TLT-1 binds to CD3ε on T cells and suppresses CD8 T cells but not CD4 T cells. (A) The initial functional changes in primary human T cells incubated with recTLT-1 was studied by evaluating the expression of p-CD28 and Bax, as shown by representative Western blots (left) and their quantitation (right). (B) The heatmap obtained by qPCR array shows recTLT-1–induced gene regulation (arithmetic mean) in CD4 and CD8 T cells isolated from healthy subjects (n = 4). The fold regulation (scale on the right) shows fold change upon recTLT-1 exposure for 24 h in culture with respect to vehicle control group. The qPCR dCt values were using a mean dCt of five constitutive genes as per recommendation of the array manufacturer. (C) Co-IP experiment using Jurkat cells incubated with Fc-tagged recTLT-1 or recIgG followed by Protein A/G bead pull down and probed with CD3ε, TLT-1, CD3γ, or CD3δ antibodies separately as shown by representative Western blots (n = 4 for each Co-IP). The band for CD3ε is observed at the position of TLT-1 in IP samples. The representative blots showed the higher (than in non-IP lysate) molecular weight band at ∼80–85 and ∼160–180 kD in immunoblotting of CD3ε (predicted ∼23 kD), and also when probed for TLT-1 on separate blot (predicted ∼55 kD) only in the co-IP sample. (D) The gene expression heat map for NF-κB pathway showing the fold change upon recTLT-1 exposure for 24 h in culture with respect to vehicle control group. The qPCR dCt values were using a mean dCt of five constitutive genes as per recommendation of the array manufacturer. We combined array data from independent experiments to select a consensus set of regulated genes. Significant differences are indicated with asterisks (*, P < 0.05; **, P < 0.01). All experimental data represent a minimum of three independent experiments. Source data are available for this figure: SourceData F6.
Figure 6.

TLT-1 binds to CD3ε on T cells and suppresses CD8 T cells but not CD4 T cells. (A) The initial functional changes in primary human T cells incubated with recTLT-1 was studied by evaluating the expression of p-CD28 and Bax, as shown by representative Western blots (left) and their quantitation (right). (B) The heatmap obtained by qPCR array shows recTLT-1–induced gene regulation (arithmetic mean) in CD4 and CD8 T cells isolated from healthy subjects (n = 4). The fold regulation (scale on the right) shows fold change upon recTLT-1 exposure for 24 h in culture with respect to vehicle control group. The qPCR dCt values were using a mean dCt of five constitutive genes as per recommendation of the array manufacturer. (C) Co-IP experiment using Jurkat cells incubated with Fc-tagged recTLT-1 or recIgG followed by Protein A/G bead pull down and probed with CD3ε, TLT-1, CD3γ, or CD3δ antibodies separately as shown by representative Western blots (n = 4 for each Co-IP). The band for CD3ε is observed at the position of TLT-1 in IP samples. The representative blots showed the higher (than in non-IP lysate) molecular weight band at ∼80–85 and ∼160–180 kD in immunoblotting of CD3ε (predicted ∼23 kD), and also when probed for TLT-1 on separate blot (predicted ∼55 kD) only in the co-IP sample. (D) The gene expression heat map for NF-κB pathway showing the fold change upon recTLT-1 exposure for 24 h in culture with respect to vehicle control group. The qPCR dCt values were using a mean dCt of five constitutive genes as per recommendation of the array manufacturer. We combined array data from independent experiments to select a consensus set of regulated genes. Significant differences are indicated with asterisks (*, P < 0.05; **, P < 0.01). All experimental data represent a minimum of three independent experiments. Source data are available for this figure: SourceData F6.

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