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The Edward Jenner Institute for Vaccine Research, Compton, Newbury, Berkshire, United Kingdom
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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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Use of animal models of autoimmune disease has greatly aided studies of
induction and maintenance of T cell tolerance. One of the most
extensively analyzed of these models is experimental allergic
encephalomyelitis
(EAE).4 EAE is a T
cell-mediated disease, inducible in susceptible animals by antigenic
challenge with proteins of myelin or synthetic peptides representing
the appropriate T cell epitopes (1, 2, 3, 4). Myelin basic
protein (MBP), one of the major target Ags in EAE, has also been
implicated in the pathogenesis of multiple sclerosis. It is a
relatively abundant protein, comprising 
30% of myelin protein
within the CNS. Expression of a neonatal form in the thymus
(5) does not lead to the deletion of MBP-reactive T cells,
as responses are normally found both in animals and in humans
(6, 7, 8).
MBP itself is an intracellular protein expressed predominantly in oligodendrocytes, the cells that form myelin sheaths within the CNS (9). These cells neither express MHC class II nor can they be induced to present Ags to MHC class II-restricted T cells (10). In addition, the blood-brain barrier (11) may greatly reduce the contact between naive but potentially MBP-reactive T cells and CNS myelin in healthy individuals. Thus, tolerance could be maintained because the T cell pool remains in ignorance of the potentially damaging autoantigen. However, should these T cells become activated by some external means, the potential for initiating an autoaggressive T cell response remains, as the activated T cells are more capable of crossing the blood-brain barrier and recognizing MBP or other myelin proteins (12, 13).
In this study, we initially set out to render mice resistant to EAE by
the induction of deletion of T cells reactive to an encephalitogenic
region of MBP. Residues 84–105 of MBP, encephalitogenic in the SJL
mouse, are expressed as a transgene in the form of a fusion protein
with hen egg lysozyme (HEL), under the control of the MHC class II E
promoter (14, 15). In fact, we found only subtle effects
on the T cell response to MBP in transgenic (TG) animals, compared with
their non-TG littermates. However, expression of the transgene rendered
the mice highly resistant to the development of EAE. Thus, it appears
that a minimal reduction in the MBP response of the TG mice was
sufficient to produce a highly significant reduction in the
susceptibility of the mice to MBP-induced EAE.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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TG mice that coexpressed residues 84–105 of mouse MBP with MHC
class II have been previously described (14). This region
of MBP contains the major encephalitogenic epitopes in SJL mice
(3, 16) and is expressed as a fusion protein using HEL as
a "carrier," with the MBP peptide embedded in one of two loop
regions of HEL (17). Briefly, TG mice expressing either
mLM43 (EH43.69) or sLM43 (E43.30) were made as follows. A
PvuI/BamHI digest of the pKCR7 plasmid with the
LM fusion proteins was excised and replaced with the
PvuI/BamHI fragment of pDOI15, containing the
mouse MHC class II E promoter (15). The
PvuI/partial-XhoI fragment (as shown in Fig. 1
) was microinjected into fertilized
(C57BL/6 x SJL)F2 oocytes. Potential
founders were identified by screening of tail DNA by standard Southern
blotting techniques using an E promoter probe.
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). EH43.69 contains three copies of the
mLM43 transgene construct, shown in Fig. 1, and E43.30 contains a
single copy of the sLM43 transgene construct (without the HA tm/cyto).
S1-nuclease protection and RT-PCR could detect expression of this
transgene. However, cell-surface expression could not be detected by
conventional Ab staining and FACS analysis (data not shown). Lines were continually backcrossed with SJL/J mice (The Jackson Laboratory, Bar Harbor, ME) for the first seven or eight generations and subsequently, for the EH43.69 line only, SJL obtained from Harlan (Olac, U.K.) Initial breeding was in a conventional mouse facility, but lines were rederived into an specific pathogen-free facility for the sixth and subsequent generations. The E43.30 line is not in continuous breeding.
Peptides
HEL112–129 (RNRCKGTDVQAWIRGCRL) was synthesized by Neosystems (Strasbourg, France). Proteolipid protein (PLP) 139–151 (HSLGKWLGHPDKF) was synthesized by Research Genetics. MBP84–104 (VHFFKNIVTPRTPPPSQGKGR), MBP84–96 (VHFFKNIVTPRTP), MBP84–95 (VHFFKNIVTPRT), and MBP87–105 (FKNIVTPRTPPPSQGKGRG) had all been synthesized at Laboratorie de Génétique Moleculaire des Eucaryotes (Strasbourg, France) and were >80% pure.
