By
From the * Center for Neurologic Diseases, Brigham and Women's Hospital and Harvard Medical
School, Boston, Massachusetts 02115; and the Department of Medicine, Royal Brisbane Hospital,
University of Queensland, Herston Q4029, Australia
We previously generated a panel of T helper cell 1 (Th1) clones specific for an encephalitogenic peptide of myelin proteolipid protein (PLP) peptide 139-151 (HSLGKWLGHPDKF) that induces experimental autoimmune encephalomyelitis (EAE) upon adoptive transfer. In spite of the differences in their T cell receptor (TCR) gene usage, all these Th1 clones required W144 as the primary and most critical TCR contact residue for the activation. In this study, we determined the TCR contact residues of a panel of Th2/Th0 clones specific for the PLP peptide 139-151 generated either by immunization with the PLP 139-151 peptide with anti- B7-1 antibody or by immunization with an altered peptide Q144. Using alanine-substituted peptide analogues of the native PLP peptide, we show that the Th2 clones have shifted their primary contact residue to the NH2-terminal end of the peptide. These Th2 cells do not show any dependence on the W144, but show a critical requirement for L141/G142 as their major TCR contact residue. Thus, in contrast with the Th1 clones that did not proliferate to A144-substituted peptide, the Th2 clones tolerated a substitution at position 144 and proliferated to A144 peptide. This alternative A144 reactive repertoire appears to have a critical role in the regulation of autoimmune response to PLP 139-151 because preimmunization with A144 to expand the L141/G142-reactive repertoire protects mice from developing EAE induced with the native PLP 139-151 peptide. These data suggest that a balance between two different T cell repertoires specific for same autoantigenic epitope can determine disease phenotype, i.e., resistance or susceptibility to an autoimmune disease.
Experimental autoimmune encephalomyelitis (EAE)1, is
an example of an organ-specific autoimmune disease
that can be induced in experimental animals by immunization with several different myelin proteins, including myelin basic protein (MBP) and myelin proteolipid protein
(PLP). EAE is mediated by myelin antigen-reactive helper
T cells because elimination of MBP reactive Ly1+, but not
Ly2+, cells prevents transfer of EAE (1, 2), and monoclonal antibodies specific for CD4 can inhibit and/or reverse ongoing disease (3, 4). Direct evidence for the role of CD4+
T cells in EAE induction has come from adoptive transfer
studies in which MBP- and PLP-reactive CD4+ T cell
lines or clones were shown to induce chronic relapsing encephalomyelitis and paralysis after transfer (2, 5, 6).
Subpopulations of CD4+ helper T cells (Th) produce
distinct patterns of cytokines, and this has led to the concept of functional heterogeneity among Th cells (7, 8).
Upon antigenic stimulation, naive CD4+ T cells (Th precursor cells) differentiate into at least two populations: Th1
and Th2. Th1 produce IL-2 and/or IFN- Immunization of SJL mice with the encephalitogenic
PLP peptide 139-151 leads to the generation of Th1 cells,
and these clones recognize tryptophan in the center of the
peptide (W144) as the primary TCR contact residue (17).
The initial observation that the PLP 139-151-specific encephalitogenic Th1 clones require W144 as the primary
TCR contact residue and most critical residue for activation has now been confirmed by three other laboratories (18). We recently generated a panel of Th2/Th0 clones/
lines specific for PLP 139-151 and transfer of these Th2
clones both prevented and reversed PLP 139-151-induced
EAE (15, 16). Preimmunization with the altered peptide
Q144 prevents development of EAE and transfer of T cell
lines specific for the Q144 peptide confers protection
against EAE induced with the native PLP 139-151 peptide. In analyzing the cellular basis for this protection, we generated T cell clones from the Q144-specific line. These
clones have two important properties: they (a) produce
Th2/Th0 cytokines and (b) they cross-react with the native
PLP 139-151 peptide (21). The PLP 139-151-specific Th2
clones included in this study were generated in three different ways. SJL mice were first immunized with PLP 139-
151 and were injected simultaneously with anti-B7-1 antibody. For the second set of clones mice were immunized
with an altered form of the encephalitogenic peptide 139-
151 in which the primary TCR contact residue was
changed from tryptophan (W) to glutamine (Q) at position
144, and T cell clones were propagated with Q144 peptide
in culture. In a third independent T cell cloning, SJL mice
were immunized with Q144, and T cell clones were propagated with the native peptide in vitro. Thus, we cloned altered peptide-specific Th2 clones from Q144 immunized
mice from two independent lines. In one case, Q144 peptide was used (Q1 clones), and in the second case, native
peptide W144 (QW clones) for in vitro propagation and as
the cognate ligands. In this study, we determined the fine epitope recognition of the panel of Th2/Th0 clones and
compared it with a panel of encephalitogenic Th1 clones,
both of which react with the PLP peptide 139-151. The
data presented here show that in contrast with Th1 clones,
Th2 clones have shifted their primary TCR contact residue
to the NH2-terminal end of the peptide. This difference in
the fine epitopic recognition of encephalitogenic and protective repertoire has been exploited to regulate EAE.
