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*
Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA 99164;
Department of Animal Sciences, University of Illinois, Urbana-Champaign, IL 61801
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, a cytokine associated with
protection against ehrlichia and needed for rapid generation of
variant-specific IgG2. The presented data support the potential
importance of a strong Th lymphocyte response to invariant MSP2
epitopes in controlling rickettsemia during persistent infection to
subclinical levels. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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)
(7). Furthermore, comparison of the amino acid sequence of
A. marginale MSP2 with that of E. phagocytophila
MSP2 in these conserved regions revealed 96% and 87% similarity in
the respective N and C termini (7). This striking
structural and sequence conservation in MSP2s from organisms with
different animal host and cell tropisms suggests that these proteins
share similar functions that are important for survival.
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Acquired immunity to ehrlichial pathogens includes both inflammatory
responses by activated macrophages and neutrophils, and development of
neutralizing Ab (16, 17, 18, 19). The development of
variant-specific IgG2 responses during persistent A.
marginale infection indicates the requirement for T lymphocyte
help, and specifically T lymphocytes expressing IFN- (20, 21). As in other species, IFN- plays a key role in activating
bovine macrophages to produce toxic molecules, such as NO and its
derivatives (22), and also augments production of the
opsonizing IgG2 subclass (21, 23).
A. marginale MSP2 and related proteins induce protective immunity and are targets for vaccine development against ehrlichial pathogens (16, 24, 25). Immunization with affinity purified MSP2 resulted in reduced levels of acute A. marginale rickettsemia following challenge with homologous or heterologous strains, indicating the importance of conserved T and B lymphocyte epitopes in immunity (16, 24). During persistence that results after natural infection or challenge of MSP2 vaccinates with A. marginale, the kinetics of the rickettsemia, where peak levels of organisms are 102–103 less than those observed during acute infection, suggests the development of an accelerated, acquired immune response to the newly emerging antigenic variants. Such a response could result from a strong memory CD4+ T lymphocyte response as a consequence of T lymphocyte priming during the acute infection against epitope(s) present in conserved regions of MSP2. T lymphocytes specific for highly conserved epitopes, which are not subject to antigenic variation, could act as effector cells to promote macrophage activation and as helper cells for primary B cells responding at each new peak of rickettsemia to the antigenically variant surface-exposed regions in MSP2.
We previously identified Th lymphocyte responses directed against MSP2 and a dominant MSP2-specific IgG2 response in cattle immunized with A. marginale OMP and protected against challenge infection (20, 26). PBMC and CD4+ T lymphocyte clones responded to native MSP2 protein affinity purified from the immunizing Florida (FL) strain and to several different strains of A. marginale, consistent with conservation of the epitopes on MSP2 recognized by these T lymphocytes. To test the hypothesis that highly conserved regions of MSP2 contain Th cell epitopes, the current study was undertaken to determine the presence of CD4+ T lymphocyte epitopes on highly conserved N- and/or C-terminal regions of MSP2, and to determine whether these epitopes are recognized by individuals with different MHC class II haplotypes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The A. marginale strains used in this study are designated by their original location of isolation, and include FL, Washington’ Clarkston (WA’C), Virginia (VA), South Idaho (SI), and St. Maries (St. M) Idaho strains. A strain of A. ovis isolated in Idaho was also used. These have been described or referenced previously (20, 27, 28). All Anaplasma strains were maintained as liquid nitrogen-cryopreserved stabilates of infected bovine erythrocytes in DMSO-PBS. Anaplasma organisms were isolated from thawed, infected bovine erythrocytes by sonication and differential ultracentrifugation as previously described (29). To prepare Ag for in vitro assays, the organisms were resuspended in PBS containing the protease inhibitors antipain and E-64 (Boehringer Mannheim, Indianapolis, IN) at 25 µg/ml and PMSF (Sigma, St. Louis, MO) at 300 µg/ml, and were homogenized by two passages though a French pressure cell (SLM Instruments, Urbana, IL) at 1500 lb/in (2). Protein concentrations were determined by the Bradford assay (Bio-Rad, Hercules, CA). For the FL strain, either homogenate or membranes prepared from the homogenate by sucrose density gradient centrifugation (20) were used. MSP2 was affinity purified from A. marginale FL strain organisms and stored at -80°C as described (30). Thirty amino acid peptides spanning the highly conserved N and C domains of MSP2 were synthesized by Gerhardt Munske (Laboratory for Biotechnology and Bioanalysis I, Washington State University, Pullman, WA). Peptides were dissolved in PBS and stored at -20°C.
