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Laboratory of Molecular Immunology, Center for Neurologic Diseases, Department of Neurology, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115
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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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GA consists of the four amino acids alanine (A), lysine (K), glutamic acid (E), and tyrosine (Y), at an A:K:E:Y molar ratio of 4.5:3.6:1.5:1 and an approximate length of 40–100 amino acids. Directly labeled GA efficiently binds to a variety of murine H-2 I-E molecules as well as to the human counterpart, HLA class II DR molecules, but not to HLA DQ or HLA class I molecules, in vitro (12). Biochemical studies revealed that GA also binds directly and with high affinity to purified HLA DR1, DR2, and DR4 molecules (13), suggesting that GA contains multiple epitopes, enabling it to bind promiscuously to HLA class II molecules, where it could potentially be recognized by CD4+ T cells. Although the majority of GA molecules were able to interact with DR1, only two-thirds were able to bind to DR2 and DR4 (13). In a follow-up study, partial binding motifs for GA on HLA DR1, DR2, and DR4 molecules were derived by sequencing of the bound fractions (14). A preference for lysine at the N-terminal end of the binding peptide, tyrosine (tyrosine and alanine for DR2) in the first MHC binding pocket (P1) and alanine at the following positions was shown.
Cross-reactivity between GA and MBP has been postulated as one of the possible mechanisms of action to explain the ability of GA to ameliorate disease in MS patients and in animals with EAE. GA-reactive T cell lines stimulated with MBP have been reported to secrete regulatory cytokines (4, 15). However, considering that GA has shown similar efficacy in the treatment of EAE induced by myelin Ags other than MBP, such as proteolipid protein and MOG, and the difficulty a number of laboratories have had in demonstrating cross-reactivity between MBP and GA-reactive T cells, it would appear that another mechanism(s) of action may be possible for the efficacy of GA reported in patients with RR-MS.
A recently emerging concept in the field of T cell biology has been the
exquisite specificity yet at the same time degeneracy of the TCR
(16, 17, 18, 19). This degeneracy has been observed in humans with
the use of T cell clones recognizing the 84–102 immunodominant epitope
of MBP using peptides with multiple substitutions of TCR contact
residues (20, 21). These findings suggest that a random
polymer of amino acids with common MHC and TCR contact residues
containing frequent spacer, nonbulky amino acids might act as a
universal peptide Ag, engaging the TCR in such a manner that the
proliferation of many T cells are induced. In this regard, T cell lines
and clones with reactivity to GA in vitro have been generated from
rats, mice, and other animal species primarily on immunization of the
animals with the Ag (10). Interestingly, in retrospect a
study using PBMCs from healthy control subjects showed a strong
proliferative and IFN-
response to GA (22). It was also
of interest that this response was reported only in humans and not in
rodents. In light of the strong reactivity in non-GA-primed humans and
the lack of responses in other species, without primary immunization,
it was of obvious interest to investigate further the mechanism of GA
induced activation of T cell proliferation in non-GA-primed human and
murine T cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Healthy control volunteers were recruited from our laboratory staff. All adult peripheral blood and neonate cord blood samples were obtained in compliance with the regulations of the Institutional Review Board of the Brigham and Women’s Hospital.
Mice
Female SJL/J (H-2s), C57BL/6 (H-2b), BALB/c (H-2d), B10.S (H-2s), and (SJL/J x PL/J)F1 (H-2sxu) mice (4–6 wk old) were housed under virus-free conditions and generously provided by Drs. Lindsay Nicholson and Ruth Maron (Center for Neurologic Diseases, Harvard Medical School, Boston, MA). Mice were maintained in accordance with the guidelines of the Committee on Animals of Children’s Hospital and Harvard Medical School.
Cell lines
The EBV-transformed DR homozygous B cell lines MGAR (DRB1*1501), JESTHOM (DRB1*0101), and BURK (DRB1* 0301) were kindly provided by Dr. Gerald Nepom (Virginia Mason Research Institute, Seattle, WA), and JECO (DRB1* 0701) was obtained from the Immunogenetics Laboratory of The Johns Hopkins University (Baltimore, MD).
