NK cell flow cytometric assay In vivo DC viability and migration assay
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1 NK cell flow cytometric assay 6 NK cells were purified, by negative selection with the NK Cell Isolation Kit (Miltenyi iotec), from spleen and lymph nodes of 6 RAG1KO mice, injected the day before with poly I:C (1µg in 2µl of PS, i.p.). The method employed for the NK cell flow cytometric assay is based on the technique developed by K. Kane et al., with minor modifications [K.L. Kane et al. Determination of Natural Killer cell function by flow cytometry. Clin. Diagn. Lab. Immunol. 1996; 3(3):295 3]. riefly, target DCs were labeled with 7µM CFSE and then incubated in triplicates, for 4h at 37 C in V-shaped 96 well plates (2, DCs / well) alone, or with increasing concentrations of unlabeled NK cells. Cells were then collected, fixed in 4% paraformaldehide, labeled with the LIVE/DEAD fixable red dead cell stain kit (Invitrogen ) and analyzed by flow cytometry. Percent of dead target cells was calculated with the following equation: 1 [(# dead DCs in sample / # total DCs in sample) (# dead DC by spontaneous lysis in sample containing only target DCs / # total DCs in sample containing only target DCs)]. In vivo DC viability and migration assay To deplete NK cells in vivo, CD mice were injected the day before the experiment with the NK cell-depleting Ab NK1.1 (2µg, i.p.). The following day, mice were injected in the footpad (s.c.) with PS (control) or a cellular mix composed of the following M-derived DCs (16 of each): (i) unlabeled (CD45.2) AL/c Im-DCs, (ii) (CD45.2) AL/c labeled with.7µm CFSE, and (iii) (CD45.2) 6 stained with 7µM CFSE. Thirty six h later, the draining popliteal LN cells were labeled with CyC-CD11c and PE-CD45.2 mab and the percentages and absolute numbers of each CD DC subset quantified by FACS.
2 Figure S1. Response of VD-treated maturation-resistant DCs () to LPS or CD4- signaling M-derived generated in vitro with VD3, or not (control-dcs) were cultured for 48 h alone (control), with LPS (5ng/ml), agonistic CD4 IgM Ab (HM4-3, 1μg/ml), or irrelevant (control) hamster IgM (1μg/ml). (A) FACS-analysis of the surface phenotype of control- and untreated or following 48 h stimulation with LPS, CD4 Ab, or irrelevant IgM. () Assessment by 3 day-mlcs of the T cell allo-stimulatory ability of control- and untreated or after 48 h stimulation with LPS, CD4 Ab, or irrelevant IgM. * p <.5, p <.1 (C) Detection by ELISA of IL-12p7 in culture supernatants of control- and following 48 h culture alone or with LPS, CD4 Ab, or irrelevant IgM (mean ± SD). As expected, DCs stimulated by CD4 signaling (in the absence of IFN-γ) did not increase secretion of the IL-12p7 heterodimer. : not detected. (A C) Representative data from 3 independent experiments is shown. Figure S2. Effect of donor-derived immature (not MR) DCs (Im-DCs) on the indirect CD4 T-cell response 6 mice, adoptively transferred with CFSE-labeled Thy1.1 1H3.1 CD4 T cells, were injected i.v. (or not) 1 day later with control syngeneic (6) Im-DCs, or AL/c Im-DCs alone or plus agonistic CD4 Ab (i.p.), the latter as a control to promote 1H3.1 cell activation. (A) Dot plots show proliferation, surface phenotype, and percentages of apoptotic cell death of splenic 1H3.1 CD4 T cells (gated on Thy1.1 cells) analyzed by FACS, 3 days after Im-DC administration. () Absolute numbers of 1H3.1 Thy1.1 CD4 T cells in the spleen, 3 and 14 days after 6 (control) or AL/c Im-DC (with and without CD4 Ab, i.p.) administration in host 6 mice, in the absence or presence of AL/c cardiac allografts transplanted 7 days after Im-DC injection. Absolute numbers of 1H3.1 cells did not change significantly in lymph nodes (cervical, axilar, mesenteric, inguinal), quantified by FACS, 3 days after Im-DC administration. (C) Amounts of IFN-γ (by