Cattle used in this study
Holstein steers 59, 60, and 61 were immunized six times with MSP2 using IL-12 and alum as an adjuvant (30). Age-matched Holstein steers 56 and 98 were inoculated with IL-12 and alum (30), and together with steer 63 were used as negative control donors of PBMC or APC. The bovine lymphocyte Ag (BoLA)-A class I alleles of the cattle were defined by serological typing (31) and DRB3 alleles were defined by PCR-RFLP analysis of exon 2 (32). The BoLA-DQ haplotypes were inferred from BoLA-A and DRB3 typing on the basis of haplotypes defined in the Seventh International BoLA Workshop (Ref. 33 ; BoLA nomenclature web site: http://www2.ri.bbsrc.ac.uk/bola/). The class II haplotypes for 59 are as follows: DRB3 22/7, DQA 9B/2, DQB 9B/2; for 60 are as follows: DRB3 22/23, DQA 9B/7D, DQB 9B/7A; for 61 are as follows: DRB3 8/8, DQA 12/12, DQB 12/12; for 63 are as follows: DRB3 11/8, DQA 3A/12, DQB 3A/12; and for 56 are as follows: DRB3 22/7, DQA 9B/2, DQB 9B/2.
MSP2 sequences
Ten MSP2 cDNA clones were isolated from the A. marginale FL blood stabilate from which MSP2 was purified for use in these studies as described (7). Similarly, seven MSP2 cDNA clones were obtained and sequenced from A. ovis (34). GenBank accession numbers for MSP2 are as follows: genomic DNA of the FL strain pCKR11.2 clone (UO7862; Ref. 4), cDNAs of the St. M (AF290590-599) or SI (AF107766-67) strains of A. marginale, and cDNAs from A. ovis (AF299052-58).
A. marginale-specific T lymphocyte lines and clones
Short-term T lymphocyte lines were established from PBMC of
A. marginale MSP2-immunized cattle 59, 60, and 61 1 mo
following the fifth immunization or more than 1 year following the last
immunization with MSP2. Additionally, cell lines were established from
cryopreserved, prescapular lymph node cells (LNC) removed 3 days after
the last immunization (30). In all experiments, cell lines
were propagated by stimulation with the FL strain of A.
marginale. Briefly, 4 x 106 PBMC or
LNC were cultured per well in 24-well plates (Costar, Cambridge, MA) in
a volume of 1.5 ml complete RPMI 1640 medium with 1–3 µg/ml A.
marginale homogenate. PBMC-derived cell lines were also propagated
using 5 µg/ml native MSP2 as Ag. After 7 days and weekly thereafter,
cells were subcultured to a density of 5 x
105 cells/well and cultured with 2 x
106 irradiated (3000 rad) autologous PBMC as a
source of APC with or without Ag, which was often given on alternate
weeks to lower background proliferation. T lymphocyte lines were
maintained for up to 5 wk, and cells were assayed for Ag-dependent
proliferation 7 days following the last stimulation. In some
experiments, 
T lymphocytes were depleted by incubating the cell
lines for 30 min at 4°C with mAb CACT 61A diluted to 15 µg/ml in
PBS, washing once, incubating for 30 min at 37°C with rabbit
complement (Sigma) diluted 1:8 in PBS, and washing three times.
T lymphocyte clones were obtained from cell lines by limiting dilution. In experiment 1, T lymphocyte lines were established from PBMC of high responder cattle 60 and 61 after five immunizations with MSP2 by using 10 µg/ml A. marginale. The lines were restimulated one time per week for 2 weeks. Cells were cloned by plating 1 or 0.3 cells per well (cpw) in the presence of APC, 5 µg/ml A. marginale, and 10% bovine T cell growth factor. Frequencies of positive wells were 9.4–10.4% (0.3 cpw) and 11.5–12.5% (1 cpw). In experiment 2, T lymphocyte lines were established from PBMC of these cattle 1 year after immunization by stimulating cells for 2 wk with A. marginale and then for 2 wk with MSP2 before cloning. Clones were stimulated with 1 µg/ml A. marginale and T cell growth factor. Frequencies of positive wells for animal 60 were 3.1% (0.3 cpw) and 15.6% (1 cpw), and for animal 61 were 18.8% (0.3 cpw).
Cell surface phenotypic analysis
Differentiation markers on T lymphocyte lines and clones were
analyzed by FACS (26). The mAb used were specific for
bovine CD2 (mAb MUC2A), CD3 (mAb MM1A), CD4 (mAb CACT 138A), CD8 (mAbs
CACT 80C and BAT 82B), and the -chain of the TCR (mAb CACT
61A), purchased from the Washington State University Monoclonal Ab
Center.
