mouse anti nkg2d antibody  (R&D Systems)

Journal: bioRxiv

Article Title: NK Cells Effectively Mediating Antibody-Mediated Kidney Allograft Rejection Requires a Specific Activation Receptor and Graft Expression of the Ligand

doi: 10.64898/2026.03.03.709363

Figure Lengend Snippet: (A) Experimental design and protocol: AJ or AJ.Rae-1 ⁻/⁻ donor kidneys were transplanted into B6.CCR5 ⁻/⁻ recipients, B6.CCR5 ⁻/⁻ NKG2D ⁻/⁻ recipients, or B6.CCR5 ⁻/⁻ recipients treated with anti-NKG2D mAb (250 µg/mouse administered on days 8, 13, 18, 23, 30, and 40 after transplantation). Nephrectomy of the native kidney was performed on day 4. (B) AJ (n=6) or AJ.Rae-1 ⁻/⁻ (n=5) kidney allografts were transplanted to groups of B6.CCR5 -/- recipients and allograft survival was monitored. All AJ.Rae-1e ⁻/⁻ kidneys survived for more than 55 days after transplantation with significantly prolonged graft survival compared with A/J kidney allografts by Kaplan–Meier analysis with log-rank test (p = 0.0018). (C) Serum from wild-type C57BL/6 or B6.CCR5 -/- recipients of A/J or AJ.Rae-1 ⁻/⁻ kidney grafts was obtained from individual recipients at the indicated times after transplant and the DSA titers. Data indicate mean DSA titer for each graft recipient group ± SD. (D) AJ kidney allografts were transplanted to groups (n = 6/group) of B6.CCR5 -/- recipients and allograft survival was monitored. Kaplan–Meier analysis with log-rank test was used to analyze graft survival in B6.CCR5 ⁻/⁻ , B6.CCR5 ⁻/⁻ NKG2D ⁻/⁻ , and B6.CCR5 ⁻/⁻ recipients treated with anti-NKG2D mAb transplanted AJ kidney allografts. Both genetic deletion of NKG2D and antibody-mediated blockade significantly prolonged graft survival compared with B6.CCR5 ⁻/⁻ controls (p=0.0007 and p=0.0159, respectively). (E) Serum DSA titers in B6.CCR5 ⁻/⁻ , B6.CCR5 ⁻/⁻ NKG2D ⁻/⁻ and B6.CCR5⁻ / ⁻ treated with anti-NKG2D mAb transplanted AJ kidney allografts., and AJ→B6.CCR5⁻ / ⁻ recipients at the indicated time points. Data indicate mean titer for each graft recipient group ± SD

Article Snippet: In some experiments, B6.CCR5 -/- recipients of A/J kidney allografts were treated i.p. with 250 μg rat anti-mouse NKG2D monoclonal antibody (catalog BE0351, clone HMG2D, Bio X Cell, NH) on days 8, 13, 18, 23, 30 and 40 after transplantation.

Techniques: Transplantation Assay

Journal: bioRxiv

Article Title: NK Cells Effectively Mediating Antibody-Mediated Kidney Allograft Rejection Requires a Specific Activation Receptor and Graft Expression of the Ligand