Measurement of GA-mediated primary in vitro human T cell proliferation
PBMCs were isolated from freshly drawn heparinized whole blood of healthy individuals by Ficoll-Paque (Amersham Pharmacia Biotech, Uppsala, Sweden) gradient centrifugation according to the manufacturer’s protocol. Primary in vitro cultures were established by culturing PBMCs at 1.5 x 105 cells/well in RPMI 1640 containing 10 mM HEPES (BioWhittaker, Walkersville, MD), 5% heat-inactivated pooled human AB+ serum (Pel Freeze, Brown Deer, WI), sodium pyruvate, nonessential amino acids, and glutamine (referred to as complete medium) with a variety of concentrations of GA in a final volume of 0.2 ml in 96-well round-bottom microtiter plates (Corning, Corning, NY) and incubated at 37°C in a humidified 8% CO2 incubator. At each time point evaluated, 1 µCi [3H]thymidine, diluted in complete medium, was added to each well for the final 24 h of the culture period. After further incubation at 37°C for 24 h, cells were harvested by using a TOMTEC (Orange, CT) cell harvester, and [3H]thymidine incorporation was determined by using a scintillation counter (LKB 1205 Betaplate; LKB Pharmacia, Wallac, Finland).
Measurement of GA-specific primary in vitro murine spleen cell proliferation
Spleens were removed from a variety of inbred strains of mice expressing different H-2 haplotypes. Single-cell suspensions were prepared in serum-free HL-1 media (BioWhittaker) supplemented with glutamine and 50 mM 2-ME (referred to as serum-free medium). The resulting single-cell preparation was depleted of erythrocytes by hypotonic treatment with a solution of ammonium chloride. Primary in vitro cultures were established by culturing spleen cells at 2.5 x 105 cells/well in serum free medium with a variety of concentrations of glatiramer acetate in a final volume of 0.2 ml in round bottom, 96-well microtiter plates and incubated at 37°C in a humidified 8% CO2 incubator. Approximately 16 h before termination of the culture, 1 µCi [3H]thymidine was added to each well. The wells were harvested, and [3H]thymidine incorporation was determined as described above.
Generation of GA-specific human T cell lines
Human PBMCs were obtained and isolated as described above.
GA-specific human T cell lines were generated by culturing PBMCs at
1.5 x 105 cell/well in complete medium with
40 µg/ml GA in a final volume of 0.2 ml in round bottom, 96-well
microtiter plates. Cells were cultured in a humidified 8%
CO2 incubator at 37°C. On day 5 of culture, 120
µl supernatant were removed from each well and replaced by 140 µl
complete medium containing 10% PHA-free T-stim (Collaborative
Biomedical Products, Bedford, MA). On day 7 of culture 120 µl
supernatant were removed from each well, replaced with 140 µl
complete medium without T-stim, and the cultures were returned to the
incubator for 3 days. On day 10, the contents of each well were
evaluated for GA-specific proliferation in a split well assay. The
split well assay was conducted as previously described
(23). Briefly, the split well assays were established by
incubating equal aliquots of a given T cell line with autologous PBMCs,
-irradiated with 3000 rad, either pulsed with 20 µg/ml GA or no Ag
for 1 h in 96-well round bottom microtiter plates in an 8%
CO2 37°C humidified incubator. When DR
homozygous EBV-transformed B cells were used, the cells were pulsed
with Ag for 3 h at 37°C, 8% CO2. To block
their proliferation, EBV/B cells were treated with 100 µg/ml
mitomycin (Sigma, St. Louis, MO) for the last hour of the incubation.
The cells were then washed three times with complete medium to remove
residual Ag and mitomycin C. After the final wash, EBV/B cells were
resuspended in complete medium and counted, and an aliquot was
transferred to a 96-well round bottom microtiter plate. The split well
plates were incubated for 72 h. Proliferation was determined by
measuring incorporation of [3H]thymidine as
described above.
CD8+ T cell depletion of PBMCs
Freshly isolated PBMCs were depleted of CD8+ T cells by magnetic separation with Dynabeads (Dynal, Oslo, Norway) coupled to CD8-specific Abs according to the manufacturer’s protocol. To confirm successful depletion, the negatively selected cells were stained with directly labeled Abs to CD3, CD8, and CD4, and T cell populations were assessed by flow cytometry.
Limiting dilution analysis of GA-reactive T cells
GA-reactive T cell lines were established as described above,
using 105, 5 x 104,
104, 5 x 103,
103, or 5 x 102
viable PBMCs/well, and adjusted to a total amount of 1.5 x
105 cells/well with -irradiated (3000 rad)
autologous PBMCs. Ten identical wells were established for each cell
concentration. The T cell lines were cultured as described above and
tested for proliferation by standard split well assay, with irradiated
autologous PBMCs, in duplicate on day 9. The percentage of positive
lines was determined using a minimum stimulation index of 3 and >1500
cpm difference between wells with and without Ag. An exponential
regression was performed against the number of cells/well separately
for each individual at each concentration of Ag tested, resulting in
the a and b values for the equation
y = a cbx.