Lymphocyte proliferation assays
Proliferation assays were conducted in replicate wells of
round-bottom 96-well plates (Costar) for 5–6 days when using PBMC, or
for 3–4 days when using short-term T lymphocyte lines or T lymphocyte
clones, as described (20, 26). PBMC (2 x
105) were cultured in triplicate wells with Ag in
a total volume of 100 µl complete RPMI 1640 medium. T lymphocyte
lines and clones (3 x 104 cells) were
cultured in duplicate or triplicate wells in a total volume of 100 µl
complete medium containing 2 x 105 APC and
Ag. Ags consisted of 0.2–25.0 µg/ml of homogenate prepared from
different strains of A. marginale or A. ovis,
native MSP2 protein, and 0.1–10 µg/ml overlapping 30-mer peptides
spanning the conserved regions of MSP2. Membranes prepared from
uninfected RBC (URBC) and recombinant GST were used as a negative
control Ags. Cells were radiolabeled for the last 18 h of culture
with 0.25 µCi [3H]thymidine, harvested using
an automated cell harvester (Tomtec, Orange, CT) and counted with a
liquid scintillation counter. In one experiment with T lymphocyte
clones, mAb to bovine MHC class II molecules (35, 36)
DR
(mAb TH14B) or DQ (mAb TH22A) were used to block presentation
of A. marginale or peptide. These IgG2a mAb and an isotype
control mAb (AV213) were obtained from the Washington State University
Monoclonal Ab Center, and purified by affinity to protein G using the
Equilibrate Hi Trap Protein G Column (Pharmacia Biotech, Piscataway,
NJ) according to the manufacturer’s protocol. Before adding T
lymphocytes and Ag, APC were incubated for 1 h with 20 µg/ml
anti-class II mAb or the isotype control AV213 mAb. Higher
concentrations of mAb were inhibitory. Results are presented as the
mean cpm of replicate cultures ± one SD, or as the stimulation
index, which represents the mean cpm of replicate cultures of cells
plus Ag/the mean cpm of replicate cultures of cells plus medium or
URBC. A stimulation index of 
3.0 was considered statistically
significant. The Student’s t test was used to determine
statistically significant differences in proliferation induced by
different APC.
Detection of IFN- and NO2- in
supernatants of T lymphocyte lines
Just before the addition of
[3H]thymidine to proliferation assay wells,
supernatants (50 µl/well) were harvested from triplicate wells of T
lymphocyte lines stimulated with 10 µg/ml A. marginale Ag
or MSP2-derived peptides. Pooled supernatants were stored at -20°C.
The bovine IFN- assay was performed using an ELISA kit (Bovigam;
CSL, Parkville, Victoria, Australia) according to the manufacturer’s
protocol. The IFN- activity in culture supernatants diluted
1:4–1:3000 was determined by comparison with a standard curve obtained
with a supernatant from a Mycobacterium bovis PPD-specific
Th lymphocyte clone that contained 440 U IFN- per ml (previously
determined by the neutralization of vesicular stomatitis virus; Refs.
21 and 26). In our assay, 0.6 U corresponds
to 1 ng IFN- (37). The results are presented as U/ml
IFN-. NO2- levels were
determined by the Griess assay (38).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To investigate the presence of Th lymphocyte epitopes in the
highly conserved N- and C-terminal regions of MSP2, T lymphocyte
responses of MSP2-immunized cattle against the immunizing FL and
different geographical strains of A. marginale were
compared. Cell lines established from MSP2-immune LNC of animal 61
proliferated similarly to the FL, WA’C, and VA strains and to A.
ovis (Fig. 2A). Similar
results were obtained from cattle 59 and 60, and all lines were tested
at three time points with similar results (data not shown). These cell
lines consisted of 94–99% CD4+ T lymphocytes,
but only 1–4% CD8+ or T lymphocytes,
showing that the CD4+ lymphocytes are the
responder population. This recall response to conserved T lymphocyte
epitopes was still present 1 year following the last immunization.
A. marginale-propagated T lymphocyte lines derived from PBMC
of cattle 60 (Fig. 2B), 59, and 61 (data not shown)
responded to the immunizing native MSP2 protein and to A.
marginale when tested at 1–3 wk after in vitro stimulation.
Furthermore, CD4+ T cell lines obtained 1 year or
more after immunization responded to different A. marginale
strains and A. ovis (data not shown). Control Ags consisting
of membranes from URBC (Fig. 2A) or recombinant GST (Fig. 2B) were not stimulatory. PBMC-derived cell lines contained
a mixture of CD4+ T lymphocytes and T
lymphocytes, with few CD8 T lymphocytes. Depletion of T
lymphocytes by lysis with specific mAb and complement did not diminish
the A. marginale-specific response, indicating that the
responder population is CD4+ T lymphocytes (data
not shown).