Proliferation assays
Mice were primed with the appropriate peptide or Ag dissolved in PBS, mixed in an emulsion of CFA together with H37-RA (Difco, Detroit, MI), at a final concentration of 4 mg/ml. Mice were primed in two sites s.c., on the dorsum of the foot and the tail base. After 10 days, (HEL assays), or 11 days (MBP assays), the popliteal lymph node (LN) on the side of injection and inguinal LN were removed under aseptic conditions. LN from individual mice were pooled, disrupted to a single-cell suspension, and resuspended in RPMI 1640 medium (Life Technologies, Grand Island, NY) supplemented with glutamine, penicillin and streptomycin, sodium pyruvate, 5 x 10-5 M 2-ME, and 1% normal mouse serum. Cells were plated out at 5 x 105 cells per well in flat-bottom 96-well plates in quadruplicate, with the appropriate dilutions of Ag. Assays were incubated for a total of 96 h at 37°C 5% CO2 with the addition of 1 µCi [3H]TdR for the final 18–20 h of culture, and proliferation was measured. Results are expressed as stimulation index, being the mean of experimental cpm for quadruplicate wells divided by the mean cpm in the absence of Ag. Background counts varied but were normally <15,000 cpm.
For IFN-
estimation, 48 h supernatants from proliferation
assays were tested by standard ELISA using the Quantikine M Mouse
IFN- Immunoassay (R&D Systems, catalogue no. M1F00). Dilutions of
supernatant were prepared at 1:2 and 1:20 in assay diluent.
Concentration of IFN- was calculated using a standard curve. IL-4
and IL-10 concentrations were established in the same manner.
T hybridomas
The HEL-specific I-As-restricted T cell
hybridoma SHEL9 was derived from a fusion of an HEL-specific T cell
line to the mouse BW58
-ß- fusion partner.
The cell line was originally grown from draining LN of SJL mice primed
with HEL (Sigma, St. Louis, MO) and restimulated in vitro with HEL.
This hybridoma recognizes the C-terminal peptide of HEL, residues
112–129, as well as native HEL.
The panel of MBP-specific T cell hybridomas was derived from two female
EH43.69 TG mice and two of their non-TG littermates. The mice were
primed with 50 µg MBP84–104 peptide (HPLC
purified >95% pure) as for a proliferation assay (14).
On day 11, draining LN were removed and a conventional proliferation
assay set up with a proportion of the cells (Fig. 4A). The
remainder of the cells were depleted of IgM-positive cells and adherent
cells by panning. Cells for fusion were restimulated in a 24-well
tissue culture plate with 2 x 106 responder
cells and 3 x 106 mitomycin c-treated SJL
spleen cells per well, in the presence of 100 µg/ml bovine MBP (BMBP)
(Sigma). Cells were fused to the BW58
-ß- fusion partner
at day 6 following addition of IL-2 to the cultures at day 3 and plated
out in limiting dilution. On an initial screen, 146 of 169, 77 from the
TG mice and 69 from the non-TG mice, recognized whole MBP presented on
I-As-transfected L cells. Of these, 63 were
stable enough to continue growing for further analysis. Vß usage was
determined by standard staining techniques using Vß-specific Abs. All
of these hybridomas were reactive to both MBP and to the longest
peptide MBP84–104 and restricted through
I-As. Statistics were performed using Arcus
Pro-II.
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T cell hybridomas were plated out in duplicate in 96-well
flat-bottom plates at 2–3 x 104 cells/well
together with stimulator cells and Ag where appropriate. Stimulator
cells were either various L cell transfectants at 2–3 x
104 cells per well, or spleen cells at 3 x
105 cells per well, or at the cell numbers shown
for the assay (Fig. 2). After 20–24 h
incubation, 50 µl supernatant was assayed for IL-2 production in a
standard assay: supernatant was transferred to a round-bottom 96-well
plate together with 1–2 x 104 CTLL-2 cells
and incubated overnight then pulsed with 1 µCi
[3H]TdR per well for the final 4–6 h of
incubation. Results are expressed as cpm proliferation of CTLL-2
cells.
