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Institute of Veterinary Virology, University of Berne, Berne, Switzerland
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Two distinct Theileria species have been extensively studied (3). Theileria parva, which is transmitted by Rhipicephalus appendiculatus, is the cause of East Coast fever, extending from Central to Eastern Africa. Theileria annulata, the causative agent of tropical theileriasis, is transmitted by ticks of the genus Hyalomma and is widespread, affecting cattle from North Africa to China. Both diseases are characterized by the proliferation of infected as well as uninfected hemopoietic cells followed by widespread cellular lysis, and are lethal if not treated (4).
Theileria parasites have a strict cell tropism. T.
parva infects and transforms preferentially, if not exclusively,
bovine T cells (5, 6, 7, 8). In contrast, T. annulata infects B
cells and macrophage (M
)5
lineage cells rather than T cells (5, 6). Whereas T.
parva-transformed T cell lines were the subject of extensive
phenotypic and functional investigations (7, 8, 9, 10, 11, 12), the properties of
T. annulata-transformed cell lines are much less well known.
Due to their multilineage origin, the T.
annulata-transformed cell lines characterized to date are
potentially heterogeneous and therefore inappropriate for detailed
analyses. Moreover, transformation may lead to dedifferentiation; we
recently showed that differentiated M progressively lose
lineage-specific surface markers and functions after infection with
T. annulata (13). This makes an identification of the
progenitor cell, B cell or monocyte-M, difficult. In this study we
used a clonal approach to analyze T. annulata-transformed
cell lines. We now show that a majority of transformed cells can be
traced back to the cell lineage of origin. We developed cloned cell
lines sharing properties with either B cells or M. The B cell clones
were analyzed further with regard to rearrangement of Ig genes and
expression of surface IgM.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Bovine PBMC were isolated according to a Ficoll-Hypaque
procedure as previously described (14). PBMC contained, on the average,
10% monocytes, based on light scatter properties and staining for the
DH159 epitope (see below) in flow cytometry. Neutrophil content was
<1%. Fetal bovine bone marrow cells (FBMC) were cultured as
previously described (15), using a Teflon bag system and high serum
concentrations (20% FCS) but no exogenous growth factors. Under these
culture conditions, M were positively selected, whereas all other
cells were negatively selected. This resulted in progressive loss of
non-M-type cells. For example, 100% of cells harvested on day 35 of
culture were M, as determined by morphology. Cells collected at
earlier intervals were heterogeneous with regard to cell populations,
as outlined in our previous study (15).
T. annulata-transformed cell lines
Various primary cell isolates derived from PBMC and FBMC were transformed with T. annulata sporozoites as described in a previous report (13). The established cell lines were cultured in 25-cm2 tissue culture flasks. The medium was Iscove’s modified DMEM (enriched with HEPES (10 mM), nonessential amino acids (1%), vitamins for MEM (1%), streptomycin (100 ng/ml), penicillin (100 IU/ml), amphotericin B (2.5 µg/ml), L-glutamine (2 mM), 2-ME (50 µM), and 10% FCS)). Bulk cultures of bone marrow-derived and of monocyte-enriched PBMC cell lines were used for generation of single cell colonies using limiting dilution techniques.
Magnetic cell sorting
B cells within PBMC were purified by magnetic cell sorting using
two procedures. In the first, CD21-positive cells were selected. In the
second, cells negative for CD3 and CD14 were collected. Cells
(108) were treated with Ab DU2–54 (see Table I
; twofold diluted tissue culture
supernatant). In parallel, the same number of cells was exposed to 10
µg of MM1A and CAM36A (see Table I). Each of the Ab-loaded
preparations was washed, followed by the addition of microbead-labeled
rat anti-mouse Abs (Miltenyi Biotec, Bergisch Gladbach, Germany).
Cells were passed through columns exposed to a magnetic field as
recommended by the manufacturer (Miltenyi Biotec). Nonretained and
retained cells were collected. Cells positively selected for CD21 or
negatively selected for CD3 and CD14 were used directly for
transformation by T. annulata.