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The conserved T lymphocyte responses to different strains and
species of Anaplasma in cattle immunized with MSP2 from the
FL strain indicated that T lymphocyte epitopes would most likely be
present in the N and C termini that are highly conserved between
otherwise variant MSP2 proteins. Previous studies defined the variable
and conserved regions of MSP2 by comparing sequences of more than 50
full-length cDNA clones obtained during acute and persistent
rickettsemia of cattle, or from ticks infected with FL, St. M, or SI
strains of A. marginale (Refs. 7 and
14 , and unpublished data). Similar analysis was performed
with MSP2 cDNA clones obtained from E. phagocytophila
(2, 7, 8). In all transcripts, MSP2 has a single, central
hypervariable region corresponding to aa 190–271 (numbering based on
the original genomic msp2 11.2 clone; Ref. 4)
and flanking regions that are highly conserved among cDNA clones of a
strain (Fig. 1
). Comparison of multiple cDNAs from different strains of
A. marginale including the FL stabilate used to prepare
MSP2, SI, and St. M strains revealed that the predicted amino acid
sequences are also nearly identical in these regions (Refs.
7 and 14 , Table I, and data not shown). Analysis of seven
cDNA clones of A. ovis MSP2 showed that the sequences in the
conserved N- and C-terminal regions were very similar to those of
A. marginale MSP2 cDNA (7), with only seven
predicted amino acids in the N terminus and four in the C terminus that
varied in all cDNA clones (Table I).
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Short-term T lymphocyte lines from MSP2-immunized cattle were used
to identify stimulatory peptides spanning the conserved N and C termini
of MSP2. The cell lines were propagated with either A.
marginale or MSP2, and tested after 1–4 wk of culture (Fig. 3 and Table II). Several interesting findings
emerged. First, the cell lines recognized multiple peptides from both N
and C termini of MSP2. Some of these were dominant, eliciting strong
proliferation by PBMC (animal 61, data not shown) or by 1-wk cultured
cell lines, whereas other peptides were subdominant, stimulating weaker
levels of proliferation and only after 2 or more weeks of culture
(39). Second, the patterns of responses were generally
very similar whether MSP2 or A. marginale Ag was used to
propagate the cell lines. Third, responses by the individual animals
that expressed different sets of class II alleles differed in the
pattern of peptides recognized. For example, animal 61 uniquely
responded to peptides 4 and 16. The C-terminal region (amino acids
372–409) spanning peptides 15 and 16, which overlap by 20 aa, contains
at least two T lymphocyte epitopes; one recognized by animals 59 and
60, which respond to peptide 15 but not 16, and one recognized by
animal 61, which responds to peptide 16 but not 15. This pattern is
consistent with the sharing of MHC class II alleles by animals 59 and
60 but not 61. Similarly, animals 59 and 60, but not 61, responded to
peptide 7. Finally, T lymphocytes from all cattle responded strongly to
peptide 12 and, to a lesser extent, peptide 13.
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T cells and tested against all peptides. The
cell lines, which contained 
2% TCR+
cells, proliferated to MSP2 and to subdominant and dominant peptides
with levels and patterns of response similar to those of undepleted
lines (data not shown). Furthermore, T cell-depleted line 61
contained only 6% CD8+ T cells. Together, the
data indicate that CD4+ T lymphocytes are the
primary responder cells in the lines. PBMC from animal 61 responded to peptides 6, 10, 11, 12, 13, and 16 with a stimulation index of >10 (data not shown). This is the same pattern of response observed with cell lines from this animal, showing that the short-term T lymphocyte lines are representative of whole PBMC. PBMC from control cattle 56 and 98, which were inoculated with IL-12 and alum, did not respond to any peptide (data not shown). PBMC from cattle 59 and 60 repeatedly had such high background proliferation that a specific response to A. marginale or peptides was not detectable. The use of short-term T lymphocyte lines solved this latter problem.
APC from cattle either sharing one haplotype (half-matched) or mismatched at MHC class II were used to present Ag to responder 60 and 61 T lymphocyte lines. Half-matched APC were clearly more effective at presenting A. marginale. However, use of mismatched APC resulted in some proliferation of lines 60 and 61, although the levels were significantly lower than those using autologous or half-matched APC (data not shown). Because APC donor cattle 60 and 61 responded to many of the same MSP2 peptides, the epitopes present on these could possibly be presented to the T lymphocyte lines following processing of A. marginale or MSP2. In fact, half-matched and mismatched APC were comparable at presenting peptide 12 to lines 60 and 61 (data not shown).
