Immunogenicity of Peptide-25 of Ag85B in Th1 development: role of IFN-
Ai Kariyone1,
Toshiki Tamura1,
Hideyuki Kano1,
Yoichiro Iwakura2,
Kiyoshi Takeda3,
Shizuo Akira3 and
Kiyoshi Takatsu1
1 Division of Immunology, Department of Microbiology and Immunology, and 2 Laboratory of Cell Biology, Center for Experimental Medicine, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan 3 Institute of Microbial Diseases, Osaka University, Osaka 565-0871, Japan
Correspondence to: K. Takatsu; E-mail, takatsuk{at}ims.u-tokyo.ac.jp
Transmitting editor: S. Koyasu
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Abstract
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Ag85B (also known as
antigen or MPT59) is immunogenic, and induces expansion and differentiation of TCRVß11+CD4+ T cells to IFN-
-producing cells in C57BL/6 (I-Ab) mice. We reported that Peptide-25 (amino acids 240254) of Ag85B is a major T cell epitope, and its amino acid residues at position 244, 247, 249 and 252 are I-Ab contact residues. Here we examined roles of IFN-
in the generation of Peptide-25-reactive CD4+ TCRVß11+ T cells and the efficacy of mutant peptides of Peptide-25 for Th1 development in mice other than C57BL/6 mice. Immunization of C57BL/6 mice with Peptide-25 included in incomplete Freunds adjuvant led to preferential induction of CD4+ TCRVß11+ IFN-
- and tumor necrosis factor-
-producing T cells. Compared with other I-Ab-binding peptides such as Peptide-9 of Ag85B, 50V of pigeon cytochrome c and ovalbumin (OVA)265280 peptide, only Peptide-25 was capable of inducing enormous expansion of TCRVß11+ IFN-
-producing T cells. Treatment of C57BL/6 mice with anti-Vß11 antibody before Peptide-25 immunization reduced the development of CD4+ IFN-
-producing T cells. Furthermore, B10.A(3R) mice, I-Ab-positive and TCRVß11 strain, showed remarkably lower response to Peptide-25 immunization than C57BL/6 mice. Peptide-25-primed IFN-
/ cells showed significantly decreased expansion of CD4+ TCRVß11+ T cells as compared with wild-type cells. Interestingly, Peptide-25-primed cells from MyD88-deficient mice responded to Peptide-25 and differentiated into IFN-
-producing cells to a similar extent as wild-type mice, indicating Toll-like receptor-independent IFN-
production. These results imply that IFN-
plays important roles for the generation and expansion of CD4+ TCRVß11+ T cells in response to Peptide-25. Although Peptide-25 was non-immunogenic in C3H/HeN mice, a substituted mutant of Peptide-25, 244D247V, capable of binding to I-Ak, induced Th1 development. These results clearly demonstrate important roles of IFN-
in the expansion of CD4+ TCRVß11+ T cells, and will provide useful information for delineating the regulatory mechanisms of Th1-cell development and for analyzing mechanisms on Th1-dominant immune responses.
Keywords: Ag85B, mycobacterium, subunit vaccine, TCRVß11, tuberculosis
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Introduction
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After encountering antigen, Th cells undergo differentiation to effector cells that can produce IFN-
, IL-4, IL-10 and other cytokines/chemokines. Naive Th cells can differentiate to at least two functional classes of cell during an immune responseTh1 and Th2 (1,2). Th1 cells produce IFN-
and lymphotoxin [tumor necrosis factor (TNF)-ß] in addition to IL-2, and are responsible for directing cell-mediated immune responses leading to the eradication of intracellular pathogens such as mycobacteria, viruses and parasites (14). Th1 cells also regulate IgG2a and IgG3 antibody production via IFN-
production that is involved in the opsonization and phagocytosis of particulate microbes. Th2 cells secrete IL-4 and IL-5 as effector cytokines, and are responsible for extracellular immunity (3).
The process by which an uncommitted Th cell develops into a mature Th1 or Th2 is a matter of fact for regulating immune response to various antigens. There is a body of evidence to indicate that the cytokines IL-12 and IL-4 are key determinants of the Th1 and Th2 response respectively (4). The strength of interaction mediated through the TCR and MHCpeptide complex directly affects the lineage commitment of Th cells to Th1 and clonal expansion (57). Antigen doses, co-stimulators, genetic modifiers and other non-cytokine factors have crucial roles in determining the polarization of a Th cell response. Critical cytokines that promote the appearance of Th1 cells include IL-12 and IL-18. The canonical Th1 cytokine IFN-
has also been implicated in Th1 cell development through its ability to optimize IL-12 production from macrophages and IL-12 receptor expression on activated T cells. As naive T cells do not produce IFN-
until after activation, the contribution of T-cell-derived IFN-
in Th1 development is still obscure.