Antigens.
PLP 139-151 (HSLGKWLGHPDKF) peptide, altered Q144 peptide (HSLGKQLGHPDKF), and alanine-substituted peptides on the native backbone were synthesized in the
laboratory of Dr. R. Laursen (Boston University, Boston, MA)
using FMOC chemistry on a Milligen synthesizer (model 9050;
PerSeptive Biosystems, Framingham, MA). The control peptide
PLP 43-64 (EKLIETYFSKNYQDYEYLINVI) was synthesized by
Dr. D. Teplow (Brigham and Women's Hospital, Boston, MA).
An I-As-binding peptide ADLIAYLKAQTAK of PCC 81-104
(synthesized by Quality Controlled Biochemicals,, Hopkinton,
MA) was used as a control in some of the experiments.
T Cell Clones and Hybridomas.
The generation of PLP 139-
151-specific Th1 (13) and Th2 (15) clones has been described. In
brief, the Th1 clones were generated from the lymph nodes of
SJL mice after immunization with the PLP peptide 139-151 in
CFA (Difco, Detroit, MI). The lymph node cells were obtained
7-10 d after immunization, activated with PLP 139-151 peptide,
and propagated in the presence of IL-2 as described previously
(15). For the epitope analysis of the Th1 clones, T cell hybridomas were also generated by the fusion of Th1 clones with
TCR, elicit delayed
type hypersensitivity (DTH) responses, activate macrophages, and have been implicated in inducing tissue injury
in many organ-specific autoimmune diseases. Th2, on the
other hand, produce IL-4, IL-5, and IL-10, are especially
important for IgE production and eosinophilic inflammation, and may suppress cell-mediated immunity. Different roles of these two subsets of Th in various immunopathological conditions like leprosy, leishmaniasis, and schistosomiasis, has been demonstrated (9). All encephalitogenic
T cell clones examined thus far have been of the Th1 type
(12, 13). In contrast, the transfer of myelin antigen-reactive
Th2 cells do not induce disease, and in some cases, have
been shown to transfer protection against EAE (14).
BW1100 (17, 22). The hybridomas and the parent
Th1 clones expressed identical TCRs and showed similar fine antigenic specificities.
Activation of T Cell Clones and Hybridomas. T cell clones or hy- bridomas (5 × 104) were cultured in triplicate in 96-well plates with 5 × 105 irradiated SJL spleen cells and various concentrations of synthetic peptides in a final volume of 0.2 ml. After 24 h, 0.1 ml of the supernatant from the hybridomas was transferred to 96-well flat-bottomed plates to which 104 HT-2 (IL-2/IL-4- dependent) cells were added. The ability of HT-2 cells to proliferate was determined by thymidine incorporation. The plates with T cell clones were incubated for 48 h at 37°C and then pulsed with [3H]thymidine for 16-18 h, after which they were harvested and the mean thymidine incorporation in triplicate wells was calculated. The [3H]thymidine incorporation was determined in a scintillation counter (model LS 5000; Beckman Instrs., Fullerton, CA). The data are presented as mean cpm incorporated into triplicate wells.