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Cell lines in exponential growth were treated with the theilericidal agent BW720c (Buparvaquone, Pitman-Moore, Uxbridge, U.K.) essentially as previously described (13, 16) in concentrations of 100 ng/ml of medium. This was Iscove’s modified DMEM containing 20% (v/v) FCS and 20% (v/v) dialyzed bovine lung fibroblast cell culture supernatant. Medium containing the drug was renewed every 48 h from the first 8 days. After this time, medium containing 20% FCS and fibroblast dialysate, but no BW720c, was added at intervals of about 10 days, depending on the pH shift.
Flow cytometry
mAbs used were provided by Dr. W. C. Davis, Washington
State University (Pullman, WA), and by Dr. Wayne Hein, Basel Institute
for Immunology (Basel, Switzerland; Table I
) (13, 17, 18, 19, 20, 21). They were
used for indirect staining in flow cytometry as previously described
(13). Goat anti-mouse IgG and IgM, both conjugated with
phycoerythrin and absorbed against human and bovine serum, were
obtained from Jackson ImmunoResearch Laboratories (West Grove, PA).
Adherent cells were removed with a rubber policeman, and cells were
washed before labeling. Labeled cells were measured directly, without
fixation, in a FACScan flow cytometer and were analyzed with PC-LYSYS
software (Becton Dickinson, San Jose, CA).
A flow cytometric procedure was used to monitor apoptosis. Washed cells were lysed with 0.1 M citrate/1% Triton X-100 to liberate nuclei, followed by treatment with propidium iodide. The percentage of cells with reduced DNA content, as determined in histograms, was taken as the apoptotic subset.
RT-PCR
Cells were harvested from 25-cm2 culture flasks, pelleted, and resuspended in TRIzol (Life Technologies, Basel, Switzerland). Total RNA was isolated according to the manufacturer’s instructions. RT was performed with 1 µg of total RNA of each sample in the presence of oligo(dT) primers and 200 U/ml AMV-RT (Promega, Madison, WI) in a total volume of 50 µl. The mixture was incubated for 2 h at 42°C. Ten microliters of the RT product were used in the following PCR. The primers used were for detection of expression of Ig heavy chain. The following primers were used: variable region of heavy chain 5' (position 5)-CTGTGGACCCTCCTCTTTGT-3' (sense) and 5' (position 370)-GAGACTTGGCTCTTGGAGTT-3' (antisense). They were selected based on sequences reported in GenBank: BTU55164, BTU49756, and BTU11631 (22). The indicated positions of the primers are correct for germline genes. The corresponding sequences in Ig heavy chain mRNA are 82 bp shorter due to elimination of an intron (GenBank BTU55164ff). The PCR conditions were as follows: annealing for 30 s at 56°C, polymerization for 30 s at 72°C, and denaturation for 1 min at 94°C. Thirty PCR cycles were run.
PCR
To obtain information about the VDJ rearrangement status of the investigated clones, genomic DNA was isolated using a modified method basing on the Qiagen DNA isolation protocol (Qiagen, Chatsworth, CA). A combination of primers located in the V and the J regions of the heavy chain allowed differentiation between germline and rearranged genes. The primers used were the following 5'-CTGTGGACCCTCCTCTTTGT-3' (sense; BTU55164, BTU49756, and BTU11631) (22) and 5'-GACGGTGACCAGGAGTCCTA-3' (antisense; GenBank BTU63637ff). The primers were created after an alignment procedure with the listed sequences and were expected to anneal to the majority of, if not all, Ig heavy chain variable sequences reported in GenBank. In the case of successful gene rearrangement, bands with a size of 500 to 600 bp could be expected. The PCR conditions were the following: annealing for 1 min at 55°C, polymerization for 1 min at 72°C, and denaturation for 1 min at 94°C. Thirty-five cycles were performed. Some of the RT-PCR products obtained were sequenced and were found to be >92% identical with known bovine Ig heavy chain variable region sequences, as determined by the BLAST (basic local alignment search tool) program accessing the National Center for Biotechnology Information data bank.