Precursor frequencies were calculated using the zero term of the Poisson distribution at 37% of negative wells and with the above constants a and b (24). The dose dependence of the precursor frequency can be approximated using a power regression model resulting in the equation y = a xb, where b is the slope of the dose response on a double logarithmic plot. b values were similar in normal healthy adults and cord blood samples, revealing a similar dose response pattern in all groups.
Cloning of GA-specific human CD4+ T cells
GA-specific T cell lines were cloned at 0.3 cell/well in 96-well round bottom microtiter plates with 1 x 105 heterologous irradiated (3000 rad) PBMCs in complete medium containing 2 µg/ml PHA-P and 5% T-stim. T cell clones were restimulated with PHA-P and irradiated heterologous PBMCs in complete medium containing 5% T-stim every 2–3 wk. Clonality was confirmed by flow cytometric analysis of TCR Vß usage. The TCR Vß analysis was conducted with a panel of unlabeled Abs against the available variable regions of the TCR ß chain (anti-TCR Vß 2, 3, 5.1, 5.2, 5.3, 6, 8, 9, 11, 12, 13.1, 13.6, 14, 16, 17, 18, 20, 21.3, 22, 23, kindly provided by Immunotech, Marseille, France) and secondary FITC labeled Abs. The staining was done as suggested by the manufacturer’s protocol.
Determination of GA-specific T cell cytokine phenotype by enzyme-linked immunospot (ELISPOT)
Millipore Multiscreen ELISPOT plates (Millipore, Bedford, MA)
were coated with 100 µl of a 5-µg/ml primary anti-IL-5 Ab
(Endogen, Woburn, MA) or 100 µl of a 2-µg/ml primary
anti-IFN- Ab (Endogen) diluted in 0.1 mM sodium carbonate
buffer, pH 8.3, overnight at 4°C. The plates were blotted dry, washed
three times with PBS, blocked with 1% BSA in HBSS for 1 h at
37°C, and washed three times with PBS. APCs and Ag were preincubated
for 1 h at 37°C in the Ab-coated plates. After the 1-h
preincubation, responder T cells were added to the appropriate wells,
and the plates were placed in an 8% CO2, 37°C
humidified incubator for 24 h. The next day the plates were washed
three times with TP buffer (PBS containing 0.05% Tween 20), 100 µl
0.3 µg/ml biotinylated anti-IL-5 Ab (Endogen) or 100 µl of a
0.5 µg/ml biotinyleted anti-IFN- Ab (Endogen) diluted in TP
buffer were added to each well, and the plates were incubated overnight
at 4°C. The next day the plates were washed three times with TP
buffer and 100 µl of a 1/1000 dilution of streptavidin alkaline
phosphatase (Sigma, St. Louis, MO) was added for 2 h at room
temperature. At the end of the incubation period the plates were washed
three times with PBS, and spots were visualized by adding 100 µl
BCIP/NBT substrate (Sigma) for 10–20 min at room temperature. The
reaction was stopped by washing the plate with distilled water. Spots
were enumerated using a Macroviewer MS3 plate reader (Optimax,
Hollis, NH).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous studies have demonstrated that GA-reactive T cell lines
can be generated when PBMCs are isolated from non-GA-primed normal
healthy individuals and incubated with GA in primary 7- to 14-day
primary in vitro cultures followed by subsequent analysis in secondary
in vitro proliferation assays (22). We have extended these
observations by demonstrating that GA induces significant
dose-dependent proliferation of PBMCs incubated in primary in vitro
cultures for 6 days (Fig. 1
). As with the
establishment of T cell lines, PBMCs examined in primary in vitro
cultures were isolated from non-GA-primed normal healthy individuals
and RR-MS patients. We did not detect a significant proliferative
response above background during the first 3 days of culture (data not
shown). Between days 4 and 7 of culture, we observed a
progressively greater dose-dependent proliferative response to GA. On
the basis of studies with 25 randomly selected normal healthy
individuals, we found that the concentration of GA required to
stimulate optimal primary in vitro proliferative responses varied
between 100 and 400 µg/ml (data not shown).