doi: 10.64898/2026.03.03.709363

Figure Lengend Snippet: (A) Representative C4d immunohistochemical staining of kidney allografts at day 15 POD. Immunohistology demonstrates diffuse strong deposition of C4D in peritubular and glomerular capillaries of AJ kidney allografts to wild-type C57BL/6, B6.CCR5 ⁻/⁻ , B6.CCR5 ⁻/⁻ NKG2D ⁻/⁻ and AJ.Rae-1 ⁻/⁻ kidney allografts to B6.CCR5 ⁻/⁻ recipients with capillary dilation observed in A/J to B6.CCR5 ⁻/⁻ recipients. (B) Periodic acid–Schiff (PAS) staining of kidney allografts reveals cortical edema and dilation of peritubular capillaries in AJ graft to B6.CCR5 ⁻/⁻ . In contrast, AJ.Rae-1 ⁻/⁻ grafts to B6.CCR5 ⁻/⁻ showed attenuated tissue injury with reduced edema. Scale bars, 100 μm (low power) and 50 μm (high power). (C) Quantitative PCR analysis of NKG2D ligands (Rae-1e, Rae-1d, and H60b) in kidney grafts on day 15 POD. Relative mRNA expression levels in each group are shown in comparison with a B6 graft to B6 recipient. Rae-1e expression was significantly reduced in AJ grafts to B6.CCR5 ⁻/⁻ NKG2D ⁻/⁻ and B6.CCR5⁻ / ⁻ treated with anti-NKG2D mAb and AJ.Rae-1 ⁻/⁻ grafts to B6.CCR5 ⁻/⁻ recipients compared with expression in AJ grafts to B6.CCR5⁻ / ⁻ recipients; whereas, Rae-1d and H60b expression showed no significant differences in allograft groups. Data indicate the mean ± SD. *P < 0.05 as determined by one-way ANOVA test.

Article Snippet: In some experiments, B6.CCR5 -/- recipients of A/J kidney allografts were treated i.p. with 250 μg rat anti-mouse NKG2D monoclonal antibody (catalog BE0351, clone HMG2D, Bio X Cell, NH) on days 8, 13, 18, 23, 30 and 40 after transplantation.

Techniques: Immunohistochemical staining, Staining, Real-time Polymerase Chain Reaction, Expressing, Comparison

Journal: bioRxiv

Article Title: NK Cells Effectively Mediating Antibody-Mediated Kidney Allograft Rejection Requires a Specific Activation Receptor and Graft Expression of the Ligand

doi: 10.64898/2026.03.03.709363

Figure Lengend Snippet: Quantitative PCR analysis of transcripts encoding mediators associated with antibody-mediated rejection (ABMR) was performed in kidney grafts harvested on day 15 POD. The expression levels of IFN-γ, Gzmb (GZB), Perforin, TNFα, CCL2, CXCL9, CXCL10, CX3CR1, MyBL1, and SH2D1B1 were compared between B6 isografts to B6 recipients, AJ allografts to B6.CCR5 ⁻/⁻ , A/J allografts to B6.CCR5 ⁻/⁻ NKG2D ⁻/⁻ , and AJ.Rae-1 ⁻/⁻ grafts to B6.CCR5 ⁻/⁻ recipients. Relative mRNA expression levels in each group are shown in comparison with a B6 graft to B6 recipient. Data are presented as mean ± SD. Statistical significance was determined by one-way ANOVA with appropriate post hoc comparisons. Data indicate the mean ± SD. *P < 0.05, **P < 0.01 as determined by one-way ANOVA test.

Article Snippet: In some experiments, B6.CCR5 -/- recipients of A/J kidney allografts were treated i.p. with 250 μg rat anti-mouse NKG2D monoclonal antibody (catalog BE0351, clone HMG2D, Bio X Cell, NH) on days 8, 13, 18, 23, 30 and 40 after transplantation.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Comparison

Journal: bioRxiv

Article Title: NK Cells Effectively Mediating Antibody-Mediated Kidney Allograft Rejection Requires a Specific Activation Receptor and Graft Expression of the Ligand

