biodistribution in mice. (A) Anti-human 89 Zr-CD25 IgG activity in whole blood, plasma, and blood cells of SUDHL1 lymphoma mice following intravenous injection (left). Data are the mean ± SD of five mice for each time group. Representative maximum intensity projection PET images at 5 days (right). (B) Representative coronal (top left) and transaxial (top right) PET images. Biodistribution at 5 days in mice at baseline or after three intraperitoneal PMA injections. Data are the mean ± S.E from two independent experiments (n = 5 per group).

Article Snippet: Mouse monoclonal IgG2a anti- CD25 Ab (7G7/B6) that is specific against human CD25 and does not react with mouse CD25, rat monoclonal IgG1 Ab against mouse CD25 (PC-61.5.3), and IgG2a isotype control Ab were from BioXcell (West Lebanon, NH).

Techniques: Drug discovery, Imaging, Activity Assay, Clinical Proteomics, Injection

Journal: Frontiers in Immunology

Article Title: Cysteine-specific 89 Zr-labeled anti-CD25 IgG allows immuno-PET imaging of interleukin-2 receptor-α on T cell lymphomas

doi: 10.3389/fimmu.2022.1017132

Figure Lengend Snippet: Target Specificity of tumor uptake in vivo . Representative coronal (left) and transaxial (right) PET images at 5 days of SUDHL1 lymphoma mice co-injected with anti-human 89 Zr-CD25 IgG and 0.8 mg of unlabeled anti-CD25 IgG for blocking (top), or with 89 Zr-labeled IgG2a isotype control Ab (middle). Biodistribution data are shown (bottom) as the mean ± S.E of values obtained from two independent experiments (n = 5 per group).

Article Snippet: Mouse monoclonal IgG2a anti- CD25 Ab (7G7/B6) that is specific against human CD25 and does not react with mouse CD25, rat monoclonal IgG1 Ab against mouse CD25 (PC-61.5.3), and IgG2a isotype control Ab were from BioXcell (West Lebanon, NH).

Techniques: In Vivo, Injection, Blocking Assay, Labeling, Control

Journal: Frontiers in Immunology

Article Title: Cysteine-specific 89 Zr-labeled anti-CD25 IgG allows immuno-PET imaging of interleukin-2 receptor-α on T cell lymphomas

doi: 10.3389/fimmu.2022.1017132

Figure Lengend Snippet: 89 Zr-CD25 IgG uptake in mouse cells in vitro and in mouse lymphoma in vivo . (A) CD25 expression (left) and anti-mouse 89 Zr-CD25 IgG binding (right) in mouse thymus Treg lymphocytes and EL4 mouse lymphoma cells. Cell uptake was compared by using equivalent amounts of cells. Blocking was done by adding 0.5 μM of unlabeled anti-CD25 IgG. (B) Representative coronal (left) and transaxial (right) PET images at 5 days of EL4 tumor-bearing immunocompetent C57/Bl6 mice administered with anti-mouse 89 Zr-CD25 IgG alone (top) or co-injected and 0.8 mg of unlabeled anti-CD25 IgG for blocking (middle). Biodistribution data are shown (bottom) as the mean ± S.E of values obtained from two independent experiments (n = 6 per group) N.S., not significant.

Article Snippet: Mouse monoclonal IgG2a anti- CD25 Ab (7G7/B6) that is specific against human CD25 and does not react with mouse CD25, rat monoclonal IgG1 Ab against mouse CD25 (PC-61.5.3), and IgG2a isotype control Ab were from BioXcell (West Lebanon, NH).