Southern blotting
The electrophoretically separated PCR products were blotted onto a positively charged nylon membrane (Boehringer Mannheim, Mannheim, Germany) and immobilized by heating to 80°C for 2 h. Hybridization with a bovine heavy chain V region-specific digoxigenin (DIG)-labeled RNA probe was performed at 50°C overnight. The hybridized blot was washed several times in 2x SSC/0.1% SDS and 0.1x SSC/0.1% SDS both at room temperature and at 68°C (slightly modified from Boehringer Mannheim method). Staining was performed with anti-DIG-alkaline phosphatase conjugate (Boehringer Mannheim) according to the instructions of the manufacturer. CDP-Star (Tropix, Bedford, MA) was used as substrate. A high performance autoradiography film (Hyperfilm-MP, Amersham, Aylesbury, U.K.) was exposed for 30 s.
Staining of cell monolayers by crystal violet
The survival of cells subjected to BW720c treatment was determined by an indirect procedure. Cells were dispensed into 96-well plates (105/well), followed by treatment with BW720c. Cell death was assessed by monitoring the decrease in numbers of adherent cells. Cells dislodged from the culture vessel and floating in the supernatant were found to take up trypan blue and were therefore characterized as dead cells. Monolayers were washed at predetermined times after the onset of redifferentiation, followed by staining with crystal violet (0.75% in 50% ethanol/0.25% NaCl/1.75% formaldehyde). OD was read in an ELISA reader at 590 nm. Since viable cells were found exclusively in the adherent cell fraction, OD was proportional to the number of surviving cells.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Various types of cells were infected with T. annulata
sporozoites, a procedure resulting in the onset of exponential growth
within 1 wk. Some of the lines were subjected to limiting dilution
either directly following infection or up to 2 wk after infection. Cell
lines and clones were screened first for expression of B cell markers
(Table II), followed by a more detailed
analysis of selected cell lines and clones for general surface marker
expression, using a broad panel of mAbs (Table I). All cell lines and
clones investigated showed a uniform phenotype with respect to the
majority of surface markers (Table III
and data not shown). Thus, all cell lines expressed MHC class I
molecules, MHC class II molecules, CD44, CD45, CD9, CD11a/CD18, and the
epitopes recognized by the Abs DH59, DH16, and LCTB22. They were
uniformly negative for CD3, CD4, CD5, CD8, CD11b, and CD14. CD2 and
CD11c expression was variable, but always weak. On the other hand, cell
surface expression of B cell markers was highly variable (Table III).
In all cases tested, there was coexpression of CD21 and the B-B2 and
B-B8 epitopes. Cell surface expression of Ig µ-, 
-, and 
-chains
was always negative on cell lines lacking these B cell markers. Cells
expressing B cell markers were either positive or negative for Ig µ
expression. Usually (with one exception), positive lines were derived
from peripheral blood cells, and negative lines were derived from the
fetal bone marrow. Lines positive for Ig µ coexpressed either or
, with two exceptions. In one, both and were coexpressed; in
the other, neither nor was coexpressed with µ. Lines negative
for µ but positive for B cell markers were negative for and ,
with the exception of one -expressing line. All in all, cells had a
characteristic, T. annulata-dictated phenotype and varied
exclusively with respect to B cell markers. Only B cell marker-positive
lines showed evidence of Ig surface expression. The three major
patterns of B cell surface marker expression are shown in Figure 1.