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The results of previous studies conducted by Fridkis-Hareli and Strominger (13) demonstrated GA binding to all alleles of HLA-DR molecules tested but detected no GA binding to any of the alleles of HLA-DQ molecules examined. These results would predict that GA-reactive CD4+ T cells are HLA-DR restricted. Our preliminary findings in an earlier study supported this hypothesis. We found that the GA-mediated proliferation of GA-reactive T cell lines was inhibited by the monoclonal anti-DR Ab LB3.1 but not by the monoclonal anti-DQ Ab IVD12 (23). Both LB3.1 and IVD12 have previously been shown to recognize nonpolymorphic determinants present on DR and DQ molecules, respectively, and to inhibit the proliferation of DR- and DQ-restricted T cell responses.
To more thoroughly address the HLA class II restriction of GA-reactive
T cells, we examined the ability of LB3.1 and IVD12 to inhibit the
GA-mediated proliferation of 80 GA-reactive T cell lines derived from
four unrelated individuals. The results of a representative analysis of
20 T cell lines derived from one individual are shown in Fig. 2. Every GA-reactive T cell line tested
was inhibited by the anti-DR Ab LB3.1 but not by the anti-DQ Ab
IVD12. The activity of IVD12 was confirmed in flow cytomeric binding
studies as well as by the inhibition of control HLA-DQ-restricted T
cell responses (data not shown). These results strongly suggest that
GA-reactive T cells are HLA-DR not -DQ restricted.
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We next examined whether the T cell population(s) stimulated
to proliferate by GA in primary in vitro cultures were restricted by
unique HLA class II DR molecules or whether GA could be presented by
any HLA class II molecule and perhaps function as a "superantigen".
Accordingly, the HLA class II phenotype of six normal healthy
individuals was determined by RFLP mapping (Table I). From two of these individuals,
subject A (DR1/DR2) and subject B (DR3/DR7), a panel of T cell clones
were generated, at 0.3 cell/well, from GA-stimulated primary in vitro
PMBC cultures. From each individual, four GA-reactive T cell clones
were isolated, and by flow cytometry all clones were
CD3+CD4+CD8-TCR
ß+
and CD56- (data not shown). The specific TCR
ß-chain used by six of the eight T cell clones could be determined by
flow cytometry (Table II). Whereas two of
the T cell clones isolated failed to react with any of the commercially
available anti-TCRß-chain Abs, the relative clonality of the
other six T cell populations was inferred by reactivity with only one
of the anti-TCR ß-chain Abs.
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and summarized in Table II
demonstrate
that each T cell clone proliferated and secreted cytokine in the
context of one of the subject’s two DR alleles. In addition, a second
set of T cell clones isolated from subject B were tested and found to
secrete both IL-5 and IFN-, suggesting that they are Th0 cells (Fig. 4), as we previously observed
(22).
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In vitro binding data indicated that GA is not bound to purified
MHC class I molecules and is therefore not likely to be recognized by
CD8+ T cells (12). Furthermore, flow
cytometric analysis of our own GA-specific T cell lines and clones
found that all the T cells were
CD4+CD8-. Nonetheless, it
was possible that GA could be processed in a MHC class I pathway and
that CD8+ GA-specific T cells could be generated.
To investigate the role of CD8+ T cells in the
generation of GA-reactive T cell responses, PBMCs from a healthy
control individual were isolated and subjected to depletion of
CD8+ T cells by magnetic bead separation. The
purity of the CD4+ population, as determined by
flow cytometry, was between 98 and 99% (data not shown). As shown in
Fig. 5, no difference was observed
between the GA dose response of the T cell lines generated with whole
PBMCs vs the PBMCs depleted of CD8+ T cells in
secondary in vitro stimulation. These results suggest that GA does not
stimulate the in vitro proliferation of CD8+ T
cells, nor are CD8+ T cells required for
CD4+ T cells to respond to GA.