To compare the effects of a particular substitution on T cell proliferation and to make data readily comparable, the data has also been presented as percentage of control, in which case the mean T cell proliferation with the cognate antigen (native peptide) was taken as 100% and response to the substituted peptide was expressed as percent response of the cognate antigen (percent control). Proliferation of >10% of the native peptide is taken as positive.In Vitro Proliferation Assays. Mice were injected subcutaneously at five sites with antigen emulsified in CFA (Difco) containing a total of 250 µg Myobacterium tuberculosis H37 RA. Mice immunized with a single peptide received a total of 100 µg of antigen (native PLP 139-151 or A144) or a mixture of these two peptides. On day 10, lymph nodes were removed and LNC prepared from them. LNC (4 × 105/well) were cultured in triplicate in 96-well round-bottomed plates (Falcon, Becton Dickinson, Lincoln Park, NJ), in the presence of antigen, for 48 h and then [3H]thymidine (1 µCi/well) was added for the last 16 h before harvesting the cells. The [3H]thymidine incorporation was determined in a scintillation counter (model LS 5000; Beckman Instr.). Supernatants from the lymph node cells activated with antigenic peptides were harvested at 40 h for determining the type and amount of cytokines accumulated in the culture supernatant.
Cytokine ELISA.
T cell clones were activated with syngeneic
spleen cells as APCs and 50 µg/ml of antigen. Supernatants were
harvested 48 h later and tested for the presence of cytokines by
ELISA as described (15). IL-2, IL-4, and IL-10 ELISA components (antibodies and cytokine standards) were obtained from
PharMingen (San Diego, CA). In brief, ELISA plates were coated
with purified rat mAb to mouse IL-2 (clone JES6-1A12), IL-4
(clone BVD4-1D11), and IL-10 (clone JES5-2A5). Biotinylated
rat mAb to mouse IL-2 (clone JES5H4), IL-4 (BVD6-24G2), and
IL-10 (SXC-1) were used as secondary detecting antibodies. IFN-
ELISA components were obtained from Genzyme (Cambridge,
MA). Monoclonal hamster anti-mouse IFN-
was used as the
primary antibody; polyclonal goat anti-mouse IFN-
was the
secondary antibody. Assays were developed with TMB Microwell peroxidase substrate (Kirkegaard and Perry Labs., Inc.,
Gaithersburg, MD) and read after the addition of stop solution at
450 nm using a model 3550 Microplate Reader (BioRad Labs.,
Hercules, CA).
In Vivo Treatment with PLP 139-151 Analogues. SJL mice obtained from Jackson Labs. (Bar Harbor, ME) were pretreated with 100 µg of each of the following peptide analogues in 100 µg CFA: A141, A142, A144, A147, and native W144. After 1 wk, these mice were immunized with 50 µg of the native PLP 139- 151 peptide (W144) and pertussis vaccine as described (15). Mice were observed regularly for signs of disease. Clinical course of the disease is scored as follows: 1, tail atony; 2, hind limb weakness; 3, hind limb paralysis; 4, paralysis of all four limbs; 5, moribund.
We previously generated a panel of T cell clones specific
for PLP 139-151 peptide, of which five clones (2E5, 4E3,
5B6, 7A5, and SPL1.1) were tested in detail for epitope
specificity (17), cytokine production (13), and ability to
transfer EAE (6) upon adoptive transfer. All of these clones
were of the Th1 phenotype in that they produced IFN-,
but not IL-4 or IL-10, upon activation with the PLP 139-
151 peptide (13). The structure of the epitope recognized
by the T cell clones was defined, and all of them showed a
critical requirement for W144 in the center of the peptide
as a TCR contact residue for activation (17). The finding
that the encephalitogenic PLP 139-151-specific Th1 clones
require W144 as the primary and the most critical TCR
contact residue for activation has since been confirmed by
other groups (18). We subsequently generated a panel
of T cell clones by immunization with PLP 139-151 peptide by administration of anti-B7-1 antibody in vivo, and
upon cytokine analysis, the vast majority of these clones
turned out to be of the Th2 phenotype. We have now analyzed in detail the cytokine production and epitope specificity of four of the clones (1E3, 1E6, 1E10, and 1A8) generated with anti-B7-1 antibody treatment in vivo. As a
control, we also used a T cell clone (1D9) that was generated in parallel by immunization of SJL mice with PLP 139-151, followed by administration of hamster Ig in vivo
(15). In a third cloning, we generated PLP 139-151-reactive Th2/Th0 clones by immunizing SJL mice with the altered peptide Q144. Q144 altered peptide induces PLP
139-151-reactive Th2/Th0 cells, which mediate protection upon adoptive transfer. Three T cell clones (Q1.1B6,
Q1.3F4, and Q1.4A11) that showed stable growth in culture were included in the study and compared with other
PLP 139-151-reactive Th1 or Th2 clones. In a fourth independent T cell cloning, we generated Th2 clones by immunizing mice with Q144 altered peptide and the lymph
node cells from the immunized mice were activated with
the native peptide 139-151 to expand only the native peptide cross-reactive clones. Three clones from this cloning
(QW.1D4, QW.6D10, and QW.9E1) were included in
this study. Thus, 10 PLP 139-151-reactive Th2/Th0 clones
generated by three independent cloning attempts were included in this study.