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Mice for EAE induction had been backcrossed at least nine times to SJL. Mice were between 8–12 wk of age. On day one, mice were challenged with MBP84–104 peptide in CFA, 100 µg/mouse, or PLP131–155 (as for a proliferation assay). Then, 24 and 72 h after challenge, mice were given an i.v. injection of pertussis toxin (catalogue no. 180, List Biological Laboratories, Campbell, CA) 0.125 µg/100 µl PBS per injection per mouse. Mice were assessed for general health status, weight, and EAE score from day 9 (day 6 for PLP peptide), every 1–2 days. EAE was scored as follows: 0, normal; 1, flaccid tail; 2, evidence of hind limb paralysis; 3, complete hind limb paralysis; 4, fore and hind limb paralysis. Animals were sacrificed 60 days after induction or earlier if severe symptoms developed. Before the commencement of experiments, tail DNA from experimental animals was stored, untyped. At the termination of the experiment, animals were sacrificed, and a further DNA sample was taken. Both sets of DNA were analyzed for the presence of the transgene by standard Southern blotting techniques, to assign the genetic status of each animal. Statistics were performed using Arcus Pro-II. Where histological sections were required, mice were sacrificed 12 days after induction, and brains and spinal cords were removed and fixed in 4% paraformaldehyde. Paraffin-embedded sections were stained with hematoxylin and eosin and analyzed by light microscopy.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Spleen cells from a number of the TG lines previously described
(14) were able to present MBP or HEL, originating from the
transgene protein, to selected T cell hybridomas without addition of
exogenous Ag. Fig. 2A shows the recognition of the EH43.69
TG spleen cells by one of a subset of MBP-specific
I-As-restricted T cell hybridomas. For
comparison, recognition of spleen cells from another transgenic line,
E43.30, which carries very low copy numbers of the transgene construct
with the secreted form (no HA transmembrane region) of the same
protein, is also shown. Fig. 2B shows recognition of the
secretor line spleen cells only by the
I-As-restricted, HEL-specific T hybridoma SHEL9.
Spleen cells from all the lines were equally efficient at presentation
of OVA to an OVA-specific T cell hybridoma (data not shown).
HEL/MBP TG mice have dramatically reduced responses to HEL
To study the effect of the transgene on development of the T cell
repertoire, mice were challenged with native HEL and the draining LN
proliferative responses assessed. Fig. 3,
A and B shows responses to either native HEL or a
synthetic peptide, HEL residues 112–129, respectively. This peptide
contains a dominant I-As-restricted T cell
epitope. Fig. 3, C–E shows the results of two separate
experiments where animals were primed with the
HEL112–129 peptide. Fig. 3, C and
E shows the in vitro proliferative response to native HEL,
and, as can be seen, there is a profound reduction of the response of
the TG mice when compared with their non-TG littermates. Fig. 3D shows the response to in vitro challenge with the
peptide. In this case, we consistently found an anti-peptide
response, which was not cross-reactive to native HEL. The lack of an
anti-HEL response in the TG mice is not due to an overall defect in
their ability to make immune T cell responses, as purified protein
derivative controls in these assays were equivalent between TG and
non-TG littermates, and TG animals were able to respond at comparable
levels to a third party Ag, OVA (data not shown).
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MBP contains a number of epitopes recognized in SJL mice
(19). Therefore, to ensure that we were looking only at
responses to our chosen region, animals were primed in vivo with
peptide MBP84–104, followed by an in vitro
challenge with native BMBP. Fig. 4,
A–C shows results of three separate experiments comparing
the anti-MBP response of the TG animals with that of non-TG
animals. Although there was a trend to a higher response in the non-TG
mice in some assays, this result was inconsistent. Fig. 4D
shows IFN- release in supernatants of proliferation assays of the
animals depicted in 4C. The levels of IFN- released were
highly variable, but there was no correlation seen with transgene
status. There was virtually no IL-4 and little IL-10 secretion in TG
and non-TG mice (data not shown). Fig. 5A shows the combined results
of stimulation indexes of seven experiments, using a total of 16 TG and
15 non-TG mice, looking at responses to MBP after priming with MBP
peptide. Although there is a trend to a higher response in the non-TG
animals, this does not reach significance for these numbers. In
contrast, looking at the equivalent comparison in the HEL response
following priming with HEL peptide, there is a highly significant
difference between TG and non-TG animals (Fig. 5B).
