Figure S2.
The mouse platelet and T cell assays with recTLT-1 and B16 tumor in vivo and ex vivo. (A) The immunoblot showing comparison of native mouse platelet TLT-1 (left lane, mouse platelets) and recTLT-1 (right lane, purified mouse TLT-1), where the sTLT-1 (left lane, soluble form, lower band) and soluble recTLT-1 give bands at the same position. This also confirms the shared antibody binding extracellular domain presence in both. (B) Representative FC plots for gating T cell population (left) in mouse blood and comparative FC zebra plots showing CD8+ T cell % of total T cells (right) and recTLT-1–bound T cell % (inside gate) in mice administered with three different amounts of recTLT-1 or Veh. The WT control mice (n = 4) were injected with three amounts (15, 60, or 150 µg/kg body weight: 1×, 4×, or 10× doses, respectively) and 200 μl of blood was collected after 6 h. The citrated mouse blood was processed for direct staining with conjugated antibodies for T cell (anti-mouse TCRb-FITC), CD8 (anti-mouse CD8-BV420), and recTLT-1 (anti-his tag-A-647). The recTLT-1 has a small His-tag at the C terminus, which was used for staining recTLT-1–bound cells on the surface. The cells were not lysed, and cell surface binding of recTLT-1 to T cells was analyzed. (C) Platelet purity and gating for platelets in freshly drawn mouse blood samples. The representative FC contour plot shows gating of CD41+ cells for TLT-1 expression analysis. (D and E) Quantitation (n = 6 per group) of platelet surface TLT-1 by FC assay in samples (D) or platelet counts (E; n = 6) from mice with (tumor) subcutaneous melanoma or without (control) after 4 wk of tumor induction. (F) Soluble TLT-1 quantitation as measured by ELISA after 4 wk of tumor growth. (G) The IFNg + CD8 T cell proportions as measured by FC before (week 1) and after (week 4) Veh or TLT-1 injections. (H) The representative FC gating strategy for splenic cells after isolation and culture of harvested spleens. The same strategy was followed for all the groups for spleen culture. The live cells were gated using fixable viability dye as shown. (I) Representative FC contour plots for resting or CD3-CD28 stimulated splenocytes gated on live CD4+ve cells. (J) The IFNg + ve cells are subgated for showing % positive CD4 T cells producing IFNg. and their mean quantitation. (K) The representative images (n = 4) show the mouse tumor cryosections immunostained by anti-CD68 and anti-CD34 (secondary Alexa-647) antibodies followed by respective secondary antibodies (Alexa-488 antibody for CD68; Alexa-647 for CD34; scale bar equals 50 μm). All the data in vertical bars shows mean ± SEM. Statistical significance tested by two-tailed unpaired Mann–Whitney, except in G where paired t test was applied between time points. Significant differences are indicated with asterisks (*, P < 0.05). The data are result of minimum of two independent experiments. Source data are available for this figure: SourceData FS2.

The mouse platelet and T cell assays with recTLT-1 and B16 tumor in vivo and ex vivo. (A) The immunoblot showing comparison of native mouse platelet TLT-1 (left lane, mouse platelets) and recTLT-1 (right lane, purified mouse TLT-1), where the sTLT-1 (left lane, soluble form, lower band) and soluble recTLT-1 give bands at the same position. This also confirms the shared antibody binding extracellular domain presence in both. (B) Representative FC plots for gating T cell population (left) in mouse blood and comparative FC zebra plots showing CD8+ T cell % of total T cells (right) and recTLT-1–bound T cell % (inside gate) in mice administered with three different amounts of recTLT-1 or Veh. The WT control mice (n = 4) were injected with three amounts (15, 60, or 150 µg/kg body weight: 1×, 4×, or 10× doses, respectively) and 200 μl of blood was collected after 6 h. The citrated mouse blood was processed for direct staining with conjugated antibodies for T cell (anti-mouse TCRb-FITC), CD8 (anti-mouse CD8-BV420), and recTLT-1 (anti-his tag-A-647). The recTLT-1 has a small His-tag at the C terminus, which was used for staining recTLT-1–bound cells on the surface. The cells were not lysed, and cell surface binding of recTLT-1 to T cells was analyzed. (C) Platelet purity and gating for platelets in freshly drawn mouse blood samples. The representative FC contour plot shows gating of CD41+ cells for TLT-1 expression analysis. (D and E) Quantitation (n = 6 per group) of platelet surface TLT-1 by FC assay in samples (D) or platelet counts (E; n = 6) from mice with (tumor) subcutaneous melanoma or without (control) after 4 wk of tumor induction. (F) Soluble TLT-1 quantitation as measured by ELISA after 4 wk of tumor growth. (G) The IFNg + CD8 T cell proportions as measured by FC before (week 1) and after (week 4) Veh or TLT-1 injections. (H) The representative FC gating strategy for splenic cells after isolation and culture of harvested spleens. The same strategy was followed for all the groups for spleen culture. The live cells were gated using fixable viability dye as shown. (I) Representative FC contour plots for resting or CD3-CD28 stimulated splenocytes gated on live CD4+ve cells. (J) The IFNg + ve cells are subgated for showing % positive CD4 T cells producing IFNg. and their mean quantitation. (K) The representative images (n = 4) show the mouse tumor cryosections immunostained by anti-CD68 and anti-CD34 (secondary Alexa-647) antibodies followed by respective secondary antibodies (Alexa-488 antibody for CD68; Alexa-647 for CD34; scale bar equals 50 μm). All the data in vertical bars shows mean ± SEM. Statistical significance tested by two-tailed unpaired Mann–Whitney, except in G where paired t test was applied between time points. Significant differences are indicated with asterisks (*, P < 0.05). The data are result of minimum of two independent experiments. Source data are available for this figure: SourceData FS2.

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