The gene for a T lymphocyte triggering factor from African trypanosomes

Abstract

An early and essential event in the protective immune response against most viruses and protozoa is the production of interferon-gamma (IFN-gamma). In contrast, during infection with African trypanosomes, protozoan parasites that cause human sleeping sickness, the increased levels of IFN-gamma do not correlate with a protective response. We showed previously that African trypanosomes express a protein called T lymphocyte triggering factor (TLTF), which triggers CD8(+) T lymphocytes to proliferate and to secrete IFN-gamma. Here, we isolate the gene for TLTF and demonstrate that the recombinant version of TLTF specifically induces CD8(+), but not CD4(+), T cells to secrete IFN-gamma. Studies with TLTF fused to the green fluorescent protein show that TLTF is localized to small vesicles that are found primarily at or near the flagellar pocket, the site of secretion in trypanosomes. TLTF is likely to be only the first example of a class of proteins that we designate as trypanokines, i.e., factors secreted by trypanosomes that modulate the cytokine network of the host immune system for the benefit of the parasite.

Figure 1

Predicted amino acid sequence of TLTF and comparison to a mouse growth arrest–specific protein. The trypanosome cDNA sequence predicts a hydrophilic, 453-amino acid protein of 54 kD. In the alignment with the mouse protein (GenBank accession No. U19589) similar amino acid residues, including conservative substitutions, are outlined and positions of identity are shaded. The two proteins exhibit 59% amino acid similarity and 35% identity. The deduced protein encoded by a human expressed sequence tag (dbEST accession No. W21172) is nearly identical (87%) to the mouse protein and also displays 34% identity with TLTF (data not shown). The trypanosome TLTF sequence data are available from EMBL/GenBank/DDBJ under accession number AF012853.

Figure 2

Antisera directed against the two different forms of recombinant TLTF inhibit interfere with the activity of native TLTF. (A) Results obtained with antisera raised against THIO–TLTF. P and I denote preimmune and immune serum, respectively. α-THIO–TLTF #1 is serum directed against a fusion protein containing the entire TLTF molecule. α-THIO–TLTF #4 is serum directed against a fusion protein containing only the NH₂-terminal 145 amino acids of TLTF. α-gp63 is serum directed against an unrelated Leishmania chagasi protein, gp63 (30); α-Tc is serum directed against a recombinant version of an unrelated Trypanosoma cruzi antigen, gp72 (31) (the gift of K. Otsu and L.V. Kirchhoff, University of Iowa, Iowa City, IA). The dilution of serum used in each case was 1:5,000. (B) Results obtained with α-GST–TLTF #1, a rabbit antiserum directed against a GST fusion protein containing the entire TLTF protein (1:1,000 dilution).

Figure 2

Antisera directed against the two different forms of recombinant TLTF inhibit interfere with the activity of native TLTF. (A) Results obtained with antisera raised against THIO–TLTF. P and I denote preimmune and immune serum, respectively. α-THIO–TLTF #1 is serum directed against a fusion protein containing the entire TLTF molecule. α-THIO–TLTF #4 is serum directed against a fusion protein containing only the NH₂-terminal 145 amino acids of TLTF. α-gp63 is serum directed against an unrelated Leishmania chagasi protein, gp63 (30); α-Tc is serum directed against a recombinant version of an unrelated Trypanosoma cruzi antigen, gp72 (31) (the gift of K. Otsu and L.V. Kirchhoff, University of Iowa, Iowa City, IA). The dilution of serum used in each case was 1:5,000. (B) Results obtained with α-GST–TLTF #1, a rabbit antiserum directed against a GST fusion protein containing the entire TLTF protein (1:1,000 dilution).

Figure 3

Recombinant TLTF has biological activity. (A) Results demonstrating the specificity of GST–TLTF #1 activity on mononuclear cells from CD4⁻CD8⁺ and CD4⁺CD8⁻ mice. The concentrations of GST–TLTF #1 used in the reactions are indicated. (B) Comparison of the activities of native TLTF, GST–TLTF #1, GST–OV7, and GST alone. GST–OV7 is an unrelated GST fusion protein prepared in the same way as GST–TLTF. Assays in B were done using cells from CD4⁻CD8⁺ mice. The concentration of the recombinant proteins and GST in the assays shown in A and B was 10 μg/ml. Conconavalin A was used at a final concentration of 5 μg/ml.

Figure 3

Recombinant TLTF has biological activity. (A) Results demonstrating the specificity of GST–TLTF #1 activity on mononuclear cells from CD4⁻CD8⁺ and CD4⁺CD8⁻ mice. The concentrations of GST–TLTF #1 used in the reactions are indicated. (B) Comparison of the activities of native TLTF, GST–TLTF #1, GST–OV7, and GST alone. GST–OV7 is an unrelated GST fusion protein prepared in the same way as GST–TLTF. Assays in B were done using cells from CD4⁻CD8⁺ mice. The concentration of the recombinant proteins and GST in the assays shown in A and B was 10 μg/ml. Conconavalin A was used at a final concentration of 5 μg/ml.

Figure 4

Monoclonal antibody MO1 directed against native TLTF inhibits the biological activity of both native TLTF and recombinant GST– TLTF. The IFN-γ assays were conducted in the absence (closed bars) or presence (cross-hatched bars) of MO1.

Figure 5

TLTF targeting signals direct a heterologous reporter protein (GFP) to vesicles that are localized to the trypanosome flagellar pocket. Procyclic T. brucei cells were transiently transfected with a T. brucei expression plasmid containing the GFP gene alone (top) or a plasmid encoding a GFP–TLTF fusion protein (bottom). The transfected cells were examined by laser scanning confocal microscopy and the transmitted and fluorescent images were superimposed on one another. GFP fluorescence is green. Cells in the bottom panel were additionally stained with ethidium bromide to visualize the nucleus and kinetoplast (red staining). The white bar corresponds to 10 μm.