The cytokine profile of the panel of 139-151-reactive T
cell clones is shown in Table 1. The 1D9 T cell clone produced large amounts of IFN- (>5 ng) and small amounts of
IL-10 (<1 ng) upon activation with PLP 139-151. The
clones 1E3, 1E6, 1E10, and 1A8 that were generated after
immunization with PLP 139-151 together with the anti-B7-1
in vivo showed a typical Th2 phenotype, in that they produced large amounts of both IL-4 and IL-10. The only exception was that the clone 1E6 produced only IL-10, and
not IL-4, after activation. The PLP 139-151-reactive T cell
clones generated after immunization with Q144 and in
vitro propagation with the Q144 peptide demonstrated a
mixed cytokine profile of Th0 and Th2 cytokines when activated with the cognate antigen Q144. The clone Q1.1B6
(Th0) produced IL-4 and IFN-
together; clone Q1.3F4 produced IL-4 (with low levels of IFN-
), whereas Q1.4A11
clone produced both IL-4 and IL-10 together with IFN-
in
some experiments. The three clones (QW.1D4, QW.6D10,
and QW.9E1) generated by immunization with Q144 peptide followed by in vitro propagation with the native PLP
peptide 139-151 showed a typical Th2 cytokine profile in
that they produced both IL-4 and IL-10 together except
for clone QW.1D4, which also produced small amounts of
IFN-
(Table 1).
To compare further MHC and CD4 requirements of the PLP-specific Th1 and Th2 clones, we added mAbs specific for I-As and CD4. Like the PLP 139-151-specific Th1 clones (13), the antigen-specific proliferative response of all clones was blocked significantly by addition of an antibody to I-As (10.2.16), but not by a control anti-I-Ek antibody (14-4-4S), suggesting that the clones recognize the antigen in the context of I-As. Furthermore, the addition of anti-CD4 antibody (GK 1.5) inhibited the proliferative responses of all the clones tested (data not shown). These data demonstrate that, like the Th1 clones, the Th2 clones require I-As and CD4 for activation.
Antigen Specificity and Cross-reactivity of the PLP 139-151-specific Th1 and Th2 Clones.The PLP 139-151-specific Th1
(2E5, 4E3, 1D9, and SPL 1.1) and Th2 (1E3, 1E6, 1E10,
and 1A8), and three Th2/Th0 (Q1.1B6, Q1.3F4, and
Q1.4 A11) clones generated by immunization with Q144
peptide were tested for their antigen specificity to the cognate ligands (PLP 139-151 or Q144) in an in vitro proliferation assay. In addition, the A144 peptide was included in
the assay because our previous data showed an absolute requirement for W at position 144 for the activation of all
Th1 clones and substitution of W at position 144 with any
other residue including A144 resulted in a peptide that did
not activate Th1 clones. The PLP peptide 43-64 was used as a negative control peptide in these experiments. The
Th1 cells (1D9, 2E5, 4E3, and 5B6) demonstrated specific
proliferation only to PLP 139-151 and did not respond to
the altered peptides A144 or Q144, confirming a strict requirement for W at position 144 for activation of Th1 cells
(Fig. 1). The Th2 clones (1E3, 1E6, 1E10, and 1A8) also
demonstrated specific proliferative responses to the PLP
139-151 peptide. However, in contrast with PLP 139- 151-specific Th1 clones, when A144 was used as the antigen in the proliferation assay, all the Th2 clones responded
with varying levels of proliferation. In addition, the Th2
clone 1A8 also showed a significant proliferative response
to Q144. These data demonstrate that the PLP 139-151-specific Th2 clones do not have an absolute requirement
for W144 for activation (Fig. 1). The group of PLP 139-
151-reactive Th2/Th0 clones generated by immunization
with Q144 peptide tested similarly in the proliferative assay
showed even greater degeneracy in their antigen recognition in that they showed a significant proliferative response
to the cognate antigen (Q144) and the native PLP peptide
139-151, but also showed a significant proliferative response to A144 peptide (Fig. 1). It was interesting to note
that reactivity pattern of some of the clones generated by
PLP 139-151 plus anti-B7-1 antibody administration appeared similar to the clones generated by Q144 immunization (compare 1A8 with Q1.1B6). These data demonstrate
that unlike Th1 clones, the Th2 clones do not have a critical requirement for W at position 144, because they all respond to A144.