Th1 cells play an important role in the development of resistance to Mycobacterium tuberculosis (817), primarily through the production of macrophage-activating cytokines, such as IFN-
, TNF-
or granulocyte macrophage colony stimulating factor. Although specific antigens eliciting Th1 cell responses are not yet known for tuberculosis, one of the major protein antigens secreted from M. tuberculosis is Ag85B (also known as
antigen or MPT59) (1824). Purified Ag85B or recombinant forms of Ag85 can elicit strong Th1 responses in vitro from purified protein derivative-positive asymptomatic individuals (1820). We have shown that in vitro stimulation of lymph node cells from M. tuberculosis-primed C57BL/6 mice with Ag85B induces the production of IFN-
and IL-2, and expansion of CD4+ T cells expressing Vß11 of the TCR (TCRVß11) in an I-Ab-restricted manner (25,26). Using peptides covering the entire mature 285-amino-acid-long Ag85B protein as 15mer molecules overlapping by 5 amino acids, we identified the 15mer peptide (Peptide-25), covering amino acid residues 240254 of Ag85B, as the major epitope for Ag85B-specific TCRVß11+ T cells (25). Peptide-25 contains the motif that is conserved for I-Ab and requires processing by antigen-presenting cells (APC) to trigger Ag85B-specific TCRVß11+ T cells (25). We also have reported that amino acid residues of Peptide-25 at positions 244, 247, 249 and 252 (P1, P4, P6 and P9 sites respectively) are I-Ab contact residues (26). Active immunization of C57BL/6 mice with Peptide-25 can induce protective immunity against subsequent infection with live M. tuberculosis H37Rv (26).
MyD88 is an adaptor molecule essential for signaling via the Toll-like receptor (TLR)/IL-1 receptor family. Both TLR2 and TLR4 have been shown to mediate M. tuberculosis-induced intracellular signaling in vitro (27). Takeuchi et al. examined the responsiveness of MyD88-deficient mice to various bacterial cell wall components and demonstrated that MyD88 is essential for the cellular response to bacterial cell wall components including mycobacterial whole-cell lysates (28). However, it remains unclear whether MyD88 is involved in the Th1 development induced by proteins or peptide secreted by M. tuberculosis.
In this report, we have examined roles of TCRVß11+ T cells in the expansion of IFN-
-producing T cells in response to Peptide-25 and of IFN-
in the development of TCRVß11+ T cells. Our results clearly demonstrate that immunization of TCRVß11+ C57BL/6 mice, but not TCRVß11 B10.A(3R) mice, with Peptide-25 is capable of inducing remarkably potent oligoclonal development of IFN-
-producing T cells including TCRVß11+ T cells. This is not observed in C57BL/6 mice immunized with Peptide-9 of Ag85B, 50V of pigeon cytochrome c and ovalbumin (OVA)265280, all of which can bind to I-Ab. The use of anti-Vß11 mAb is effective in the prevention of the development of Th1 cells specific for Peptide-25. Reduced proportions of TCRVß11+ T cells are observed in Peptide-25-immunized IFN-
-deficient mice after Peptide-25 stimulation, indicating an important role of IFN-
. A mutant Peptide-25, 244D247V, whose amino acids critical for I-Ab-binding are substituted for amino acids capable of binding to I-Ak molecules, can induce the Th1 development in C3H/HeN mice. We will discuss the mechanisms on Th1-inducibility of Peptide-25.
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Methods
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Mice
C57BL/6, BALB/c, C3H/HeN and C3H/HeJ mice were obtained from Japan SLC (Hamamatsu, Japan). B10.A(3R) mice were kindly provided by Dr T. Yokochi (University of Tokyo Graduate School of Medicine). MyD88-deficient (MyD88/) mice and IFN-
-deficient (IFN-
/) mice were generated as described (29,30). All mice were maintained in the animal facility at the Institute of Medical Science, University of Tokyo under specific pathogen-free conditions and used at 815 weeks of age.