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Although there was little quantitative difference between the two
sets of mice in response to MBP, we wanted to determine whether there
was a qualitative response in terms of the range of epitopes seen. To
do this, the fine specificity of a panel of T cell hybridomas from two
animals of the TG mouse line EH43.69 was compared with that from two of
their non-TG littermates (14). On an initial screen, 146
of 169, 77 from the TG mice and 69 from the non-TG mice, recognized
whole MBP presented on I-As-transfected L cells
(data not shown). Of these, 65 were stable enough to continue growing
for further analysis. All of these hybridomas were reactive to both MBP
and to the longest peptide MBP84–104 and
restricted through I-As. To assess the
specificity of the hybridomas, each was tested on three different
shorter peptides within the 84–105 region,
MBP87–105, MBP84–96, and
MBP84–95. In addition, hybridomas were tested
for direct recognition of the HEL/MBP fusion protein (mLM43), either as
presented by an L cell transfectant cotransfected with
I-As and mLM43 or by EH43.69 TG spleen cells. A
summary of the results is shown in Table I. The hybridomas are grouped according
to their pattern of reactivity to the various reagents. TCR Vß usage
is also shown and does not necessarily correlate with specificity.
Hybridomas from both TG and non-TG animals were able to recognize each
of the combinations of peptide. Interestingly, roughly equal
proportions of the TG- and non-TG-derived T hybridomas were able to
recognize the transgene protein (mLM43) when presented by transfected L
cells, 15/28 TG and 21/37 non-TG. This is in contrast to the absence of
TG-derived T hybridomas capable of recognizing TG spleen, without the
addition of exogenous Ag. We have previously shown a major effect on
the range of epitopes presented from this region of MBP depending on
whether the APC also expresses the MHC class II-associated invariant
chain (Ii) (14). It is probable that we are seeing the
same type of effect here in comparing recognition of mLM43 from TG
spleen (Ii+) and the L I-As
cells, which are Ii low or negative. The L I-As
cells are unable to present native HEL to an
I-As-restricted HEL-specific T hybridoma (data
not shown).
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and Fig. 2). Interestingly,
there are TG-derived cells capable of recognizing this combination of
peptides, but none of them are able to recognize the L cell
transfectant. This appears to be the only difference in repertoire
between the TG and non-TG animals in recognition of the
MBP84–105 region of MBP. Thus, a similar
repertoire of response was obtained from both TG and non-TG animals.
The only evident difference between them was the lack of T cells
capable of recognizing the endogenously produced MBP epitope from TG
spleen. This was not the major responding population of T cells in the
non-TG mice, although it did result in an excess of non-TG hybridomas
recognizing the shortest peptide, MBP84–95. This
same subset of hybridomas was also able to recognize whole MBP,
expressed as a transgene and presented by spleen cells, whereas the
majority of T cells specific for this region of MBP could not (A.
Kruisbeek, unpublished observation). Susceptibility to EAE
Finally, we wanted to test the susceptibility of TG mice to
induction of EAE with a peptide fragment of the insert of the
transgene. Mice were challenged according to the stated protocol with
MBP84–104 peptide in CFA, followed by two
injections of pertussis toxin, 24 and 72 h after peptide priming.
Table II shows the results of three
experiments of induction in TG and non-TG mice of the EH43.69 line.
Surprisingly, TG animals proved remarkably resistant to the induction
of EAE by this regime when compared with their non-TG littermates. Over
the three experiments, only four of a total of 34 TG mice developed any
signs of EAE, while half of their non-TG littermates were affected (14
of 28). Of the four TG animals that did develop signs of EAE, three had
tail paralysis only and one had quite severe signs with total hind limb
paralysis. In these experiments, animals were monitored for a period of
60 days to look for signs of development of EAE; therefore, no
histology was performed. In another experiment, animals were left for
12 days following EAE induction and then sacrificed for histology. In
the absence of clinical signs, three of six TG and four of six non-TG
mice had minimal histological signs of EAE, with no apparent difference
in distribution between them (data not shown). Thus, although TG
animals are susceptible to EAE and like their non-TG littermates can
develop histological signs in the absence of clinical signs, there is a
highly significant difference in the incidence of clinical EAE in the
TG animals.
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shows that TG mice were equally as susceptible to disease as
their non-TG littermates. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this paper, we have presented a TG mouse model designed to test the
nature of T cell tolerance to an encephalitogenic peptide of MBP. We
have made use of the MHC class II E promoter (15) to
target expression to all MHC class II-expressing cells, some at least
of which should be competent to cause deletion of autoreactive cells.