Determination of Critical Residues for Activation of the Th1 and Th2 Clones.
Because the PLP 139-151-specific Th1 and Th2 clones showed a difference in proliferative responses when position 144 peptide-substituted analogues (Q144 and A144) were used, the question was raised of whether PLP 139-151-specific Th1 and Th2 clones recognize the PLP 139-151 peptide differently. To address this issue, we tested the PLP 139-151-specific Th1 and Th2 clones with a panel of peptides synthesized with alanine substitutions at each residue on the PLP 139-151 backbone at concentrations of 0.1-100 µg/ml in a proliferative assay. Maximal T cell responses were generally achieved between 10-100 µg/ml of the antigen, and only the maximal T cell responses at a single dose are shown (Fig. 2). The panel of Th1 clones reacted well to the native peptide, and various different clones were sensitive to alanine substitutions at positions 141, 142, 143, 144, and 147 (Fig. 2 a). In agreement with our previous results (17) and that of others (18- 20), only W at position 144 was absolutely required for the activation of Th1 clones in that an alanine substitution at position 144 in the PLP peptide 139-151 led to abrogation of T cell proliferation of all the Th1 clones tested, including the newly derived Th1 clone 1D9. Histidine at position 147 and lysine at position 143 appear to be essential for three of the four Th1 clones, whereas two of the four Th1 clones had a requirement for L141 and G142 (Fig. 2 a).
Epitopic analysis of Th2 clones specific for PLP peptide 139-151 with the panel of alanine substituted peptides is shown in Fig. 2 b. The clones 1E3, 1E6, and 1E10 showed a decrease or a complete loss of reactivity when residues at positions 141, 142, and 143 were substituted with alanine in the native peptide. Clone 1A8 also failed to proliferate when alanine substitutions were made at positions 146, 147, and 148. In contrast with the Th1 clones, all Th2 clones showed significant reactivity to the A144 peptide (Fig. 2 b). The cumulative data with the four Th2 clones suggest that alanine substitution at position 142 completely abrogated responses to the peptide in all four clones tested, and an alanine substitution at position at 141 was not tolerated by three of the four clones.
The T cell clones generated by immunization with the altered peptide Q144 are generally of Th2/Th0 phenotype, and besides recognizing Q144 peptide, these clones cross-react with the native PLP 139-151 peptide (see Fig. 1). Because the Th2 clones specific for the PLP 139-151 show an epitope shift, it was of interest to determine whether PLP 139-151-reactive T cell clones generated by immunization with Q144 peptide show a similar epitope shift, which may explain both cross-reactivity with the native peptide and the Th2 phenotype. To address this issue, we tested the panels of alanine-substituted peptides in the native PLP 139-151 backbone (Fig. 2 c). Clone Q1.1B6 has a requirement for residues 141, 143, and 147. Clone Q1.4A11 was not activated when substitutions were made at residues 140, 141, 142, 143, 145, 146, and 147. Q1.3F4 needed residues 141, 142, 143, 145, and 148 for activation. Similar to the Th2 clones described above, these clones also did not need W144 or Q144 for activation. However, the cumulative data suggested that Q144-specific T cell clones required residues 141 and 143 for activation, because alanine substitutions at these positions completely abrogated T cell activation of all the three clones. Glycine at position 142 and histidine at position 147 was critical for the activation of two out of three clones. The analysis of the third independent set of Th2 clones generated by immunization with the altered peptide Q144 and in vitro propagation with the native peptide also showed a pattern very similar to the Th2 clones described above (Fig. 2 d). Using alanine substitutions in the native peptide backbone, it was clear that this set of Th2 clones also did not show any critical requirement for W144, in that alanine substitution at this position induced significant proliferation of the clones. However, alanine substitution at positions 141 and 147 completely inhibited T cell proliferation in all the three clones and additional inhibitory effects were also seen by alanine substitution at positions 142, 143, and 146 for this set of Th2 clones. The analysis of fine epitopic specificity of the PLP 139-151-reactive Th1 versus Th2/Th0 clones, therefore, show striking differences in the requirement for critical residues for activation; while W144 is the primary and critical residue for the activation of all the Th1 clones, the Th2/ Th0 clones do not need W144 for activation.