Antigens and reagents
Ag85B was purified as described (31) and used at selected concentrations. Peptide-25 and its substituted mutant, Peptide-25 (244D247V), Peptide-9 of Ag85B (25), 50V of pigeon cytochrome c (32) and OVA265280 (33) were synthesized by Sawaday Chemicals (Tokyo, Japan) using Fmoc chemical strategies and purified using HPLC. Amino acid sequences of each peptide are shown in Fig. 1. The following mAb were used: GK1.5 (34) which recognizes CD4 (ATCC, Rockville, MD), 53-6.72 (35) which recognizes CD8 (ATCC) and 2.4G2 (36) which recognizes Fc
R (ATCC). B20.6 [anti-Vß2 (37)], 44-22-1 [anti-Vß6 (38)] and F23.1 [anti-Vß8 (39)] were kindly provided by Dr Y. Yoshikai (Kyushu University, Fukuoka). KJ-25 [anti-Vß3 (40)], KT4 [anti-Vß4 (41)], B21.5 [anti-Vß10 (42)], MR11-1 [anti-Vß12 (43)], MR12-3 (anti-Vß13) and 14-2 [anti-Vß14 (44)] were purchased from PharMingen (San Diego, CA). MR9-4 [anti-Vß5 (45)], MR10.2 [anti-Vß9 (46)] and RR3-15 [anti-Vß11 (47)] were kindly provided by Dr O. Kanagawa (Washington University, St Louis, MO) and Dr H. Nakauchi (University of Tokyo, Institute of Medical Science). The following anti-cytokine mAb were used: 11B11 (48) which neutralizes IL-4 and was kindly provided by Dr W. E. Paul (National Institutes of Health, Bethesda, MD), BVß6-24G2 (49) which recognizes IL-4 (PharMingen), RA4-6A2 (PharMingen) which neutralizes IFN-
and XMG1.2 [anti-IFN-
(49)] which was kindly provided by Dr. M. Howard (DNAX Research Institute, Palo Alto, CA). For intracellular cytokine staining, we used the following phycoerythrin-conjugated mAb (PharMingen): TRFK5 which recognizes IL-5, C.15.6 which recognizes IL-12, MP6-XT22 which recognizes TNF-
, S4B6 which recognizes IL-2 and R3-34 for rat IgG1 isotype control.

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Fig. 1. Amino acid sequence of peptides. (A) Peptide-25 and its substituted mutant, 244D247V, (B) Peptide-9 of Ag85B, (C) 50V of pigeon cytochrome c and (D) OVA265280. We searched for the prediction of Peptide-9 and OVA265280 binding to the I-Ab molecule by using the RANKPEP program (Molecular Immunology Foundation). Amino acid residues shown in red are predicted binders to the I-Ab molecule.
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Immunization
Each strain of mice was immunized by s.c. injection at the base of the tail with 110 µg of each peptide in incomplete Freunds adjuvant (IFA). In some experiments, mice were boosted with 10 µg of relevant peptide 7 days before in vitro culture.
Cell culture and ELISA
A single-cell suspension was prepared from inguinal lymph nodes of peptide-immunized mice. In some experiments, CD4+ T cell clones specific for Peptide-25 were used as described (26). For proliferation assay, cells (5 x 105) were cultured in 96-well flat-bottom microtiter plates (Nunc, Roskilde, Denmark) in the presence of selected concentrations of synthetic peptides. Each well was filled with 200 µl of complete medium consisting of RPMI 1640 medium (Gibco) supplemented with 8% FCS, penicillin (100 IU/ml), streptomycin (50 µg/ml) and 5 x 105 M 2-mercaptoethanol. Cultures were set up in triplicate. The cells were pulse labeled with [3H]thymidine (1 µCi/well) during the last 8 h of a 72 h culture period and the incorporation of radioactivity was measured as previously described (26). The amounts of IFN-
secreted in supernatants of 3-day culture were quantitatively analyzed by ELISA. For FACS analysis, cells (5 x 106 in 2 ml) were cultured in the presence of peptides in 24-well flat-bottom microtiter plates. Representative results are shown from a series of three separate experiments.
Flow cytometric analysis of intracellular cytokine production
CD4+ cloned T cells (2 x 105/ml) specific for Peptide-25 were stimulated with plate-coated anti-CD3. Various peptide-primed lymph node cells were stimulated with each peptide for 4 days. In both cases, 4 h before cell harvest, GolgiStop (PharMingen) was added to block intracellular transport processes. Cells were harvested, washed and stained with FITC-labeled anti-CD4, anti-CD8 or biotin-labeled anti-Vß mAb. Anti-Fc
R mAb (10 µg/ml) was added over the duration of the incubation period for cell staining to block non-specific binding of labeled mAb. Then the cells were stained with streptavidinallophycocyanin in 7-amino-actinomycin D buffer (2 µg/ml) to distinguish viable cells from dead cells. After washing, cells were fixed with 4% formaldehyde. Then the cells were washed and stained with phycoerythrin-labeled anti-cytokine mAb. For intracellular staining by anti-cytokine mAb, we added 1% FCS and 0.1% saponin to all staining reagents and washing buffer, and kept them at 4°C. Fluorescence intensity of blastic T cells was analyzed on a FACSCalibur (Becton Dickinson, San Jose, CA). To assess particular TCRVß11+ cells, we calculated the percentage of TCRVß11+ cells from total CD4+ cells.