doi: 10.64898/2026.03.03.709363

Figure Lengend Snippet: AJ kidney allografts were transplanted to groups (n = 5/group) of B6.CCR5 -/- , B6.CCR5 ⁻/⁻ NKG2D ⁻/⁻ , B6.CCR5 ⁻/⁻ treated with anti-NKG2D mAb recipients and in AJ.Rae-1 ⁻/⁻ allografts transplanted to B6.CCR5 ⁻/⁻ recipients. (A) Representative flow cytometric analysis of infiltrating NK cells in kidney allografts on day 15 post-transplant. Single-cell suspensions were prepared from graft tissues and gated on CD45⁺ leukocytes, followed by exclusion of CD3⁺ cells. NK cells were defined as NK1.1⁺DX5⁺ cells within the CD45⁺CD3⁻ population. (B) Quantification of total NK cell numbers per mg of graft tissue in the indicated groups. Absence of recipient NKG2D or donor Rae-1 suppresses NK cell accumulation. Data indicate the mean ± SD. *P < 0.05, **P < 0.01 as determined by one-way ANOVA test. (C) Representative flow cytometric analysis of BrdU incorporation in graft-infiltrating NK cells. BrdU was administered i.p. on POD 14 and allografts were harvested and cell aliquots stained to detect BrdU + NK1.1 + cells (D) Quantification of BrdU⁺ NK cell numbers per mg of graft tissue. NKG2D deficiency or blockade and absence of donor Rae-1 decreased NK cell proliferation within the graft. Data indicate the mean ± SD. *P < 0.05 as determined by one-way ANOVA test. (E) ELISA analysis of cytokine levels in graft tissues on day 15 post-transplant. IL-21 levels were increased in AJ grafts to B6.CCR5 ⁻/⁻ recipients compared with the other groups, whereas IL-15 and IL-7 levels showed no significant differences among groups. Data indicate the mean ± SD. *P < 0.05, **P < 0.01 as determined by one-way ANOVA test.

Article Snippet: In some experiments, B6.CCR5 -/- recipients of A/J kidney allografts were treated i.p. with 250 μg rat anti-mouse NKG2D monoclonal antibody (catalog BE0351, clone HMG2D, Bio X Cell, NH) on days 8, 13, 18, 23, 30 and 40 after transplantation.

Techniques: Single Cell, BrdU Incorporation Assay, Staining, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: NK Cells Effectively Mediating Antibody-Mediated Kidney Allograft Rejection Requires a Specific Activation Receptor and Graft Expression of the Ligand

doi: 10.64898/2026.03.03.709363

Figure Lengend Snippet: (A) Representative histograms of CD107a + NK cells in kidney allografts. CD107a expression was assessed within graft-infiltrating NK cells defined as CD45⁺CD3⁻ NK1.1⁺DX5⁺ cells among the groups of AJ grafts to B6.CCR5 /- , B6.CCR5 ⁻/⁻ NKG2D ⁻/⁻ , B6.CCR5 ⁻/⁻ treated with anti-NKG2D mAb recipients and AJ.Rae-1 ⁻/⁻ grafts to B6.CCR5 ⁻/⁻ recipients. (B) Quantification of CD107a⁺ NK cell numbers per mg of graft tissue in the groups. NKG2D deficiency or blockade and absence of donor Rae-1 reduced the number of CD107a⁺ NK cell compared with AJ grafts to B6.CCR5 ⁻/⁻ . Data indicate the mean ± SD. *P < 0.05, **P < 0.01 as determined by one-way ANOVA test. (C) Representative flow cytometric analysis of NKG2D expression within graft-infiltrating NK cells (CD45⁺CD3⁻NK1.1⁺DX5⁺) and (D) CD107a + NK cell subset in the indicated groups. The expression of NKG2D was reduced in recipient NKG2D-deficient or donor Rae-1-deficient conditions.

Article Snippet: In some experiments, B6.CCR5 -/- recipients of A/J kidney allografts were treated i.p. with 250 μg rat anti-mouse NKG2D monoclonal antibody (catalog BE0351, clone HMG2D, Bio X Cell, NH) on days 8, 13, 18, 23, 30 and 40 after transplantation.

