By
§
From the * Department of Biochemistry, University of Iowa, and the Howard Hughes Medical
Institute, Iowa City, Iowa 52242; Department of Medicine, Unit for Molecular Medicine,
Karolinska Hospital, Stockholm, Sweden; § Division of Neurology, Huddinge University Hospital,
Karolinska Institute, Stockholm, Sweden; and the
Department of Neuroscience (Division of
Neurodegenerative Disease Research), Karolinska Institute, Stockholm, Sweden
An early and essential event in the protective immune response against most viruses and protozoa is the production of interferon- (IFN-
). In contrast, during infection with African trypanosomes, protozoan parasites that cause human sleeping sickness, the increased levels of IFN-
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-
. 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-
. 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.
African trypanosomiasis is a widespread, fatal disease in
Africa that is commonly called sleeping sickness in humans and ngana in cattle. These diseases are caused by several different species or subspecies of Trypanosoma, which are
transmitted by tsetse flies to the bloodstream of their mammalian hosts where they circulate extracellularly and can
eventually affect the central nervous system. Mortality occurs from either massive parasitosis or secondary infections
due to immunosuppression, an important trait of the disease.
Previously, we showed that trypanosomes release a T
lymphocyte triggering factor (TLTF)1 that induces CD8+
T cells to secrete IFN- Molecular Biology Procedures.
The TLTF cDNA was identified by immunoscreening a (1, 2); subsequent studies indicated that TLTF binds to the CD8 molecule on T cells (3).
When rodents are infected with Trypanosoma brucei, IFN-
production in the spleen increases markedly. Moreover, in
rats depleted of CD8+ T cells by injection of anti-CD8
monoclonal antibody or in knockout mice carrying a deletion of the CD8 gene, a trypanosome infection does not
induce as much IFN-
production, the parasitemia is decreased and the infected animals survive longer (3). In passive immunotherapy experiments, a mouse monoclonal antibody directed against TLTF greatly reduces parasite levels
in, and increases survival of, animals infected with T. brucei
(4, 5). Here, we identify the gene for TLTF and show that
bacterially expressed TLTF has T cell stimulatory activity
that is inhibited by monoclonal antibodies raised against the
native protein. We further show that TLTF has sequence
similarity with a mammalian protein and that the subcellular localization of a green fluorescent protein (GFP)-TLTF
fusion protein is consistent with that expected for a secreted protein.
ZapII cDNA library of bloodstream
T. b. rhodesiense RNA (6) with rabbit antiserum against affinity-purified TLTF (4) at a 1:2000 dilution. The antiserum was preadsorbed with Escherichia coli lysate before use. The secondary antibody was a horseradish peroxidase-linked donkey anti-rabbit IgG
(Amersham Corp., Arlington Heights, IL) used at a 1:5000 dilution. At all stages, a 5% solution of nonfat powdered milk was used to
block nonspecific binding. Potentially positive plaques were detected
using the enhanced chemiluminescent system (Amersham Corp.,
Arlington Heights, IL) and taken through two more rounds of
screening. The cDNA sequence was determined by standard sequencing procedures using the Sequenase 2.0 kit (US Biochemicals,
Cleveland, OH). Homology searches of databases were performed
using the BLAST algorithm (7). Protein sequence alignments were
done using the University of Wisconsin GCG software package (8).
DNA Transfections. Procyclic trypanosomes (clone YTAT1.1, obtained from E. Ullu, Yale University, New Haven, CT) were maintained in Cunningham's SM medium (12) supplemented with 20% FCS. Cells were harvested from mid-log phase cultures (2-4 × 106 cells/ml), washed once with electroporation medium (120 mM KCl, 0.15 mM CaCl2, 10 mM K2HPO4, 25 mM Hepes, 2 mM EDTA, 5 mM MgCl2, pH 7.6) and resuspended in the same buffer at 1-3 × 108 cells/ml. DNA (50 µg) was introduced into cells by electroporation with a BioRad Gene Pulser using two pulses of 1,500 V at 25 µF in 0.4 cm cuvettes. Transfected cells were transferred to fresh culture medium and examined for GFP expression 16-24 h after transfection. For ethidium bromide staining, cells were incubated with 2 mg/ml ethidium bromide for 15 min, then washed twice with PBS. Cells were examined using a BioRad MRC-1024 Laser Scanning Confocal Microscope.
IFN- Assays.
