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The gene for a T lymphocyte triggering factor from African trypanosomes.

Vaidya T, Bakhiet M, Hill KL, Olsson T, Kristensson K, Donelson JE - J. Exp. Med. (1997)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Iowa, and the Howard Hughes Medical Institute, Iowa City, Iowa 52242, USA.

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.

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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).
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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).

Mentions: The trypanosome cDNA was cloned into two different bacterial expression systems in which the NH2-terminal fusion partner is either GST or THIO. In each case, a PCR-derived fragment of the TLTF cDNA was cloned such that the methionine start codon is in frame with the last amino acid codon in the GST or THIO gene. In the THIO case, an additional clone was constructed in which the TLTF segment terminates at codon 145. The recombinant proteins were used to generate polyclonal sera in rabbits and mice. Each serum was then tested for its inhibition of the ability of native TLTF to induce T cells to secrete IFN-γ. Fig. 2 A shows that rabbit antisera directed against either of the two versions of THIO–TLTF (#1 and #4) reduced the activity of native TLTF to the background levels seen in the absence of TLTF. Similarly, mouse antiserum raised against a separate aliquot of THIO–TLTF #1 also reduced the activity of native TLTF to background levels. The presence of the corresponding preimmune serum did not significantly diminish TLTF activity. In control experiments, rabbit antiserum against Leishmania chagasi gp63 and mouse antiserum against Trypanosoma cruzi gp72 (each of which is a surface protein of its respective parasite) had no effect on the biological activity of native TLTF. Fig. 2 B shows that similar results were obtained in parallel experiments using rabbit antiserum directed against the GST–TLTF fusion protein.


The gene for a T lymphocyte triggering factor from African trypanosomes.

Vaidya T, Bakhiet M, Hill KL, Olsson T, Kristensson K, Donelson JE - J. Exp. Med. (1997)

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).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199003&req=5

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).
Mentions: The trypanosome cDNA was cloned into two different bacterial expression systems in which the NH2-terminal fusion partner is either GST or THIO. In each case, a PCR-derived fragment of the TLTF cDNA was cloned such that the methionine start codon is in frame with the last amino acid codon in the GST or THIO gene. In the THIO case, an additional clone was constructed in which the TLTF segment terminates at codon 145. The recombinant proteins were used to generate polyclonal sera in rabbits and mice. Each serum was then tested for its inhibition of the ability of native TLTF to induce T cells to secrete IFN-γ. Fig. 2 A shows that rabbit antisera directed against either of the two versions of THIO–TLTF (#1 and #4) reduced the activity of native TLTF to the background levels seen in the absence of TLTF. Similarly, mouse antiserum raised against a separate aliquot of THIO–TLTF #1 also reduced the activity of native TLTF to background levels. The presence of the corresponding preimmune serum did not significantly diminish TLTF activity. In control experiments, rabbit antiserum against Leishmania chagasi gp63 and mouse antiserum against Trypanosoma cruzi gp72 (each of which is a surface protein of its respective parasite) had no effect on the biological activity of native TLTF. Fig. 2 B shows that similar results were obtained in parallel experiments using rabbit antiserum directed against the GST–TLTF fusion protein.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Iowa, and the Howard Hughes Medical Institute, Iowa City, Iowa 52242, USA.

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.

Show MeSH
Related in: MedlinePlus