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To obtain evidence that lines expressing B cell markers were derived
from B cells, PBMC were subjected to magnetic cell sorting with a view
to enrich for B cells. Positive selection of B cells was based on CD21
expression, since this marker was present on a slightly larger
proportion of PBMC than B-B2 and B-B8. Positive selection for CD21
expression resulted in a homogeneous population, which was 97.2%
positive for CD21. Negative selection of B cells, based on the absence
of CD3 and CD14 on the B cell surface, resulted in a population of
52.1% CD21-positive cells. Both B cell-enriched populations were
infected with T. annulata and were cloned by limiting
dilution. Positively selected, transformed cells yielded six clones,
and negatively selected, infected cells gave rise to 35 clones. All six
positively selected, transformed cells clones expressed B cell markers,
and five of the six were positive for Ig µ-chain. Five of thirty-five
negatively selected, PBMC-derived clones were positive for both B cell
markers and Ig µ-chain. This contrasts to findings with clones
derived from bone marrow cell cultures. Ten clones obtained from a
35-day-old primary bone marrow cell culture, which contained virtually
100% M, were analyzed, and none of these expressed B cell markers
or Ig µ. Likewise, bone marrow cells cultured for 14 days, a period
sufficient to select for M lineage cells in the culture system used
(15), were infected with T. annulata. None of the resulting
clones analyzed expressed B cell markers or Ig µ. This strongly
suggests that the expression of B cell markers indicated lineage
progeny.
Transcription of Ig heavy chain
To confirm Ig expression by T. annulata-transformed
lines at the mRNA level, total RNA of all clones generated was isolated
and subjected to RT-PCR, using a primer combination for the Ig V heavy
chain region defining a 293-bp fragment. The specificity of the
amplified products was confirmed by Southern blotting with a specific
probe. Evidence for Ig heavy chain mRNA expression was obtained in
clones derived from PBMC, which were positive for B cell markers as
shown in the previous flow cytometric analysis (Fig. 2A). In contrast, no
mRNA for Ig heavy chain was found in PBMC clones negative for B cell
markers or in clones derived from FBMC even if they were positive for B
cell markers (Fig. 2A). Thus, Ig V heavy chain
expression based on RT-PCR analysis completely corroborated flow
cytometry.
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Another method to determine cell progeny was to test for heavy
chain gene rearrangement in transformed clones. Genomic DNA was
isolated from clones derived from T. annulata-transformed
PBMC and FBMC and was used for PCR, which allowed the detection of
rearranged heavy chain VDJ. The primers were located in the V (forward)
and the J (reverse) regions. Detectable bands of 500 to 600 bp were
expected in clones with rearranged genes. Specificity was evidenced by
Southern blotting, using the above-mentioned probe for Ig V heavy chain
elements. As shown in Figure 2C, not only sIgM-expressing
cells but also FBMC-derived clones, which were positive for B-B2, B-B8,
and CD21 but negative for sIgM, showed bands in the expected size
range. In contrast, all clones that were negative for the B cell
markers also remained negative in the PCR. This supports the view that
the expression of B cell markers is indicative of lineage progeny.
Redifferentiation behavior of clones after elimination of T. annulata
Since surface markers characterizing monocyte-M lineage cells
are progressively down-regulated upon transformation by
Theileria (13), no cell surface markers were available,
indicating monocytoid lineage progeny. However, we recently showed that
after elimination of the parasite, M-derived lines reacquired
phenotypic characteristics of the cells from which they were derived
(13). Several clones were treated with the theilericidal drug, BW720c,
with a view to test changes in their phenotypic properties. This was
performed under culture conditions optimized for maintaining M type
cells in culture, i.e., in the presence of high concentrations of serum
and of conditioned medium derived from bovine lung fibroblasts.
Elimination of T. annulata stopped proliferation in all
clones. The majority of B cell marker-negative clones showed expression
of CD14 and CD11b and phagocytic activity 20 days after the onset of
BW720c treatment (Table IV). The ability
to redifferentiate was restricted to cells of relatively low passage
number (<10). With increasing passage numbers, the efficiency of
redifferentiation decreased, and the survival of BW720c-treated cells
under the described culture conditions was poor. In contrast, B cell
marker-positive clones invariably failed to redifferentiate, and the
cells were lost within 10 to 15 days (Fig. 3). Thus, using the described culture
conditions, the ability to acquire M properties was a characteristic
of clones obtained from M and was not shared by clones derived from
B cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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lineages. Although established lines have been
characterized extensively with regard to surface marker phenotypes (6, 8, 23, 24), for most of these lines the exact origin was not clear.