ELISA) secreted by splenocytes of each group of host/recipient 6 mice re-stimulated ex vivo (24 h) with the AL/c IE α allopeptide. (D) Percentages and absolute numbers of splenic (Thy1.1) FoxP3 + 1H3.1 CD4 T cells 14 days after Im-DC injection in each group of host/recipient 6 mice. NS: not significant. (A D) Results are representative of 2 independent experiments with 3 or more host animals and 3 transplant recipients per group (mean ± SD). Figure S3. Effect of donor-derived apoptotic (Apo) on the indirect CD4 T-cell response Host 6 mice that received CFSE-labeled Thy1.1 1H3.1 CD4 T cells, were injected i.v. (or not), the next day with control Apo syngeneic (6), or Apo AL/c alone or plus agonistic CD4 Ab (i.p.), the latter used as a control to promote full 1H3.1 T cell activation. (A) Dot plots show proliferation, surface phenotype, and percentages of apoptotic cell death of splenic 1H3.1 CD4 T cells (gated on Thy1.1 cells) analyzed by FACS, 3 days after Apo MR-DC administration. () Absolute numbers of 1H3.1 Thy1.1 CD4 T cells in the spleen, 3 and 14 days after Apo 6 (control) or Apo AL/c MR-DC (with and without CD4 Ab, i.p.) administration in host 6 mice, in the absence or presence of AL/c cardiac allografts transplanted 7 days after Apo MR-DC administration. Absolute numbers of 1H3.1 cells did not change significantly in lymph nodes (cervical, axilar, mesenteric, inguinal), assessed 3 days after Apo MR-DC injection. (C) Amounts of IFN-γ (by ELISA) secreted by splenocytes of each group of host/recipient 6 mice re-stimulated ex vivo (24 h) with the AL/c IEα52 68 allopeptide. (D)
3 Percentages and absolute numbers of splenic (Thy1.1) FoxP3+ 1H3.1 CD4 T cells 14 days after Apo MR-DC injection in each group of host/recipient 6 mice. NS: not significant. (A D) Results are representative of 2 independent experiments with 3 or more host animals and 3 transplant recipients per group (mean ± SD). Figure S4. Targeting of therapeutic donor-derived by NK cells in vitro (A) Example of the assessment of killing of AL/c by 6 NK cells in vitro by flow cytometric analysis. Target CFSE-labeled were incubated (4h, 37 C) alone (control) or with increasing numbers of unlabeled 6 NK cells, then fixed, labeled with the LIVE/DEAD fixable read dead cell stain kit, and analyzed by flow cytometry. Double positive cells correspond to dead. Numbers represent percentages of. () Percentages of target AL/c killed by 6 NK cells in vitro. Target Yac-1 cells were used as positive controls, and syngeneic (6) M-derived DCs matured (CD86 + ) in vitro with poly I:C, as negative controls. (,C) Results are representative of 2 independent experiments (mean ± SD). Figure S5. Viability of (A) Viability of AL/c before administration (i.v.) into 6 mice, assessed by flow cytometry and compared to control 6 and to control AL/c Im-DCs, the latter not exposed to exogenous VD. Numbers represent percentages of cells. () In vivo comparison of survival, susceptibility to NK cell targeting, and trafficking to lymph nodes between AL/c and control 6 and AL/c Im-DCs. Host (CD45.1) 6 mice, untreated or injected the day before with the NK cell-depleting Ab NK1.1, were injected in the footpad (s.c.) with PS (control), or a cellular mixed composed of the following M-derived DCs (16 of each, per injection): (i) unlabeled (CD45.2) AL/c Im-DCs, (ii) CFSElow (CD45.2) AL/c, and (iii) CFSEhigh (CD45.2) 6. Thirty six h later, the percentages and absolute numbers of each DC subset injected were assessed by FACS-analysis in the draining popliteal lymph node. AL/c survived and migrated to draining lymph nodes similarly to control DCs, and in all cases their survival was significantly impaired by the presence of host NK cells. This phenomenon was detected even when syngeneic (6) Im-DCs were injected, likely due to their low level of expression of surface (self) MHC-I.