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Our finding that GA stimulates a vigorous dose-dependent
HLA-DR-restricted primary in vitro T cell proliferative response
suggests that PBMCs, isolated from normal healthy adults, contain a
high frequency of GA-reactive T cells. It was unclear whether the
GA-reactive T cell repertoire was present in the naive T cell
repertoire or is expanded during life, perhaps in response to
environmental antigenic stimulation(s). In an effort to address these
questions, limiting dilution analysis was conducted on freshly isolated
PBMCs obtained from four neonatal cord bloods as well as five normal
healthy adult volunteers. Primary in vitro cultures were established at
1, 4, 10, and 40 µg/ml GA. Plotting of the percentage of negative
wells, i.e., those wells not proliferating in response to GA against
the number of cells/well on a semilogarithmic scale, revealed a linear
relationship which enabled us to make use of the Poisson
distribution for further analysis (data not shown). Precursor
frequencies were calculated for each concentration of GA tested (Fig. 6). We found that when cultures were
established with 40 µg/ml GA, the precursor frequencies among
non-GA-primed healthy adults ranged from 1:5,000 to 1:125,000. When the
concentration of GA was decreased, the precursor frequency decreased in
all adults tested from 1:15,000 to >1:1,000,000. At 40 µg/ml GA,
PBMCs isolated from neonate cord blood had a precursor frequency
ranging between 1:15,000 and 1:1,000,000. Again, the precursor
frequency of neonate responses decreased with lower concentrations of
GA. There was no statistically significant difference in the median
frequency of GA-reactive cells between adult and cord blood derived
PBMCs.
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Having demonstrated that GA induced significant dose-dependent in vitro HLA-DR-restricted proliferative responses when cultured with PBMCs isolated from virtually any human adult or neonate, we chose to reexamine whether similar responses could be demonstrated in other species. Given the clinical interest in using GA to treat MS, the ability to demonstrate equivalent GA responses in an animal model would potentially be useful for mechanism of action studies. Previous studies by Fridkis-Hareli et al. (12) suggested that GA binds to murine class II I-E molecules with a promiscuity similar to that observed with human HLA class II DR molecules. Accordingly, we chose to examine the ability of GA to induce the primary in vitro proliferation of murine spleen cells isolated from adult SJL/J (H-2S), B10.S (H-2S), BALB/c (H-2d), C57BL/6 (H-2b), and (SJL/J x PL/J)F1 (H-2S x H-2u) mice. These inbred strains were chosen because they express different H-2 class II molecules. Furthermore, with the exception of BALB/c mice, none of the strains chosen express I-E molecules.
As shown in Fig. 7, GA induced the
dose-dependent proliferation of spleen cells isolated from every mouse
strain tested after incubation for 6 days. Similar to what we found
with primary in vitro human PBMC cultures, no GA-mediated proliferation
was seen during the first 3 days of any murine primary in vitro
cultures (data not shown). Although the kinetics of murine and human
responses to GA were similar, the dose-dependent curves were quite
different. As seen in comparing the results presented in Figs. 1 and 7,
the optimal concentration of GA required to stimulate murine spleen
cells is 
10-fold lower than that required to stimulate human PBMC.
The ability of a number of strains of mice which do not express I-E
molecules to respond to GA strongly suggests that GA induces H-2 I-A-
as well as I-E-restricted T cells. The ability of I-A/I-E mixed
molecules to serve as restriction elements was not evaluated. Finally,
murine spleen cells appear to be inhibited from proliferating at
concentrations of GA in excess of 100 µg/ml. Preliminary studies
suggest that the high dose inhibition observed with GA is due to
TCR-dependent activation induced cell death. Similar degrees of
inhibition of human PBMCs require concentrations of GA 500–800
µg/ml.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A significant secondary in vitro proliferative response was observed in healthy human subjects, confirming an earlier report (21). Surprisingly, this response was strong enough to be detectable in vitro in primary T cells, which usually occurs only with recall Ags such as tetanus toxoid in immunized individuals. In addition, and in contrast to previous unpublished work, a similarly high proliferative response to GA was also observed in primary in vitro cultures established with spleen cells isolated from a variety of H-2 strains of non-GA-primed mice. Thus, the reactivity to this Ag in both species appear to be comparable. Given the numerous precedents for the applicability of immunological findings derived from murine studies to the corresponding human situation, it should be possible to conduct GA mechanisms of action studies in mice.
The ability of GA to induce significant primary in vitro proliferative responses in cultures containing spleen cells from a variety of strains of mice as well as PBMCs isolated from human neonatal cord blood and adult peripheral blood suggests that GA is a universal Ag. The evidence that GA is recognized as a conventional Ag, and not as a mitogen or superantigen, is compelling. In agreement with biochemical data demonstrating binding of GA to multiple alleles of purified HLA class II DR molecules but not to class II DQ or class I molecules, we found that all human GA-reactive T cell lines and clones were CD4+CD8- and restricted by unique DR, but not DQ, molecules. Furthermore, PBMCs depleted of CD8+ cells responded to GA in a manner identical to that of whole PBMCs. Moreover, the wide variety of TCR Vß chains expressed by GA T cell clones offers additional evidence that GA does not behave like a superantigen. Finally, the inability of GA to stimulate the proliferation, cytokine release, or activation-induced cell death of a variety of human and murine CD4+ T cell clones specific for Ags other than GA, suggests that GA is not a conventional mitogen (J.L.K. and D.A.H., manuscript in preparation).