Response patterns by MSP2-specific CD4+ T lymphocyte clones to A. marginale and A. ovis
Because many of the peptides that stimulated dominant
proliferative responses overlapped by 10 residues, T lymphocyte clones
were obtained to try to further define the epitopes on these peptides.
Clones derived from 60 and 61 cell lines stimulated with A.
marginale or MSP2 were all of the Th lymphocyte phenotype
CD3+, CD4+,
CD8-, TCR- (data
not shown). To identify those clones that recognized highly conserved
determinants, they were screened for proliferation against different
A. marginale strains or A. ovis. Results from two
experiments are summarized in Table III.
All clones responded to multiple strains of A. marginale,
whereas only 19 of 33 (58%) of all clones responded to A.
ovis. Examination of the cytokine transcripts expressed upon
A. marginale stimulation of clones from experiment 1 showed
that all clones expressed high levels of IFN- and undetectable or
low levels of IL-2 and IL-4 mRNA (data not shown), consistent with a
Th1-type response observed previously in Ag-stimulated LNC from these
cattle immunized with MSP2 adsorbed to alum and IL-12
(30).
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. Consistent
with a Th1 response, A. marginale Ag elicited both
proliferation and IFN- production by these Th cells, although there
is variation among clones. All but one clone (2.61.4B9) was specific
for MSP2. A. ovis Ag was included because the N- and
C-terminal regions of MSP2 are highly conserved, but not identical with
A. marginale MSP2 (Table I). In this experiment, none of the
Th lymphocyte clones from animal 60 responded to A. ovis,
whereas most of the clones from animal 61 did (Table IV). Together,
these results suggested that, of the MSP2-specific Th lymphocyte
clones, the majority recognized epitope(s) highly conserved among
strains and species of Anaplasma, consistent with the
presence of epitopes in the N or C termini of the protein. However, the
finding that many clones responded to MSP2 but not to A.
ovis suggested that the T lymphocyte epitope(s) recognized by
these are not conserved between Anaplasma species. If
present in the N or C termini, they may include key amino acids that
vary in A. ovis (Table I). Alternatively, the epitopes could
reside in the central variable region of MSP2.
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Thirteen MSP2-specific Th lymphocyte clones were then tested for proliferation against the synthetic 30-mer peptides that spanned each conserved N or C terminus to define their epitopes. Surprisingly, none of the four clones tested from animal 60 recognized any peptide (data not shown). This result, plus the lack of response to A. ovis, suggested that the T lymphocyte epitopes for these clones were within the central variable region of MSP2. Of the ten MSP2-specific clones tested from animal 61, nine responded in a dose-dependent manner only to peptide 8 (data not shown). In addition, one "clone" responded to peptide 8 and additionally to peptides 10 and 11, showing that this was not a monoclonal cell line. The recognition of peptides 8, 10, and 11 by these CD4+ T lymphocytes is consistent with the response of T lymphocyte lines from animal 61, but does not reflect the whole repertoire of peptides that are capable of stimulating strong proliferation. In separate studies, we have obtained A. marginale- and MSP2-responsive CD4+ T cell clones specific for peptides 10 and 11 from cell lines by propagating cell lines with peptide 11 (W.C.B., G.H.P., H.A.L., and T.C.M., unpublished observations).
MHC class II restricted response of peptide 8-specific T lymphocytes
To determine that the response to peptide 8 was MHC class
II-restricted, T lymphocyte clones were stimulated with either peptide
8 or A. marginale in the presence of autologous APC or APC
from cattle that were half-matched or mismatched at MHC class II
alleles. Of four clones tested, all responded to A.
marginale presented by class II half-matched APC from animal 63,
whereas none responded when mismatched APC from animal 60 were used
(Fig. 4). In contrast, some stimulation
occurred when peptide 8 was presented by mismatched APC, although this
was less than that with autologous or class II half-matched APC. This
may be due to the binding of the peptide to class II expressed on
mismatched APC or on the responding bovine T cells. Stimulation of
bovine Th lymphocyte clones specific for Theileria parva was
also observed when fixed, T. parva-transformed T lymphocytes
were used as Ag in the absence of exogenous APC (40).
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or DR were used to block stimulation by
peptide 8 or A. marginale (Fig. 5). The results showed that the response
to peptide 8 is restricted by DQ, and not by DR molecules. Similar
results were obtained using A. marginale as Ag (data not
shown). In other studies using these mAb and T lymphocyte clones
specific for A. marginale MSP1a, we have identified both DQ-
and DR-restricted responses, showing that both Abs are capable of
blocking Ag presentation (our unpublished observations). Although DQ
molecules are not commonly used for presenting Ag to human T cells,
Glass et al. (41) recently described DQ- and DR-restricted
responses in cattle to a viral peptide Ag used for vaccination.
