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Blood, Vol. 110, Issue 12, 3926-3935, December 1, 2007
Rapidly induced, T-cell–independent xenoantibody production is mediated by marginal zone B cells and requires help from NK cells
Blood Li et al.
110: 3926
Supplemental materials for: Li et al
Files in this Data Supplement:
- Table S1. Effector B cells for xenograft rejection in BALB/c nude mice receiving C57BL/6 splenic tissue transplant are contained within the BALB/c nude recipient MZ B cell compartment (PDF, 13.6 KB) -
As rejection is prevented by treating recipient BALB/c mice with splenectomy or 2 Gy TBI
* Experiments were terminated at 60 days after hamster heart transplantation.
Following transplantation on day -7 of spleen tissue from lethally irradiated C57BL/6 nude splenic tissue, 9 of 11 xenohearts were rejected (cfr. Table II group d). Following transplantation on day -7 of spleen tissue from naïve (n=6) or lethally irradiated C57BL/6 mice (n=4), recipient splenectomy and concomitant xenoheart transplantation on day 0 resulted in the abrogation of xenograft rejection. Specific MZB-cell depletion in BALB/c nude recipients, followed by transplantation of lethally irradiated C57BL/6 nude splenic tissue and xenoheart transplantation on day 0 again resulted in permanent graft survival and absence of IgM Xab production (n=6).
- Table S2. BALB/c nude NK cells fail to lyse allogeneic C57BL/6 target cells (PDF, 13.9 KB) -
DX5+ MACS purified cells from BALB/c or C57BL/6 mice were used as effector cells in a classical NK 51Cr-release assay, with C57BL/6 blast cells, YAC-1 and hamster blast cells as target cells. Results are shown from one of two independent experiments with identical results.
- Figure S1. Immunohistochemical stainings showing that acute vascular rejection of hamster xenoheart grafts is accompanied by IgM and complement deposition and manifest expression of P-selectin (PDF, 37.5 KB) -
Immunohistochemical staining on tissue sections from xenoheart grafts were performed for IgM (upper panel), C3 (lower left) and P-selectin (lower right). In C57BL/6 nude mice, rejected grafts stained positive for IgM (upper left), whereas permanently accepted xenografts in BALB/c nude mice did not show IgM deposition (upper right). IgM deposition in rejected xenografts in C57BL/6 nude mice was accompanied by Complement deposition (lower left) and manifest expression of P-selectin (lower right), which was not the case in BALB/c nude recipients (not shown).
- Figure S2. Overview of immunohistochemical stainings, performed on frozen sections of spleens of C57BL/6 nude and BALB/c nude mice (PDF, 72 KB) -
Similar staining profiles for markers of B cell subsets (B220, IgM, IgD, CD21/CD35, CD5), marginal zone metallophilic macrophages (MOMA-1), follicular dendritic cells (FDC-M1), stromal cells (CD169), T cells (CD3, not shown) and/or NK cells (DX5) and macrophages (F4/80) were found.
- Figure S3. Following adoptive transfer of C57BL/6 TCRβ−/− spleen cells into BALB/c nude mice, at least a fraction of C57BL/6 NK cells migrates to the spleen and survives until the day of xenografting (PDF, 48.9 KB) -
CD19− C57BL/6 TCR −/− spleen cells were transferred to BALB/c nude mice. On day 7, spleen cells of these mice were used for flowcytometric detection of C57BL/6 NK cells, identified as NK1.1+DX5+ (left) or H2Kb+DX5+ cells (right). Plots are shown from a naïve BALB/C nude mouse (A), a BALB/c nude mouse given 32 × 106 CD19− C57BL/6 TCR−/− spleen cells, a BALB/c nude mouse given 5 × 106 CD19− C57BL/6 TCR−/− spleen cells (C), and a naïve C57BL/6 TCR−/− mouse (D, positive control). Percentages indicate the frequency of C57BL/6 NK cells (upper right quadrant) in total spleen cells.
- Figure S4. In vivo T-independent IgM xenoantibody production in C57BL/6 nude mice does not involve NK-mediated lysis of xenogeneic target cells (PDF, 56 KB) -
(A) Cytotoxic lysis by purified NK cells from C57BL/6 euthymic mice (n=pool of 20 mice), in the presence of blocking anti-Ly49D MoAb (5 µg/106 cells) or control Ig (5 µg/106 cells), and of BALB/c euthymic mice (n=pool of 3 mice) was tested against Golden Syrian Hamster blasts or YAC-1 tumor cells. Graphs represent % specific lysis at different effector to target ratio’s. B-D: C57BL/6 nude mice were treated with blocking anti-Ly49D MoAb (n=3) or control Ig (n=1) in vivo. On day 7 after initiation of treatment, full blockade of Ly49D receptors was obtained (dot plots from one representative animal are shown) (B), selective inhibition of ex vivo NK cytotoxic lysis of hamster targets and not Yac cells was documented in antiLy49D-treated C57BL/6 mice (results as obtained in one representative animal are shown) (C), but the capacity to produce IgM xenoantibody in vivo was maintained (histograms from one representative animal are shown) (D).
- Figure S5. Histograms illustrating the TNP-Ficoll-specific IgM and IgG2a antibody response in C57BL/6 and BALB/c nude mice (PDF, 50.3 KB) -
On day 7 after TNP-Ficoll immunization of naïve or splenectomized mice, serum was obtained for flowcytometric analysis of TNP-Ficoll specific IgM and IgG2a levels. Mean serum IgM and IgG2a levels of experimental groups are shown in Fig 1 of the manuscript. Here, histograms are shown from one representative animal from each group. MFI is indicated.
- Figure S6. Immunohistochemical stainings showing Syndecan-1, CD21/CD35, and DX stainings, performed on frozen sections of naïve or xenoheart grafted C57BL/6 nude or BALB/c nude mice (PDF, 41.4 KB) -
(A) Syndecan-1- and CD21/35 staining, performed on frozen sections from a naïve (1-2) or a xenoheart rejecting (3-4) C57BL/6 nude mouse. (B) Immunohistochemical staining for DX5 in (1) a naïve C57BL/6 nude mouse, (2) a C57BL/6 nude mouse that rejected the hamster xenoheart graft on day 6 post-transplantation, (3) a naïve BALB/c nude mouse and (4) a BALB/c nude mouse that failed to reject a hamster xenoheart graft on day 6 post transplantation.
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