Purification of native TLTF, isolation of mononuclear cells and the spot assay for IFN-
secretion were performed as described previously (1, 3, 4). The final concentration
of concanavalin A in the assays was 5 µg/ml. The final concentration of monoclonal antibody MO1 was 5 µg/ml. All assays
were conducted using cells from CD4
CD8+ mice except where
indicated. The origin of the CD4
CD8+ and CD4+CD8
mice
were described earlier (13). Most of the assays for IFN-
secretion that involved the recombinant fusion proteins were conducted blind using protein prepared in the United States and sent
in coded tubes to Sweden, and were done at least in triplicate.
A monoclonal antibody, called MO1, was used to affinity purify TLTF from bloodstream trypanosome extracts.
Monovalent polyspecific antiserum directed against the affinity-purified protein was generated in rabbits and used to
immunoscreen a cDNA expression library of total RNA
isolated from bloodstream forms of T. b. rhodesiense, a subspecies that causes the human disease (6). A cDNA clone was identified that remained immunopositive through multiple rounds of screening. The nucleotide sequence of this
cDNA revealed that it was a partial length clone lacking the
39 nucleotide sequence of the 5 spliced leader, a universal
feature of trypanosome mRNAs. A full-length coding sequence was obtained from the product of a reverse transcriptase-PCR amplification of total trypanosome RNA
using an internal 3
primer derived from the TLTF partial cDNA sequence and a 5
primer matching the 5
spliced
leader sequence. Fig. 1 shows the deduced 453-amino acid
sequence of the encoded protein, a 54-kD hydrophilic
polypeptide.
The amino acid sequence of TLTF bears no obvious
similarity to IGIF (IFN--inducing factor), a protein recently identified in mouse liver that induces IFN-
production by CD4+ T lymphocytes (14). The lack of similarity between these two proteins may be because IGIF is
thought to target CD4+ cells, whereas TLTF interacts specifically with the CD8 molecule. However, computer
searches did identify similarities to two other sequences that
have been deposited in GenBank without further characterization. One encodes a mouse protein of 489 residues designated as a growth arrest-specific protein. The other
protein is encoded by a randomly sequenced human cDNA
from a fetal lung cDNA library. Comparison of these proteins with TLTF reveals several regions of high sequence
identity (Fig. 1). Although the properties and functions of
these two mammalian proteins are yet to be described, their percent identity to TLTF (35% identity, 58% similarity) appears too large to be fortuitous.
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-.
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 THIO and GST fusion proteins were also tested for
TLTF activity, i.e., the ability to induce T cells to secrete
IFN-. Fig. 3 A shows that GST-TLTF induces IFN-
secretion from CD8+, but not CD4+, T cells. The splenocytes used in these assays were obtained from gene knock-out mice that lacked either the CD4 gene or the CD8 gene
(13). Similar to native TLTF, the GST-TLTF fusion protein induced IFN-
secretion only from the population
containing CD8+ cells and not from the cells derived from
CD8 knockout mice. Control experiments in which concanavalin A was used as a mitogen demonstrated that both
populations of cells were intrinsically capable of secreting
IFN-
in response to an appropriate signal. Fig. 3 B demonstrates in a separate series of experiments that the ability
of GST-TLTF to induce IFN-
secretion is specifically due to the TLTF portion of the molecule. Neither GST
alone nor another unrelated GST fusion protein, GST-
OV7, possessed an activity significantly greater than the
background level. In similar experiments not shown, THIO-
TLTF, also possessed a similar level of TLTF activity.
Next, we examined whether antibodies that specifically
recognize the native TLTF molecule can inhibit the activity of recombinant TLTF. We previously demonstrated
that the monoclonal antibody MO1 reduces the ability of
native TLTF to induce IFN- secretion from CD8+ cells
(4). Fig. 4 shows the results obtained when the activity of
GST-TLTF was measured in the presence of MO1. Under
the conditions of the assay, the activity of native TLTF was
reduced by 62% in the presence of MO1. Consistent with
the interpretation that GST-TLTF is the recombinant version of this protein, a concomitant decrease of 60% in the
activity of GST-TLTF occurred in the presence of MO1.
In the case of the unrelated GST-OV7, the number of
IFN-
-secreting cells detected in the assay was unaffected
by the presence or absence of MO1.
