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Blood, Vol. 112, Issue 3, 661-671, August 1, 2008
Natural killer cells recruited into lymph nodes inhibit alloreactive T-cell activation through perforin-mediated killing of donor allogeneic dendritic cells
Blood Laffont et al.
112: 661
Supplemental materials for: Laffont et al
Files in this Data Supplement:
- Document 1. Supplemental materials and methods (PDF, 60.8 KB)
- Figure S1. NK cell depletion induces strong alloreactive CD4 T-cell priming in allogeneic DC-immunized but not naïve mice (JPG, 43.7 KB)
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B6 CD8−/− mice treated or not with anti-NK1.1 PK136 mAb, were injected subcutaneously into the hind foot pads with either PBS (A) or with allogeneic BALB/c BM-DCs (0.5 × 106/mouse) (B). Six days after immunization, draining lymph nodes were harvested and purified CD4 T cells were cultured (2 × 105 cells/well) in the presence or absence of irradiated allogeneic BALB/c splenocytes (1 × 105 cells/well) for 72 hours. CD4 T-cell proliferation was evaluated by 3H-TdR incorporation and results are expressed as mean ± SEM of quadruplicate cultures. Data are from one representative experiment of two performed.
- Figure S2. Selectivity of Ly49D+ NK cell depletion (JPG, 104 KB)
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B6 CD8−/− mice were treated with anti-Ly49A/D 12A8, anti-Ly49D 4E5, isotype control rat IgGa or anti-NK1.1 PK136 mAb as described in Materials and Methods. Mice were then injected subcutaneously into the hind footpads with allogeneic BALB/c DCs (106/mouse). At 24 h after immunization, draining popliteal lymph nodes (A) and spleen (B) were collected and stained for TCRβ, NK1.1, CD127 and Ly49D expression. The frequency of TCRβ- NK1.1+ NK cells and the expression of CD127 and Ly49D on NK cells (TCRβneg NK1.1pos) are shown for one representative animal per group.
- Figure S3. Allogeneic BALB/c DCs persistence in draining lymph nodes is impaired in the presence of host Ly49D+ NK cells (JPG, 29.7 KB)
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Representative CFSE profiles of injected DCs obtained at 48 hours from untreated or PK136-treated mice are shown. B6 CD8−/− mice were treated, or not, with anti-NK1.1 PK136 mAb or anti-Ly49D 4E5 mAb, and injected with a mixture of CFSElow-B6 DCs and CFSEhigh-BALB/c DCs. 48 hours after immunization, the presence of CFSE positive DCs (gated on CD11cpos MHC IIhigh) was analyzed in the draining lymph nodes and the percentage of CFSEhigh BALB/c DCs normalized to control CFSElow B6 DCs was determined (%BALB/c DCs : %B6 DCs) × 100.
- Figure S4. Perforin-mediated killing, but not IL-12Rβ2-signaling, is critical for allogeneic DC elimination by host NK cells in vivo (JPG, 59.1 KB)
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(A) B6 WT, B6 pfp−/− or (B) IL-12Rβ2−/− mice, were all injected with anti-CD8 mAb, and then treated or not with anti-NK1.1 PK136 mAb before immunization with equal numbers of CFSElow-B6 DCs and CFSEhigh-BALB/c DCs. 48 hours after immunization, the presence of CFSE positive DCs (gated on CD11cpos MHC IIhigh) was analyzed in the draining lymph nodes and the percentage of BALB/c DCs among control B6 DCs was determined. Representative CFSE profiles of injected DCs obtained at 48 hours from untreated or PK136-treated mice are shown. Results are expressed as mean ± SEM of 3 to 4 mice per group.
- Figure S5. Absence of allogeneic DC killing in perforin-deficient B6 CD8−/− mice despite normal recruitment of NK cells in draining lymph nodes (JPG, 52.7 KB)
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(A) B6CD8−/− or B6CD8−/−pfp−/− mice were injected with a mixture of CFSE-labeled BALB/c DCs (green) and CMTMR-labeled B6 DCs (red). Draining lymph nodes were removed at 48 h. Acetone fixed frozen sections were stained with goat NKp46-specific antibodies followed by donkey anti-goat Alexa 633-conjugated antibodies. (B) Histograms indicate the percentage of CFSE BALB/c cells among control CMTMR B6 cells (left) and the number of NKp46+ cells per lymph node sections (right). Results are expressed as mean ± SEM of individual lymph nodes (3 mice per group).
- Figure S6: Alloreactive CD4 T cell priming in response to allogeneic H-2d DCs is not affected by host NK cells in pfp-deficient mice (JPG, 39.6 KB)
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B6 CD8−/− (A) or B6 CD8−/−pfp−/− (B) mice, treated or not with anti-NK1.1 PK136 mAb, were injected s.c. into the hind foot pads with allogeneic BALB/c DCs (106/mouse). Six days after immunization, CD4 T cells were purified from draining lymph nodes, and restimulated (at 2 × 105 cells/well) with the indicated amounts of irradiated allogeneic BALB/c splenocytes for 72 hours. CD4 T-cell proliferation was evaluated by 3H-TdR incorporation during the last 8 hours of culture. Results are expressed as mean ± SEM of 3–4 mice per group. Data are from one representative experiment of three performed.
- Figure S7. Adoptively transferred NK cells reduces allogeneic DC numbers in perforin-deficient B6 CD8−/− mice (JPG, 47.5 KB)
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(A) B6CD8−/−pfp−/− mice were reconstituted or not with purified wild-type NK cells i.v. (5 × 106/mouse, see supporting Materials and Methods below) before immunization with 1.5 × 106 CMTMR-labeled BALB/c DCs (red). Draining lymph nodes were removed at 72 hours. Acetone fixed frozen sections were stained with APC conjugated anti-B220 (blue) mAb and goat NKp46-specific antibodies followed by donkey anti-goat Alexa 488-conjugated antibodies (green). (B) Histograms indicate the numbers of CMTMR BALB/c DCs cells per lymph node sections and results are expressed as mean ± SEM of individual lymph nodes (2–3 mice per group).
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