Moreover, most T. annulata-transformed cell lines are of
polyclonal origin. This is the first study in which individual clones
of T. annulata-transformed lines were characterized with
respect to lineage progeny. Our study allows the following conclusions
to be made. 1) Confirming earlier reports, both B cells and cells of
monocyte-M lineage can be transformed by T. annulata (6).
Under the described culture conditions, cloning efficiency for
M-derived lines was higher than that for B cell-derived lines. 2)
Cells of monocyte-M lineage lose phenotypic properties upon
transformation, but reacquire some of these when the parasite is
eliminated. 3) Cells of B cell lineage maintain expression of
lineage-specific markers such as CD21, B-B2, and B-B8, and they show
evidence for Ig heavy chain gene rearrangement. 4) B cell lines derived
from the peripheral blood, but not those derived from the fetal bone
marrow, transcribe and express Ig.
The observation that M-derived cell lines lose their phenotypic
properties upon transformation confirms our earlier report in which a
detailed analysis of M functions was presented (13). It raises the
question of why monocytoid cells, but not B cells, dedifferentiate upon
transformation. One explanation would be that monocytes and M are
postmitotic cells, whereas B cells physiologically may undergo cell
division without loss of their properties. In this context it is
interesting that two other ruminant cell lines of monocyte-M
lineage, Bo-Mac (25) and M617 (26), also lack expression of
M-specific surface markers and functions (H. Sager and T. M.
Jungi, in preparation). In our view, all these lines poorly represent
the phenotype of differentiated monocytes or M. On the other hand,
T. parva-transformed cell lines exhibit properties of T
cells and maintain surface marker characteristics of mature T cells
(23). Thus, the effects imparted on lymphocytes by the two
Theileria species are clearly distinct from those on
monocytoid cells.
Hitherto, the loss of lineage-specific surface markers and functions
precluded an identification of the progenitor cells for most existing
T. annulata-transformed lines. The present study suggests
that the expression of B cell markers is indicative of B cell progeny.
Even if existing T. annulata-transformed cell lines are
heterogeneous with respect to cell of origin, a simple cloning
procedure of cells selected for the presence or the absence of B cell
markers should allow generation of lines of defined progeny. This is
supported by the absence of B cell marker-expressing lines obtained by
transformation of pure M cultures, by the high proportion of B cell
marker-expressing lines obtained by transforming highly purified B
cells, and by the evidence that Ig heavy chain gene rearrangement has
occurred in all lines expressing B cell markers.
Despite the fact that Ig heavy chain gene rearrangement could be
demonstrated, cells varied with regard to IgM surface expression.
Almost all B cell lines derived from PBMC expressed IgM at their
surface, and the majority of these coexpressed Ig or . One of 11
lines expressed Ig , but not IgM, raising the possibility that
another isotype was expressed at the surface. In the other lines,
surface IgM expression was correlated with mRNA expression for Ig heavy
chain. This supports the concept that mature B cells can be transformed
by this parasite. We did not systematically investigate whether
susceptibility to transformation is limited to certain stages of B cell
differentiation and activation. Intriguingly, however, B cell lines
obtained from fetal bone marrow cultured for 10 days or less were found
not to express Ig heavy chain on the surface. The absence of an Ig
heavy chain transcript argues against the expression of an Ig isotype
other than IgM. Expression of surrogate light chain (27) not recognized
by the light chain-specific Abs that were used is remote, since no IgM
signal was obtained. It is conceivable that a less mature stage of B
cells present in bone marrow was transformed. Indeed, it has recently
been proposed that mouse pre-B cells down-regulate Ig gene
transcription shortly after heavy chain rearrangement (28). We regard
it as improbable that expression of B cell markers by these lines is
incidental, since we showed that all these underwent Ig heavy chain
gene rearrangement, a complex process unlikely to be induced by the
transforming parasite. Whatever the reason, it will be of interest to
study the susceptibility to transformation of B cells from different
sites and isolated at different stages of maturation and/or
activation.