Using several different strategies to identify I-As binding residues in the PLP 139-151 peptide (17), we found that leucine at position 145 and proline at position 148 are the major anchor residues for binding to I-As.
Structure of the PLP 139-151 Peptide Recognized by the Th1 and Th2 Clones.From the cumulative data, using the
panel of alanine-substituted peptide analogues to identify
the structure of PLP 139-151 recognized by Th1 and Th2/
Th0 clones, it appears that the two subsets differ significantly in their recognition of PLP 139-151 peptide. The
epitope recognition data of the Th1 clones is consistent with our previous data (17) and those of three other independent laboratories (18). The data of the Th2/Th0
clones is based upon three different clonings and the generation of PLP 139-151-specific Th2/Th0 cells by different
techniques. The responses of 4 PLP 139-151-specific Th1
clones and 10 Th2/Th0 clones to alanine-substituted peptides in the native peptide backbone is summarized in Fig. 3 a. The most striking difference is that all 4 Th1 clones require W144 for T cell activation, and an alanine substitution at this position completely abrogates the response. In
contrast, all 10 Th2/Th0 clones showed a significant response to the A144 peptide, demonstrating that these
clones do not require W144 as the primary contact residue.
In the case of Th1 clones, W144 is the major TCR contact
residue, and K143 and/or H147 the secondary TCR contact residues. L141/G142 are minor TCR-binding residues
for the Th1 clones. 9 out of 10 Th2/Th0 clones seem to
require L141, and 7 of 10 clones require G142 for activation; thus, the emphasis for the primary TCR contacts has
been shifted to the NH2-terminal end of the peptide (Fig. 3
b). Although 9 out of 10 Th0/Th2 clones require leucine
at position 141, alanine at this position does activate some
Th2 clones. Like the Th1 clones, these Th2 clones also appear to have a minor requirement for the residues K143 and H147. From these data, it appears that the Th2/Th0-reactive clones, generated by three different techniques, all
do not require W144 for activation, but mostly seem to require L141/G142 for their activation. However, neither of
the two residues (L141 or G142) appear to be absolutely
critical for the clones tested. Furthermore, there appears to
be subtle differences among the Th2 clones generated by
the three techniques: anti-B7-1-derived clones show main
emphasis on residues 142 and 141, Q144-specific clones require residues 141 and 143 with a minor requirement for 142 and 147 residues, and QW clones require 141 and 147. Unlike Th1 clones where W144 is absolutely required, alanine substitution at the position 141 or 142 does not lead to
complete abrogation of PLP 139-151-reactive Th2 responses.
Effect of Preimmunization with A144 Analogue on EAE.
Because encephalitogenic Th1 and protective Th2 cells
show a dramatic difference in activation with A144 peptide
in that A144 peptide activates PLP 139-151-specific Th2
clones but not the Th1 clones, we tested the effect of preimmunization with A144 peptide on the induction of
EAE. Groups of SJL mice were preimmunized either with
the native W144, A141, A142, or A144 peptides in CFA and then challenged with the encephalitogenic PLP 139-
151 to induce EAE. As shown in Fig. 4, preimmunization
with A144 protected mice from the development of EAE.
In contrast, preimmunization with A142 did not show significant protection and A141 showed slight inhibition of
disease, which was not significant. However, in other experiments preimmunization with A141 and A142 accelerated the day of onset and enhanced severity of disease (data
not shown). These data suggest that preimmunization with
A144 probably leads to the expansion of the A144-reactive
protective repertoire that inhibited the disease induced
with the native peptide.