We observed that GA is recognized by MHC DR-restricted CD4+ T cells, and the responses can be detected in all individuals examined regardless of their particular MHC class II haplotype. This is consistent with previous work by Fridkis-Hareli and Strominger (13), who demonstrated that GA can bind in a promiscuous manner to many, if not all, DR molecules. At present, it is unclear whether GA binds to HLA class II DP molecules and if so whether HLA DP-restricted GA-reactive T cell populations can be generated from PBMCs. Our present data suggest that a large preexisting repertoire of CD4 T cells expressing TCRs capable of recognizing GA is present in virtually all normal subjects and MS patients (K. Bieganowska and D.A.H., manuscript in preparation).
The promiscuous binding of GA observed with a variety of isotypes of DR molecules is similar to that seen with several well studied natural ligands, such as the immunodominant MBP 84–102 peptide (25) and the immunodominant tetanus toxoid 838–853 peptide (26) as well as, that seen with a synthetic pan DR epitope (PADRE) (27). Although a number of highly promiscuous DR-binding peptides have been identified, none has been shown to be capable of stimulating primary or secondary in vitro proliferative responses in all individuals such as GA. There are several likely reasons for the unusually high precursor frequency of GA-reactive T cells. First, the high alanine content of GA may enhance the promiscuous binding observed with HLA class II DR molecules while not affecting T cell recognition. A number of studies have demonstrated that alanine substitutions are well tolerated within T cell epitopes (27, 28, 29). Second, tyrosine, glutamic acid, and lysine residues are preferentially found in consensus binding sequences for DR molecules and can be accommodated in the MHC class II pockets as anchor residues. Third, in many cases, these residues have also been found to function as primary TCR contact residues (30, 31). Due to the particularly high content in lysine, GA is likely to interact with a subset of T cells with accumulation of polar, acidic amino acid residues in the CDR3 region (32).
Perhaps one of the most likely explanations for the magnitude of the primary in vitro responses to GA is the potential size of the peptide pool. GA is a complex mixture with 411 (>106) possible different 11-mer sequences which may be considered a core sequence of a T cell epitope. Many of these sequences will be closely related due to the fact that only one of four amino acids can occupy each position. In accordance with the notion emerging over the past few years that Ag recognition by CD4 T cells is more degenerate than originally thought (20, 21, 33), individual GA-reactive CD4 T cells are likely to respond to many different possible epitopes. These cross-reactive epitopes may be considered altered peptide ligands for any given T cell with the potential of being superagonists, agonists, partial agonists, antagonists, or null ligands (19).
Further investigation of GA as an immunomodulatory agent would appear to be justified. Our own studies suggest that when chronically administered to patients with MS, GA is capable of inducing immune deviation, which may result in amelioration of inflammatory responses (23). The apparent universal immunogenicity of GA suggests the possibility that GA could serve as the ultimate helper epitope. GA in combination with class I epitopes could significantly enhance attempts to develop CD8-dependent vaccines. The demonstration that GA induces similar responses in murine and human systems provides the rationale for initiating such studies in the appropriate murine systems.
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Acknowledgments |
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Footnotes |
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2 P.W.D. and J.I.K. contributed equally to the work.
3 Address correspondence and reprint requests to Dr. David A. Hafler, Center for Neurologic Diseases, Harvard Institutes of Medicine, 77 Avenue Louis Pasteur, Boston, MA 02115.
4 Abbreviations used in this paper: GA, glatiramer acetate; EAE, experimental autoimmune encephalomyelitis; MBP, myelin basic protein; MOG, myelin oligodendrocyte glycoprotein; MS, multiple sclerosis; RR-MS, relapsing remitting multiple sclerosis; ELISPOT, enzyme-linked immunospot.
Received for publication June 8, 2000. Accepted for publication September 22, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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), or DR 1 and DR 2 (
) were
preincubated with various doses of GA for 1 h at 37°C, 8%
CO2. GA-pulsed (1 x 104 cells/well) EBV B
cells were cocultured with T cell clone 3.1 (A–C),
clone 3.2 (D–F), and clone 3.3b (G–I)
(5 x 104 cells/well) for 3 days to assess
proliferation or 1 day to assess IFN-