As the transgene protein is a fusion between
MBP84–105 and HEL, it is possible to compare T
cell responses to the two Ags. Thymic expression of Ags, particularly
in association with MHC class II-positive cells, would normally have
been expected to result in the deletion of potentially self-reactive
developing thymocytes. In our system, we have shown a profound loss of
T cell response to HEL in mice that express the HEL/MBP fusion protein
as a transgene. This finding is in agreement with other studies
demonstrating a high degree of tolerance to the dominant epitopes of
HEL, when expressed as a transgene (25, 26), in comparison
to their non-TG littermates. However, when we looked at the T cell
response to the MBP portion of the transgene, we found very little
effect on the overall response to this region of MBP. That this is
unlikely to be due to the site of expression of the transgene is
evidenced by the effect on HEL responses in these animals.
A further analysis of the MBP-specific T cell responses from the
EH43.69 TG line revealed a remarkably similar pattern of T cell
specificity between the transgenic animals and their non-TG
littermates, with almost the full range of specificities found in the
TG mice (Table I). The principle effect on T cell responses to
MBP84–104 appears limited to the epitope or
epitopes that are efficiently presented on the TG APC themselves,
probably due to processing of the endogenous transgene-encoded protein.
This group of T cells is represented by the T cell hybridoma 3B3 in
Fig. 2A. A T cell hybridoma with a similar specificity, 3A1,
(14), also recognizes whole MBP expressed as a transgene
presented endogenously by spleen cells, while most other
MBP84–104-specific T cells, with differing fine
specificities, do not recognize the endogenously presented molecule (A.
Kruisbeek, unpublished observations). The lack of tolerance to the
transgene protein is not necessarily due to its inability to be
presented in our in vitro assays. Neither a HEL-specific
I-As-restricted T cell line (not shown) nor T
cell hybridomas specific for HEL, such as SHEL9 (Fig. 2B),
are able to recognize the transgene protein presented by splenic APC,
yet the mice are still unresponsive. Other workers have found that, in
general, a lower amount of self-Ag may be required for tolerance
induction than for eliciting a T cell response (26, 27, 28).
Thus, it may be that there is insufficient presentation of the HEL
epitope for detection of T cell recognition in vitro, while there is
sufficient presentation for deletion of potentially reactive thymocytes
during T cell development. However, this does not explain the
persistence of response to the MBP peptide, where recognition of a
single specificity is lost, while responses to other epitopes within
the MBP84–104 region remain relatively intact.
We have previously shown major differences in the epitopes presented
from this region, depending on the presenting cell phenotype
(14), indicating that in this situation the preserved
specificities are not presented in the thymus or peripheral APC. This
is not a function of the fusion protein itself, as recombinant
Escherichia coli-produced protein is recognized by a wide
spectrum of the T cell hybridomas representing all the peptide
specificity groups (14). This raises an interesting
question in relation to susceptibility to EAE. It is thought that MBP
is presented not by the brain oligodendrocytes, which while they
synthesize the MBP do not express MHC class II (29, 30),
but by other cell types within the brain using an exogenous pathway of
presentation.
Surprisingly, given the modest reduction in MBP responses in the EH43.69 TG mice, we found a considerable degree of resistance to the development of EAE. The difference between TG and non-TG animals was highly significant (p = 0.001), in contrast to the nonsignificant reduction on overall T cell proliferative response to MBP following in vivo challenge with the peptide, MBP84–104, also used for EAE induction. In contrast, TG and non-TG mice are equally susceptible to EAE induction with an unrelated myelin-derived peptide, PLP131–155. This demonstrates that there is no intrinsic defect in function induced in the TG mice due to transgene expression.
There are a number of possibilities that could account for the observed
discrepancy between EAE susceptibility and T cell response. The most
obvious is that the development of EAE is dependent on the presence of
T cell response capable of recognition of MBP when presented as an
endogenously synthesized protein. As this T cell response appears to be
missing in the TG animals, it could contribute to their resistance.