Besides expanding a protective T cell repertoire, several
other mechanisms could explain protective effects of preimmunization with A144 peptide; for example, A144 could
mediate MHC blockade or antagonize PLP 139-151-specific Th1 cells and inhibit generation of an encephalitogenic T cell repertoire. A144 peptide does not have significantly higher binding for I-As than the native PLP 139-151
peptide. Furthermore, we coimmunized mice with A144 plus
PLP 139-151 peptides and tested the lymph node cells for
their ability to proliferate to PLP 139-151 and A144 peptides. Coimmunization of mice with A144 plus PLP 139- 151 does not inhibit the generation of PLP 139-151-specific W144-reactive T cells (Fig. 5), suggesting that A144
does not mediate MHC blockade, thereby inhibiting the
generation of W144-reactive cells. We have undertaken a
detailed analysis to test whether A144 can act as a TCR antagonist of the PLP 139-151-specific Th1 repertoire. Our studies showed that the A144 does not antagonize PLP
139-151-specific T cell clones or PLP 139-151-specific T
cell lines (17). To study whether A144 immunization expanded the protective Th2/Th0 repertoire, we immunized
SJL mice with PLP 139-151 or A144 in CFA and tested the
lymph node cells from these mice for proliferation and cytokine production by in vitro activation with native PLP 139-151 and A144 peptide (Table 2). The lymph node
cells from PLP 139-151 immunized mice proliferated specifically to the native peptide and produced large amounts
of Th1 cytokines, particularly IL-2 and IFN-. Consistent
with the Th1 clone data, PLP 139-151-immunized lymph
node cells did not show a significant proliferation or produce significant cytokines in response to A144 peptide. In
contrast, the lymph node cells from the A144-immunized
mice proliferated equally well to both the A144 and PLP
139-151 peptides as has been detected with the Th2/Th0
clones. The lymph node cells from A144-immunized mice
when activated with A144 produced little IFN-
, but produced large amounts of IL-10. We could not detect significant amounts of IL-4 in the primary lymph node cultures
after activation with A144, but small amounts of IL-4 were
detected after activation with the native peptide. These
data are consistent with the observation that in contrast
with the native peptide that induces Th1 cells, immunization with A144 even in CFA activates the alternate PLP 139-151 cross-reactive Th2/Th0 repertoire, which may be
responsible for protection against EAE. The data obtained
with immunization with the A144 peptide are similar in
many ways to those obtained with the Q144 peptide in
which we have seen that preimmunization with Q144 protects mice for the development of EAE and lymph node cells of these mice produce IL-10 upon in vitro activation
(21). However, after cloning these cells in vitro, IL-4-producing Th2 clones were uncovered (21). It is relatively difficult to detect IL-4 in primary lymph node cultures, which
may be due to very low levels of IL-4, or to its consumption or inhibition by other cytokines.
|
We have examined the structural requirements for activation of PLP 139-151-specific Th1 and Th2 clones. Although the autopathogenic Th1 clones recognize W144 as the primary and critical TCR contact residue, the PLP 139- 151-specific Th2 clones do not require this residue for activation. Instead, the Th2 clones have shifted their emphasis to L141/G142 at the NH2-terminal end of the peptide. Analysis of additional PLP 139-151-reactive Th2/Th0 clones, generated by immunization with an altered peptide in which tryptophan was substituted by glutamine at position 144 (21), also showed a similar shift in the critical TCR residues towards the NH2-terminal end of the peptide. These results suggest that Th1 and Th2 clones specific for the same peptide in the autoreactive repertoire can be dependent on different residues in the peptide for their induction and activation and that T cell differentiation may be influenced by the recognition of specific residues in an autoantigenic peptide.
The data presented here raise an important issue as to
why Th1 and Th2 clones specific for PLP 139-151 should
recognize different parts of the same peptide. It has already
been shown that T cells from TCR transgenic mice of
identical specificity can be driven to become either Th1 or
Th2 cells, depending upon the cytokines used in the primary activation culture (23, 24) or the dose of antigen (25,
26) used in initial activation. Although cytokine milieu is the
most dominant factor in determining the outcome of T cell
differentiation (27), studies with antigen dose would suggest
that strength of initial signal generated by the ligation of
TCRs would also influence the outcome. Low dose antigen
(lower T cell activation/lower strength of signal) generated
Th2 cells; relatively higher amounts of antigen (presumably
giving a higher strength of signal) promoted differentiation
of T cells in the Th1 direction; further higher concentration led to the generation of Th2 cells (25, 26). Our data
would suggest that in vivo, the PLP 139-151-specific T
cell repertoire consists of at least two populations of T cells
using either W144 or L141/G142 as the major contact residues. When these populations are exposed to PLP 139-
151 under conventional immunizing conditions, the W144-dependent cells become dominant in the expanded repertoire.