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4 IgG Isotype (control) No LPS + LPS Control-DCs Cell number H-2D d I-A d CD8 CD86 CD4 IgG Isotype (control) + Irrel. IgM + CD4 Ab Control-DCs Cell number H-2D d I-A d CD8 CD86 [ 3 H]TdR incorporation (c.p.m.x1 3 ) control-dcs control-dcs + LPS + LPS * * [ 3 H]TdR incorporation (c.p.m.x1 3 ) control-dcs + Irrel. IgM control-dcs + CD4 Ab + Irrel. IgM + CD4 Ab Stimulator : Responder cell ratio Stimulator : Responder cell ratio C 3 25 IL-12p7 (pg / ml) Control-DCs Control-DCs Figure S1
5 Im-DCs AL/c Im-DCs + agonistic CD4 Ab # of 1H3.1 T cells / spleen (x 1 3 ) C IFN-γ (pg / ml) Spleen Spleen AL/c Im-DCs CD CFSE p <.1 p <.1 p <.1 Day 3 Day 14 p <.5 p <.1 Day 3 Day 14 CD62L Tx with AL/c hearts on day Tx with AL/c hearts on day 7 p <.5 Non-treated 6 Im-DCs (d ) p <.1 D Annexin-V % of FoxP3 + 1H3.1 T cells # of FoxP3 + 1H3.1 T cells / spleen AL/c Im-DCs (d ) + CD4 Ab (d,1,2) # of 1H3.1 1 T cells (x 1 3 ) Spleen (day 14) Lymph nodes p <.1 NS Day 3 Tx with AL/c hearts on day 7 p <.3 Tx with AL/c hearts on day 7 NS AL/c Im-DCs (d ) Figure S2
6 Apo Apo AL/c + agonistic CD4 Ab # of 1H3.1 T cells / spleen (x 1 3 ) C IFN-γ (pg / ml) Spleen Spleen Apo AL/c CD CFSE p <.1 p <.1 p <.1 Day 3 Day 14 Day 3 Day 14 CD62L Tx with AL/c hearts on day 7 Non-treated Tx with AL/c hearts on day 7 p <.1 Apo 6 (d ) D Annexin-V # of 1H3.1 1 T cells (x 1 3 ) 5 % of FoxP3 + 1H3.1 T cells # of FoxP3 + 1H3.1 T cells / spleen Spleen (day 14) Apo AL/c (d ) + CD4 Ab (d,1,2) Lymph nodes Day 3 p <.1 NS Tx with AL/c hearts on day 7 p <.5 Tx with AL/c hearts on day 7 NS Apo AL/c (d ) Figure S3
7 LIVE/DEAD Fixable Red stain fluorescence NK CFSE Effector : target cell ratio 4:1 2:1 1:1.5:1 : C % Dead target cells p <.1 % Dead target cells :1 2:1 1:1.5:1 Effector : target ratio 4:1 2:1 1:1.5:1 Effector : target ratio Yac-1 cells (+ control) Yac-1 cells (+ control) AL/c AL/c 6 mature DCs (- control) AL/c immature DCs Figure S4
8 In vitro: Gated on CD11c + cells: AL/c Im-DCs 3 4 AL/c AAD Annexin-V In vivo: Gated on CD11c + CD cells: CD45.2 Untreated α-nk1.1 Ab R1 R2 R3 R1 R2 R3 CFSE # of CD11c + CD cells / popliteal LN p <.1 p <.1 AL/c Im-DCs AL/c p <.1 6 R1 (unlabeled) : AL/c Im-DCs R2 (CFSE low ) : AL/c R3 (CFSE high ) : 6 Untreated α-nk1.1 Ab Figure S5
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SUPPLEMENTARY FIGURE 1
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activation with anti-cd3/cd28 beads and 3d following transduction. Supplemental Figure 2 shows
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a surface permeabilized
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Supplementary Figures
Supplementary Figures Supplementary Fig. 1. Surface thiol groups and reduction of activated T cells. (a) Activated CD8 + T-cells have high expression levels of free thiol groups on cell surface proteins.
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Page 39 of 44. 8h LTA & AT h PepG & AT h LTA
Page 39 of 44 Fig. S1 A: B: C: D: 8h LTA 8h LTA & AT7519 E: F: 8h PepG G: 8h PepG & AT7519 Fig. S1. AT7519 overrides the survival effects of lipoteichoic acid (LTA) and peptidoglycan (PepG). (A) Human
for six pairs of mice. (b) Representative FACS analysis of absolute number of T cells (CD4 + and
SUPPLEMENTARY DATA Supplementary Figure 1: Peripheral lymphoid organs of SMAR1 -/- mice have an effector memory phenotype. (a) Lymphocytes collected from MLNs and Peyer s patches (PPs) of WT and SMAR1
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a TMRM FI (Median) b TMRM FI (Median) c 20 15 10 5 0 8 6 4 2 0 pro-b large pre-b small pre-b 0 10 20 30 40 50 60 70 80 90 100 TMRM (nm) pro-b large pre-b small pre-b 0 1 2 4 8 16 32 64 128 256 CCCP (mm)
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Supplemental materials
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Supporting Information
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Supporting Information Table of Contents
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