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: Cell Transplantation

Article Title: A novel GMP-manufactured medicinal product candidate composed of NK and γδ T cells as adjunct immunotherapy for hematopoietic stem cell transplantation

doi: 10.1177/09636897251374248

Figure Lengend Snippet: Flow cytometric analysis of cell populations during the process. Panel A. Number of cells and proportion of populations obtained at the different steps of the process starting from buffy coat (BC) or leukapheresis (LA). Panels B–D. One representative experiment using LA (#3) as staring material is shown. Panel B. CD14 + monocytes (a), CD19 + B cells (b) CD3 + T cells (c), CD3 − CD56 + NK cells (d), γδ and αβ T lymphocytes (e) as well as Vδ1 and Vδ2 subsets (f), present after Ficoll-Paque gradient in gated lympho-mononuclear cells. Panels C and D. CD14 + monocytes (a), CD19 + B cells (b) CD3 − CD56 + NK cells (c), γδ and αβ T lymphocytes (d) as well as Vδ1 and Vδ2 subsets (e), gated mononuclear cells at the end of expansion (day 14) (Panel C) and harvested after αβ T-cell depletion (Panel D). Panel E shows γδ T cell differentiation, by APC-CD27 and PEVio770-CD45RA staining.

Article Snippet: Flow cytometric analyses were performed on the GADEKILL in gated CD3 + (γδ T) and CD3 − (NK) cells using Vioblue-CD3, APCVio770-CD16, PE anti-NKG2A, PEVio770 anti-NKG2D, PEVio770 anti-NKp30, PEVio770 anti-NKp44, PEVio770 anti-NKp46, PEVio770 anti-PD1, and PE anti-TIM (all from Miltenyi).

Techniques: Cell Differentiation, Staining