This may be because precursor cells of this type are present
at higher frequency in the naive PLP 139-151-specific
pool, or because W144-dependent TCRs have a higher
avidity for antigen-MHC complex. In the latter case, such
cells will receive a stronger signal at all antigen concentrations, leading to greater growth than the L141/G142- dependent cells and also tending to promote differentiation
along a Th1 pathway. This may explain why immunization
with the native PLP 139-151 routinely induces W144-specific Th1 clones when the cells are reactivated by the native
peptide (13, 18). In the presence of anti-B7-1 antibody,
the W144 costimulation-dependent Th1 clones are presumably not activated, allowing the growth of L141/G142-recognizing cells. These L141/G142 Th2 may belong to a
memory population, stimulated in vivo by autoantigen or
cross-reactive ligand and may, therefore, have a less stringent requirement for costimulation. It has been shown that
memory cells that produce TGF- or Th2 cytokines may
play a critical role in maintenance of peripheral tolerance to
self-antigens (28).
We (21) and others (29, 30) have recently shown that altered peptides generated by the substitution of TCR contact residues selectively affect T cell differentiation and alter cytokine balance. In our studies, replacement of the primary contact residue of tryptophan with glutamine at position 144 in the PLP 139-151 peptide leads to the induction of T cells that have two important properties: (a) the clones are of Th0/Th2 phenotype and (b) besides recognizing Q144, they also recognize the native PLP 139-151 peptide. Although it is possible that Q144-specific T cell clones could recognize Q at position 144 as the primary contact residue in the altered peptide and be Q144-specific, in that case the T cells would not cross-react with the native peptide. How does a single residue substitution in the primary T cell contact point of the encephalitogenic PLP peptide generate Th2/Th0 clones that cross-react with the native peptide? Our data on the shift of primary contact residue from W144 to L141/G142 begins to explain this data. The immunization with Q144 altered peptide does not activate W144-reactive Th1 cells, but promotes the growth of L141/G142-reactive cells. Because of this epitope shift, the cells are of a Th2 phenotype, but maintain cross-reactivity with the native peptide.
The PLP 139-151-specific Th2 clones that we have reported here were generated after the administration with anti-B7-1 antibody in vivo, and one might argue that epitope shift is not unique to the PLP 139-151-specific Th2 cells but is due to the presence of anti-B7-1 antibody. Two lines of evidence argue against this hypothesis: (a) a PLP 139-151-specific Th1 clone generated in parallel by anti- B7-1 antibody administration in vivo used W144 and not L141/G142 as the primary TCR contact residue (data not shown), and (b) immunization with the altered peptides in which the primary TCR contact residue was substituted induces PLP 139-151-reactive Th2 cells with a similar shift in the primary contact residues, further suggesting that epitope shift is not specifically due to the administration of anti-B7-1 antibody in vivo.
Some studies have shown that Th2 cells are not sufficient to protect from autoimmune disease (16). Khoruts et al. (14) and Katz et al. (31) reported that Th1 populations effectively transferred autoimmunity, whereas Th2 did not. However, the Th2 did not suppress autoimmunity caused by Th1. In our previous studies (15, 21), we demonstrated the Th2 clones studied in detail here and Q144-specific lines mediated protection from EAE. These cells also have a shift in their TCR contact residue. Therefore, it is possible that epitope specificity together with Th2 cytokines may be necessary for the protective phenotype. Thus, because PLP 139-151-specific pathogenic Th1 and protective Th2 clones differ in their primary TCR contact residue, this provided us with a unique opportunity to generate altered peptides that can inhibit pathogenic Th1, selectively induce protective Th2 clones, and ameliorate autoimmunity.
Address correspondence to Vijay K. Kuchroo, Center for Neurologic Diseases, Brigham and Women's Hospital, 77 Avenue Louis Pasteur, Boston, MA 02115. E-mail: Kuchroo{at}cnd.bwh.Harvard.edu
Received for publication 28 January 1997 and in revised form 30 June 1997.
1 Abbreviations used in this paper: DTH, delayed type hypersensitivity; EAE, experimental autoimmune encephalomyelitis; LNC, lymph node cells; MBP, myelin basic protein; PLP, proteolipid protein; Q, glutamine; W, tryptophan.We thank T. Howard and V. Turchin for their technical help and Dr. H. Weiner for support.
This work was supported by grants from the National Multiple Sclerosis Society, New York (RG2571A2 and RG2320B3) and the National Institutes of Health (R29-30843, R01NS35685, P01AI39671-01A1) to V.K. Kuchroo. L. Nicholson is a postdoctoral fellow of the National Multiple Sclerosis Society.
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