We previously showed that TLTF is released as a soluble factor from T. brucei into the culture medium (15) and yet the amino acid sequence of TLTF (see Fig. 1) does not contain a classical NH2-terminal signal sequence similar to those found in most secreted eukaryotic proteins (16). Because secretion of TLTF is likely essential for its immunomodulatory function, we investigated how it is targeted to the exterior of the cell. This is an especially relevant question in trypanosomatids, because the entire surface of these organisms is covered by a very dense protein coat that restricts possible sites of endocytosis and secretion to the flagellar pocket (17). This organelle derives from a specialized invagination of the cell membrane that forms at the site where the flagellum emerges from the cell and is relatively protected from the assaults of the immune system of the host. The kinetoplast of T. brucei is also located near the base of the flagellum and serves as a marker for the subcellular location of the flagellar pocket.
To examine the cellular fate of TLTF, we fused it to
GFP from Aquorea victoria (11). Constructs encoding either
this fusion protein, or GFP alone, were expressed in transiently transfected trypanosomes, which were examined by
laser scanning confocal microscopy (Fig. 5). Because Northern blot analysis indicated that TLTF is expressed in both
procyclic and bloodstream form trypanosomes (data not
shown), we used the more readily cultured procyclic forms
for these transfection experiments. Western blots using an
-GFP polyclonal antiserum (Clonetech, Palo Alto, CA)
confirmed that transfected trypanosomes were producing a
protein of the expected size (data not shown). Cells in the
bottom panel of Fig. 5 were also stained with ethidium
bromide to visualize the nucleus and kinetoplast (red staining). As can be seen in the top panel of Fig. 5, GFP alone is
expressed evenly throughout the entire cell. The GFP-
TLTF fusion protein, on the other hand, is restricted to
vesicles that are usually, but not exclusively, located at or
near the trypanosome flagellar pocket (Fig. 5, bottom).
These results are consistent with TLTF being a secreted
protein, as shown previously (15), and demonstrate that
signals for TLTF targeting are located within the protein itself, even though it does not contain a hydrophobic NH2-terminal signal sequence. Thus, TLTF is added to the
growing list of secreted eukaryotic proteins for which the
amino acid sequence gives no indication of a readily identifiable targeting signal (18). Interestingly, many such proteins play a role in immune system function (e.g., IL-1
and adult T cell leukemia-derived factor [ADF]. IL-1
is a
monocyte-derived, proinflammatory cytokine that stimulates a broad range of cell types. ADF mediates upregulation of IL-2 receptor expression and may be involved in
the abnormal proliferation of T cells observed in some
types of leukemogenesis (19). Although some features of
export may be shared between leaderless secreted proteins, the mechanisms responsible are unknown. Recent data indicate that multiple mechanisms exist (20) and that two different proteins may utilize partially, but not completely,
overlapping pathways (21).
TLTF is likely to be the first confirmed member of a cabal of trypanosome-derived, immunomodulatory factors for
which we suggest the name trypanokines. Trypanosomal
cysteine protease, which is found in the serum of infected
animals (22), is another potential trypanokine, because
cysteine proteases of other microorganisms have been shown
to modulate cytokine activity either directly or indirectly
(25, 26). Still other unknown mitogenic trypanosome molecules have been proposed previously to be the cause of the massive nonspecific polyclonal activation of B cells during
acute trypanosomiasis (27). Parallels have already been
drawn between gene rearrangements that generate the antibody repertoires in vertebrates and those responsible for the
perpetual antigenic variation in trypanosomes (28). Trypanokines represent another example of the ability of the
trypanosome to mimic a normal host function; in this case,
the synthesis of cytokines that modulate the immune response. Recent experiments with viruses and infectious bacteria indicate that this strategy may also be commonly
used by other microbial pathogens (25, 29). In this regard,
the availability of the TLTF gene should facilitate novel approaches towards achieving protection against trypanosomiasis. In addition, recombinant TLTF could find applications in the treatment of other infectious agents, such as
Leishmania and viruses, for which IFN- production is among
the first events of a protective response.
Address correspondence to John E. Donelson, Department of Biochemistry, University of Iowa, Iowa City, IA 52242. Phone: 319-335-7889; FAX: 319-335-6764. The current address for Tushar Vaidya is Center for Cellular and Molecular Biology, Uppal Rd., Habshiguda, Hyderbad, India 500007.
Received for publication 9 April 1997 and in revised form 27 May 1997.
This research was funded in part by a grant from the United Nations Development Program/World Bank/ World Health Organization Special Program for Research and Training in Tropical Disease to M. Bakhiet, T. Olsson, and K. Kristensson.We would like to thank D. Russell for conducting preliminary immunogold localization of native TLTF. We are extremely grateful to E. Ullu for the YTAT1.1 trypanosome clone and for her assistance in our efforts to express GFP in T. brucei. We would also like to thank B. Cormack and S. Falkow for providing the GFPmut3 cDNA.
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