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In an attempt to explain the biased response of T lymphocyte
clones from animal 61 to the subdominant peptide 8, the responses by
one cell line to peptide 8 and dominant peptides 12 and 16 were
compared over time (Fig. 6). The line was
tested sequentially for 4 wk, because the peptide 8-specific T
lymphocyte clones were derived from a cell line cultured for 4 wk
before cloning. Relative to dominant peptides 12 and 16, which elicited
strong recall responses by PBMC and by cell lines at all time points,
the proliferative response to peptide 8 was undetectable by PBMC but
increased over time. In contrast, the response to peptide 12
fluctuated, and that to peptide 16 diminished from-wk 1 onward, so that
by 4 wk of culture, proliferation to all three peptides was comparable.
Thus, a relatively high proportion of T lymphocytes specific for
peptide 8 was likely present in this line at the time of cloning. This
explains why peptide 8-specific Th clones were obtained, but does not
explain the lack of Th clones specific for dominant peptides, such as
12 and 16.
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and NO production by immunodominant peptides
T lymphocyte proliferation can be down-regulated by high levels of
IFN- and NO produced during the immune response (42),
suggesting the inability to clone T lymphocytes specific for dominant
peptides was caused by production of high levels of these inhibitory
molecules. Furthermore, IFN- is associated with protective immunity
against A. marginale (20, 26) and the related
HGE agent (18). NO, produced by phagocytes in response to
IFN-, appears to be important for inhibition of E.
risticii and E. phagocytophila in a murine model
(43, 44). For these reasons, it was of interest to
determine the ability of different peptides to induce IFN- and NO. T
lymphocyte lines from the MSP2-immunized cattle were propagated with
A. marginale, and proliferative and IFN- responses were
determined in the same assay cultures of T lymphocytes and APC
stimulated with individual peptides previously identified as
immunostimulatory for a given cell line (Fig. 7). Immunodominant peptides 7 (line 59),
12 (all lines) and 10, 11, and 16 (line 61) induced very high amounts
of IFN-, which for peptide 12 was greater than the amount induced by
A. marginale. However, production of IFN- correlated
positively with proliferation to a given peptide, with
r2 values of 0.77 (line 59), 0.64 (line 60), and
0.85 (line 61). In contrast, nitrite levels were similar in the
supernatants of T lymphocyte lines cultured with APC and either no Ag
or peptide, and did not correlate with proliferation or IFN-
production (data not shown). Together, these results indicate that
proliferation is not adversely affected by the level of IFN- or NO
produced. Thus, it is unlikely that the predominant expansion of
peptide 8-specific T lymphocyte clones from animal 61 was simply the
result of production of growth-inhibitory levels of IFN- by T
lymphocyte clones specific for the more immunodominant peptides.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in response to defined
peptides, indicating that these epitopes are derived during natural Ag
processing and presentation. Cell lines depleted of T
lymphocytes and containing few CD8+ T cells also
respond to multiple peptides, showing that the response is mediated by
memory Th cells. Importantly, many immunodominant peptides reside in
the highly conserved N and C termini of MSP2 flanking the central
variable region that is responsible for variant-specific Ab responses
(7).
Control of acute infection following vaccination with outer membranes
is associated with Ab and CD4+ T lymphocyte
responses directed against MSP2 (20, 26, 45), and
immunization with MSP2 affords protection against acute rickettsemia
(24). Together, these studies indicate the importance of
MSP2 in protective immunity. However, despite a competent immune
response that controls the initial acute infection, A.
marginale maintains a state of persistence, characterized by
sequential cycles of rickettsemia that occur every 6–8 wk throughout
persistent infection and absence of clinical disease. We previously
demonstrated that B cell epitopes in MSP2 variants that arose in each
cycle of rickettsemia were unrecognized by Ab at the time of variant
emergence, but that termination of the rickettsemic cycle occurred
concomitantly with a primary Ab response to the central variable region
of MSP2 (7). However, the rickettsemic cycles associated
with new variant emergence peak at 107
organisms/ml of blood, levels far lower than those observed in acute
infection. Our new data support the hypothesis that a rapid and strong
anamnestic Th lymphocyte response to conserved MSP2 epitopes
contributes to the control of rickettsemia during persistent infection
by promoting accelerated MSP2-specific Ab responses. Further, the
presence of numerous immunodominant and conserved Th lymphocyte
epitopes on MSP2 could explain why Ab responses to this protein are
immunodominant in cattle and humans infected with A.
marginale and E. phagocytophila,
respectively.