Techniques: Expressing

Journal: bioRxiv

Article Title: NK Cells Effectively Mediating Antibody-Mediated Kidney Allograft Rejection Requires a Specific Activation Receptor and Graft Expression of the Ligand

doi: 10.64898/2026.03.03.709363

Figure Lengend Snippet: (A) Representative flow cytometric analysis of graft-infiltrating Ly6C hi CD11b hi monocytes. After exclusion of dead cells, leukocytes were gated as CD45⁺ cells, followed by exclusion of CD3⁺ cells. CD11b⁺Ly6G⁻ cells were identified, and F4/80⁻ cells were defined as monocytes. Ly6C expression was evaluated within this monocyte population. Representative contour plots were obtained from AJ grafts to B6.CCR5 /- , B6.CCR5 ⁻/⁻ NKG2D ⁻/⁻ , B6.CCR5 ⁻/⁻ treated with anti-NKG2D mAb recipients and AJ.Rae-1 ⁻/⁻ grafts to B6.CCR5 ⁻/⁻ recipients. Quantification of Ly6C⁺ monocyte numbers per mg of graft tissue and Mean fluorescence intensity (MFI) of Ly6C expression in graft-infiltrating monocytes were evaluated in the indicated groups. Rae-1 deficiency grafts reduced MFI of Ly6C expression in monocytes compared with AJ grafts to B6.CCR5 ⁻/⁻ . Data indicate the mean ± SD. *P < 0.05 as determined by one-way ANOVA test.

Article Snippet: In some experiments, B6.CCR5 -/- recipients of A/J kidney allografts were treated i.p. with 250 μg rat anti-mouse NKG2D monoclonal antibody (catalog BE0351, clone HMG2D, Bio X Cell, NH) on days 8, 13, 18, 23, 30 and 40 after transplantation.

Techniques: Expressing, Fluorescence

Journal: Nature Communications

Article Title: Aiolos restricts the generation of antigen-inexperienced, virtual memory CD8 + T cells in mice

doi: 10.1038/s41467-025-67540-8

Figure Lengend Snippet: a Heatmap showing differences in different cytokine receptors between WT and Aiolos-deficient T VM samples. b Schematic of the in vitro treatment of WT and Aiolos-deficient splenic CD8 + T cells, created in Microsoft PowerPoint. Bulk CD8 + T cells were isolated from the spleens of WT and Aiolos-deficient mice and treated with rmIL-12, rmIL-18, and IL-15C (IL-15/IL-15Rα complex) for 30 min (pY-STAT5 and pY-STAT4) or 16 h (IFN-γ, granzyme B and NKG2D) with protein transport inhibitor (PTI) added 4 h prior to harvesting the cells for flow cytometry analysis. Analysis of frequency (%) and median fluorescence intensity (MFI) fold change for ( c ) pY-STAT5 and ( d ) pY-STAT4 between WT and Aiolos-deficient T VM cells, relative to WT. Data representative of 5 independent experiments, n = 5/group, mean ± SEM; two-sided, unpaired Student’s t-test, *p ≤ 0.05 and **p ≤ 0.01. Frequency (%) and MFI fold change for e IFN-γ, f granzyme B, and g NKG2D between WT and Aiolos-deficient T VM cells, relative to WT. Data represents 6 independent experiments, n = 6/group for e , f ; and 5 independent experiments with n = 5/group for NKG2D. Data presented as mean ± SEM; two-sided, unpaired Student’s t-test, *p ≤ 0.05, ***p ≤ 0.001 and ****p ≤ 0.0001. h Percent (%) dead YAC-1 (target) cells when co-cultured with CD8 + T cells in the presence of cytokines (IL-12, Il-18, IL-15C), with or without anti-NKG2D antibody, data from 5 independent experiments with n = 5/condition. Data presented as mean ± SEM; two-way ANOVA with Tukey’s multiple comparisons test *p ≤ 0.05 and ****p ≤ 0.0001. Source data are provided as a file.

Article Snippet: For NKG2D blocking, cells were treated with anti-NKG2D antibody (10 μg/mL, clone: HMG2D; catalog # BE0111, BioXCell) or isotype control (catalog # BE0091, BioXCell) for 30 min prior to YAC-1 cells addition.

Techniques: In Vitro, Isolation, Flow Cytometry, Fluorescence, Cell Culture