However, the resistance of the TG mice was not complete; four mice
developed EAE, including one with a severe disease score of 3. An
alternate explanation could be an alteration in the cytokine profile of
responses in the TG animals. In proliferative T cell responses to MBP,
after priming with MBP84–104 peptide, we could
find no gross differences in the level of IFN- secretion between the
two sets of mice by cytokine ELISA (Fig. 4D) or RT-PCR (data
not shown). In addition, we found little secretion of IL-4 and low
levels of IL-10 in these cultures by either of the two methods (data
not shown). Although this does not exclude the possibility of local in
vivo effects, we were unable to uncover any gross differences between
the TG and non-TG animals. The third possibility is related to the
level of response to MBP. Although there is no significant difference
between the TG and non-TG response to MBP with the numbers of mice
studied, there is nonetheless a trend toward a higher response in the
non-TG animals. In our EAE induction studies, half of the non-TG
animals developed EAE following induction with
MBP84–104 peptide. In a comparison of the TG and
non-TG response to MBP following priming with the same peptide (results
represented in Fig. 5A), there are only three TG animal that
respond to 100 µg/ml MBP with a stimulation index above that of the
median of the non-TG response. Thus, if there was a threshold of
response above which clinical signs of EAE would develop, then there
could be a major difference in the susceptibility to disease due to the
wider range of MBP responses possible in non-TG animals compared with
their TG littermates. In addition, the susceptibility to disease may
also be influenced by the breadth of response to the encephalitogenic
region of MBP. We have demonstrated loss of only one minor epitope
within this region; however, this may again be sufficient to put the
majority of the TG animals below a response threshold for disease and
therefore to protect them from disease induction.
Susceptibility to autoimmune diseases is well known to be dependent not
only on the MHC type of the responding individual, but also on a wide
variety of other non-MHC genes (31). SJL/J and B10.S mice
share the same H-2 haplotype, but there is a big difference in the
susceptibility of the two strains to the development of EAE
(32, 33, 34). In our system, we are looking at the difference
in susceptibility to EAE between mice with the same genetic background,
following at least nine generations backcross to SJL (Table II, Expt.
1) or eleven generations (Expt. 2). The effect of transgene expression
is the deletion of a relatively minor epitope in the encephalitogenic
region of MBP, resulting in a relatively minor effect on the T cell
response to the inducing Ag. Thus, we are able to demonstrate that a
subtle modulation of the immune response to MBP can have a major effect
on the susceptibility to disease induction in the absence of any other
genetic effects. A further implication of this finding is that in
populations susceptible to autoimmune disease, the incidence of disease
may be greatly reduced by only a modest reduction in immune response to
relevant target autoimmune Ags.
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Acknowledgments |
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Footnotes |
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2 Current address: Department of Biophysics, University of Toronto, Ontario Cancer Institute, 610, University Avenue, Toronto, Ontario M5G 2 M9, Canada
3 Address correspondence and reprint requests to Dr. Helen C. Bodmer, The Edward Jenner Institute for Vaccine Research, Compton, Newbury, Berkshire RG20 7NN, U.K.
4 Abbreviations used in this paper: EAE, experimental allergic encephalomyelitis; HEL, hen egg lysozyme; TG, transgenic; MBP, myelin basic protein; BMBP, bovine MBP; HA, haemagglutinin; PLP, proteolipid protein; LN, lymph node; Ii, invariant chain.
Received for publication August 2, 1999. Accepted for publication April 12, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and TNF-. Neurosci. Lett. 123:45.[Medline]
monoclonal antibody treatment on the development of experimental allergic encephalomyelitis in resistant mouse strains. J. Neuroimmunol. 53:101.[Medline]
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. Results are shown
as stimulation index (proliferation with Ag/proliferation in the
absence of Ag) in A and B; in
C, results are expressed as cpm proliferation.
D, Supernatants from the proliferation assay shown in
C were assayed for IFN-
; 10 µg/ml BMBP,
; MBP84–104 peptide 10 µg/ml, 
. Stimulation index
responses to H37RA were >11 in the A and
B and 4.3–9.2 in the C. Three
representative experiments are shown.
) or sLM43 (E43.30, 
), but not
non-TG spleen cells (x). B, An
I-As-restricted HEL-specific T hybridoma is only able to
recognize spleen cells from the E43.30 TG line, and not EH43.69 or
non-TG spleen. A total of 3 x 104 cells of the T
hybridoma, either 3B3 or SHEL9, were added to a titration of the
appropriate fresh spleen cells from in duplicate as shown in 96-well
flat-bottom plates. Results are shown as CTLL-2 proliferation (cpm
x 10-3) on supernatants. Results
representative of at least two separate experiments.