Analysis of peptide-specific responses by T lymphocyte lines from three cattle demonstrated individual animal differences. Lines from animal 59, compared with animals 60 and 61, responded after 1 wk of culture to only two peptides (peptides 7 and 12), and overall consistently recognized fewer peptides in total (peptides 7, 8, 9, 12, 13, and 15). This more limited repertoire could, in part, explain the relatively weaker T lymphocyte response of this animal as previously described (30). In contrast, T lymphocyte lines from animals 60 and 61 responded to a larger number of peptides. Lymphocytes from animal 60 recognized four peptides (peptides 7, 10, 12, and 15) after 1 wk of culture, and nine peptides in total (peptides 5, 6, 7, and 10–15). Similarly, PBMC or lymphocytes cultured for 1 wk from animal 61 recognized seven peptides (peptides 4, 6, 10–13, and 16), and additionally responded to peptide 8 after longer culture, seeing eight peptides in total. The exact number and location of the Th lymphocyte epitopes in the conserved regions of MSP2 are not known, because many of the stimulatory 28- to 30-aa peptides overlap by 10 aa. Nevertheless, considering potential overlapping regions of the immunostimulatory peptides, animal 59 recognized a minimum of four, and animals 60 and 61 recognized a minimum of seven dominant or subdominant epitopes. This is a relatively large number, when compared with the limited number of one or two epitopes recognized by mice immunized against a variety of foreign proteins in adjuvant (39). In both cases, memory/effector cells were restimulated in vitro before screening with peptides. However, the larger antigenic repertoire for MSP2-specific T lymphocytes may result from having immunized the animals multiple times, compared with the single immunization given to mice. In mice, the recognition patterns remained stable for up to 6 mo postimmunization (46), indicating that the memory cell responses detected 1 year later in our cattle are representative of the pool of effector/memory cells induced after priming and boosting.
Interestingly, T lymphocytes from all cattle responded to peptides 12
and 13. This sequence, consisting of amino acids 312–361 in the
C-terminal region of A. marginale MSP2 is highly conserved
in the orthologous MSP2 protein of E. phagocytophila
(HGE-44), where 40 of 50 aa representing residues 341–390 are
identical (8). In comparison, only 16 of 38 aa are shared
between the corresponding regions of peptides 15–16. Although Th
lymphocyte responses against E. phagocytophila MSP2 have not
been reported, our results target regions in this protein to test as
potential epitopes. In light of the high levels of IFN- induced by
peptide 12, and its sequence conservation, this MSP2 region may be
important for inducing T lymphocyte-dependent protection against both
A. marginale and E. phagocytophila (16, 18). Experiments are in progress to more precisely define the
immunodominant T lymphocyte epitopes on A. marginale MSP2
and to determine which sequences may serve as "universal" epitopes
for bovine class II molecules. This information is important for
vaccine strategies using MSP2 in cattle.
Comparison of the MSP2 peptide-specific responses by short-term T
lymphocyte lines and clones demonstrated that lines were clearly more
informative, because a restricted response of the clones was observed.
For animal 61, this was directed predominantly against one of the
subdominant peptides, peptide 8. The reasons for the selective response
by the T cell clones are not known. One possibility is that over the
duration of the culture period (6 wk to screening clones), T
lymphocytes recognizing peptide 8 expanded preferentially, whereas
those that recognized epitopes stimulating a more dominant response did
not continue to expand, perhaps due to Ag-induced apoptosis
(47). Indeed, the response by lines to peptide 8 continued
to increase over time in culture, whereas the response to dominant
peptide 16 declined. The high levels of proliferation and IFN-
produced in response to the more dominant peptides are indicative of an
activated phenotype. However, neither IFN- nor NO appeared to
negatively affect proliferation, at least in the 4-day assays that were
performed.
The lack of response of peptide 8-specific T lymphocytes to peptides 7 and 9, which overlap peptide 8 by 10 residues at each end, indicated that the core T lymphocyte epitope contains the central residues TASVFLLGKE. In the 10 aa region overlapping peptide 7 at the N end of peptide 8 is a proteolytic substrate site consisting of a dibasic amino acid (KK) motif (39). These motifs play a role in determining the availability of antigenic epitopes following processing of the protein (48). Studies with a rhoptry-associated protein of Babesia bigemina identified a 29-aa peptide that contained an immunodominant bovine Th cell epitope, which also had a KK motif (49). Additional studies will be needed to define the T lymphocyte determinant within peptide 8 and the importance of the KK motif for its processing.