Based on this clonal analysis, we suggest that the effects imparted by
T. annulata on immune cells can be summarized as shown in
Table V. There was a low number of
exceptions, however. One single line did not express B cell markers and
failed to redifferentiate to M. One single line coexpressed Ig µ,
, and , suggesting that isotype suppression was not operative.
One single Ig µ-expressing line failed to stain for and ,
pointing to a possible abnormality in the light chain.
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markers after transformation,
but the survival properties after treatment with BW720c were consistent
with a monocyte origin. The relatively high proportion of non-B cell
clones could be due to the higher transformation efficiency of
monocytes compared with B cells (5, 6). When B cells were selected by a
positive procedure, however, a population containing 97% CD21-positive
cells, five of six clones showed B cell properties, and the Ig
µ-negative clone was lost after two passages. This is consistent with
a low proportion of monocytes in this subset, but it cannot be ruled
out that the selecting Ab that was used interfered with the
transformation efficiency. According to a recent study, peripheral
blood contains CD21-negative B cells that do not recirculate but have a
predilection for the spleen (29). Given the heterogeneity of the B cell
system, it will be of interest to compare different B cell subsets for
their susceptibilities to transformation by T. annulata. The present analysis can be regarded as a first step toward controlled immortalization of ruminant B cells and harvest of their products. Such cells could be useful tools to generate Ab of a desired specificity in the context of a natural infection or vaccination. Importantly, immortalization of ruminant B cells by T. annulata represents an epigenetic manipulation and is not associated with the integration of transformation-promoting genetic information into the host cell genome. It will be of interest to explore whether B cells expressing Ag receptors of defined specificity can be immortalized by the described procedure. Moreover, since most of the cell lines described are of monoclonal origin and are defined with regard to progeny, they might be useful tools for biochemical studies such as stimulus-response coupling. Such studies have been extensively performed with T. parva-transformed T cells (7, 8, 9, 10, 11, 12), but not with T. annulata-infected B cells.
We were unable to propagate B cell-derived lines upon treatment with
the theilericidal drug, BW720c. This does not necessarily imply that B
cells are intrinsically incapable of surviving after elimination of the
parasite. The conditions under which these experiments were performed
were adapted from our previous work (13). The use of conditioned medium
from bovine lung fibroblasts, possibly containing the mononuclear
phagocyte growth and survival factor, M CSF, favored monocyte-M
differentiation and enhanced survival, but the requirements for B cells
may be entirely different. It is hoped that future studies will
identify culture conditions favoring B cell long term survival and
redifferentiation, possibly even to the stage of plasma cells. Since B
cells, unlike M, are not postmitotic cells, this might be feasible.
That this will be possible is also suggested in analogy to T.
parva-transformed T cell lines (7), which survive BW720c treatment
in the presence of IL-2. Our demonstration that single B cells can be
cloned and immortalized may be a first step toward the long term
maintenance of the Ig repertoire of a given animal by an epigenetic
manipulation using a natural infectious agent.
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Acknowledgments |
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Footnotes |
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2 Current address: Institute of Parasitology, University of Berne, Berne, Switzerland.
3 Address correspondence and reprint requests to Dr. T. W. Jungi, Institute of Veterinary Virology, University of Berne, Länggass-Strasse 122, CH-3012 Berne, Switzerland. E-mail address:
4 Address correspondence and reprint requests to Dr. T. W. Jungi, Institute of Veterinary Virology, University of Berne, Länggass-Strasse 122, CH-3012 Berne, Switzerland. E-mail address:
5 Abbreviations used in this paper: M, macrophage(s); FBMC, fetal bone marrow-derived cells; sIgM, surface immunoglobulin M.
Received for publication December 15, 1997. Accepted for publication February 20, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B in bovine T lymphocytes. Mol. Cell Biol. 11:4677.
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