Techniques: In Vitro, In Vivo, Expressing, Binding Assay, Blocking Assay, Injection

Journal: Frontiers in Immunology

Article Title: Cysteine-specific 89 Zr-labeled anti-CD25 IgG allows immuno-PET imaging of interleukin-2 receptor-α on T cell lymphomas

doi: 10.3389/fimmu.2022.1017132

Figure Lengend Snippet: Autoradiography and histologic staining of microsections of SUDHL1 tumors from 89 Zr-CD25 IgG- injected mice. (Top) Photograph of a film exposed to a microsection of a small tumor showing radioactivity efficiently delivered into the inner tumor regions (left). A microscopic image of the tumor region in the boxed area demonstrates a focus of greater radioactivity (middle) that contained microvessels shown in red by alkaline phosphatase staining in an immediately adjacent microsection (right). (Bottom) Photograph of a film exposed to a microsection of a larger tumor showing greater radiation accumulated in the tumor periphery (left). A microscopic image of the tumor region in the boxed area demonstrates radioactivity distribution (middle) that correlated with immunohistochemistry staining for human CD25 (right).

Article Snippet: Mouse monoclonal IgG2a anti- CD25 Ab (7G7/B6) that is specific against human CD25 and does not react with mouse CD25, rat monoclonal IgG1 Ab against mouse CD25 (PC-61.5.3), and IgG2a isotype control Ab were from BioXcell (West Lebanon, NH).

Techniques: Autoradiography, Staining, Injection, Radioactivity, Immunohistochemistry

Journal: bioRxiv

Article Title: Binding of inhibitory checkpoints to CD18 in cis hinders anti-cancer immune responses

doi: 10.1101/2025.09.10.675342

Figure Lengend Snippet: ( A to C ) The impact of SIRPα variants defective in CD18-binding, CD47-binding or phosphatase signaling, alone or in combination, expressed in BMDMs, was analyzed. ( A ) Schematic depictions of SIRPα variants, as was done for . SIRPα R91T carried an arginine (R)-to-threonine (T) mutation at position 91 (shown by blue star), which abolished CD18-binding. ( B ) Phagocytosis assays of IgG-opsonized L1210 cells by BMDMs, as was done for . ( C ) Efficiency of phagocytosis inhibition was calculated as for , using values from . ( D and E ) Representative flow cytometry profiles ( D ) and compiled data from 3 independent experiments ( E ) of ICAM-1-binding using SIRPα KO BMDMs expressing WT SIRPα or SIRPα R91T BMDMs, in the presence or absence of FcR triggering using mouse IgG2a. ( F and G ) The impact of a SIRPα variant carrying the isoleucine-to-glycine 332 (I332G) mutation, expressed in SIRPα KO BMDMs, was analyzed. (F) Flow cytometry analyses of CD11b expression. ( G ) Compiled data from 3 independent phagocytosis assays, assessed by microscopy. ( H ) FRET assays of donor-labeled SIRPα, acceptor-labeled CD18 and unlabeled CD11b in the presence of WT CD11b or CD11b I332G , as was done for , D to F. ( I ) FRET assays of donor-labeled human SIRPα version (V) 1 or V2 with acceptor-labeled human CD18 and unlabeled human CD11b, in the presence of Ctrl IgG, human CD18 mAbs CBR LFA1/2 or TS1/18, as was done for , D to F. ( J ) Phagocytosis of human lymphoma cells Raji, which were opsonized with CD20 mAbs, by human peripheral blood monocyte (PBMC)-derived macrophages, in the presence of the indicated mAbs, was assessed by microscopy. All data are means ± s.e.m. ns, not significant; * p < 0.05, ** p < 0.01 and **** p < 0.0001. Results in ( D and F ) are representative of 3 independent experiments. Results in ( B , C , E and G to J ) are pooled from 3 independent experiments. Each symbol in ( B , E and G to J ) represents one cell, mouse or healthy donor.

Article Snippet: For opsonization of target cells with IgG, Fc-intact human CD25 mAb 7G7 (mouse IgG2a, BioXCell, Lebanon, NH) and Fc-intact human CD20 mAb rituximab biosimilar (human IgG1, BioXCell) were used.

Techniques: Binding Assay, Mutagenesis, Inhibition, Flow Cytometry, Expressing, Variant Assay, Microscopy, Labeling, Derivative Assay