The failure of MSP2-specific Th lymphocyte clones from animal 60 to recognize any conserved peptide is puzzling, because lines tested at the time of cloning (4 wk) were strongly stimulated by multiple peptides. This paradox is not likely explained by T lymphocyte recognition of epitopes in contaminating proteins in the MSP2 immunogen, because immunoblotting of this Ag with mAb specific for other MSPs of A. marginale did not detect any contamination (30). A more likely explanation is the presence of additional epitopes in the central variable region. Within this region spanning aa 190–271, there are blocks of sequence conserved among the different cDNA clones that could encode a T lymphocyte epitope (Ref. 7 , and unpublished observations). Furthermore, these regions are more conserved among cDNA clones from A. marginale (FL strain) than A. ovis (data not shown), which might explain the lack of responsiveness of these clones to A. ovis. In future experiments, recombinant proteins representing the different MSP2 central variable regions identified in the immunizing native MSP2 will be expressed and tested for the presence of T lymphocyte epitopes using the lines and clones described in this study.
In summary, we have demonstrated the presence of multiple, naturally
processed Th lymphocyte epitopes in the N- and C-terminal regions of
MSP2 that are structurally conserved among genogroup II ehrlichia. The
presence of numerous Th lymphocyte epitopes on MSP2 recognized by
cattle with different MHC class II haplotypes could explain the
serological dominance of this protein for humans and animals infected
with genogroup II ehrlichia, or with related members of genogroup I:
E. chaffeensis, E. canis, and Cowdria ruminantium
(50, 51, 52). Furthermore, during persistent infection when
antigenically variant organisms sequentially emerge, a strong
anamnestic Th lymphocyte response directed against invariant epitopes
on MSP2 could provide accelerated B lymphocyte help, leading to rapid
variant-specific Ab production. This response, in combination with
IFN- production, is hypothesized to control rickettsemia to the low
levels observed during persistence.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Wendy C. Brown, Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA 99164.
3 Abbreviations used in this paper: MSP2, major surface protein 2; OMP, outer membrane protein; HGE, human granulocytic ehrlichiosis; LNC, lymph node cells; FL, Florida; WA’C, Washington’ Clarkston; St. M, St. Maries; VA, Virginia; SI, South Idaho; URBC, uninfected RBC; BoLA, bovine lymphocyte Ag; TCGF, T cell growth factor; cpw, cells per well.
Received for publication August 21, 2000. Accepted for publication October 25, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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J. Norimine, C. E. Suarez, T. F. McElwain, M. Florin-Christensen, and W. C. Brown Immunodominant Epitopes in Babesia bovis Rhoptry-Associated Protein 1 That Elicit Memory CD4+-T-Lymphocyte Responses in B. bovis-Immune Individuals Are Located in the Amino-Terminal Domain Infect. Immun., April 1, 2002; 70(4): 2039 - 2048. [Abstract] [Full Text] [PDF]
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V. Shkap, T. Molad, K. A. Brayton, W. C. Brown, and G. H. Palmer Expression of Major Surface Protein 2 Variants with Conserved T-Cell Epitopes in Anaplasma centrale Vaccinates Infect. Immun., February 1, 2002; 70(2): 642 - 648. [Abstract] [Full Text] [PDF]
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R. R. Ganta, M. J. Wilkerson, C. Cheng, A. M. Rokey, and S. K. Chapes Persistent Ehrlichia chaffeensis Infection Occurs in the Absence of Functional Major Histocompatibility Complex Class II Genes Infect. Immun., January 1, 2002; 70(1): 380 - 388. [Abstract] [Full Text] [PDF]
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W. C. Brown, G. H. Palmer, H. A. Lewin, and T. C. McGuire CD4+ T Lymphocytes from Calves Immunized with Anaplasma marginale Major Surface Protein 1 (MSP1), a Heteromeric Complex of MSP1a and MSP1b, Preferentially Recognize the MSP1a Carboxyl Terminus That Is Conserved among Strains Infect. Immun., November 1, 2001; 69(11): 6853 - 6862. [Abstract] [Full Text] [PDF]
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J. de la Fuente and K. M. Kocan Expression of Anaplasma marginale Major Surface Protein 2 Variants in Persistently Infected Ticks Infect. Immun., August 1, 2001; 69(8): 5151 - 5156. [Abstract] [Full Text] [PDF]
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) or
IFN-
). Stimulation index was determined as
proliferation (mean cpm) to 10 µg/ml of A. marginale
or the indicated peptides compared with mean cpm of cells cultured with
medium alone, which were 13,471 cpm for 59 T cells, 550 cpm for 60 T
cells, and 1,353 cpm for 61 T cells. A stimulation index of >3.0 is
considered positive. IFN-