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Results
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Production of Th1 cytokines by Peptide-25-primed CD4+ TCRVß11+ T cells upon Peptide-25 stimulation
To set up conditions for intracellular staining and cytometric analysis of intracellular cytokine production, three different Peptide-25-reactive T cell clones (BP1, 2501 and BM2) were stimulated with plate-coated anti-CD3 mAb. Each Peptide-25-reactive T cell clone produced both IFN-
and TNF-
intracellularly upon anti-CD3 stimulation, but did not produce IL-12 and IL-4 (data not shown). Production of IL-2 and IL-5 was variable clone by clone. For example, cloned TCRVß11+ BP1 and 2501 cells showed no significant production of IL-2 and IL-5, but cloned TCRVß14+ BM2 cells produced IL-2 and IL-5 in addition to IFN-
and TNF-
.
We then investigated intracellular production of IFN-
, TNF-
, IL-4 and IL-5 in Peptide-25-stimulated, Peptide-25-primed lymph node cells by cytometric analysis. Lymph node cells of Peptide-25-primed C57BL/6 mice were stimulated in vitro with Peptide-25 and intracellular cytokine production was examined after the culture. As controls, lymph node cells stimulated with Peptide-25 from non-primed C57BL/6 mice were also stained under the same conditions. Within 5 h of stimulation of Peptide-25-immunized cells with Peptide-25 a significant proportion of IFN-
- and TNF-
-producing CD4+ T cells, and to a lesser extent IL-2-producing CD4+ T cells, became detectable (data not shown). Proportions of IFN-
- and TNF-
-producing CD4+ T cells were increased by day 4. Cells producing IL-4 or IL-5 were rarely detected. We could not detect IFN-
-producing cells in Peptide-25-stimulated non-primed cells (data not shown). These results indicate that large proportions of IFN-
-producing cells are CD4+ T cells.
To compare immunogenicity of Peptide-25 with other I-Ab-binding peptides regarding the development of TCRVß11+ IFN-
-producing cells, we immunized C57BL/6 mice with Peptide-25, Peptide-9 of Ag85B, 50V or OVA265280 included in IFA, and their lymph node cells were re-stimulated in vitro with relevant peptide. Results revealed that all of peptides tested induced development of IFN-
-producing cells to various extents (Fig. 2A). Among peptides, Peptide-25 showed the most potent ability to induce IFN-
-producing cells. Levels of IFN-
produced by Peptide-25-primed cells were 6- and 8-times higher than these produced by 50V-primed cells and OVA265280-primed cells, respectively. Interestingly, large proportions of Peptide-25-reactive IFN-
-producing cells were TCRVß11+ T cells and a significant proportion, but to a lesser extent, of IFN-
-producing cells were TCRVß11 T cells (Fig. 2B). In contrast, IFN-
-producing cells reactive to Peptide-9, 50V or OVA265280 were TCRVß11 T cells. Peptide-9-reactive T cells and 50V-reactive T cells showed an increase in TCRVß8+ and TCRVß14+ populations respectively, while OVA265280-reactive T cells displayed an increase in TCRVß4+ and TCRVß11+ populations (Fig. 2B).
CD4+ TCRVß11+ T cells of Peptide-25-immunized mice produce IFN-
and TNF-
As we described, large proportions of Peptide-25-stimulated, Peptide-25-immunized cells in C57BL/6 mice are TCRVß11+ T cells (Fig. 2). To examine the role of TCRVß11+ T cells in the development of IFN-
-producing T cells, we immunized C57BL/6 and B10.A(3R) mice, both of which are I-Ab-positive strains. B10.A(3R) mice are TCRVß11. Lymph node cells of each group of Peptide-25-immunized mice were re-stimulated in vitro with Peptide-25 for 4 days. As shown in Fig. 3(A), Peptide-25-primed C57BL/6 cells responded to Peptide-25 in a dose-dependent manner for proliferation and differentiation into IFN-
-producing cells. The response peaked at 1.0 µg/ml of Peptide-25 stimulation. In contrast, Peptide-25-immunized cells from B10.A(3R) mice showed a significant proliferation, but marginal IFN-
production was observed upon Peptide-25 stimulation even at higher doses (10 µg/ml) whose level was much lower than that produced by C57BL/6 mice (Fig. 3A). TCRVß11+ T cells play a role in the development of Peptide-25-reactive precursors for IFN-
-producing cells.
More than 85% of IFN-
-producing cells in the culture of Peptide-25-stimulated, Peptide-25-primed cells of C57BL/6 mice were CD4+ T cells (Fig. 3B, upper panel) and >65% of CD4+ T cells expressed TCRVß11 (data not shown). About 55 and 30% of IFN-
-producing CD4+ cells were assumed to be TCRVß11+ and TCRVß11 T cells respectively. Indeed, as shown in Fig. 3(B),
57 and 43% of IFN-
-producing cells were TCRVß11+ and TCRVß11 T cells respectively. Intracellular staining analyses also revealed that a small proportion of Peptide-25-primed CD4+ T cells in B10.A(3R) mice produced IFN-
upon Peptide-25 stimulation (Fig. 3B, lower panel). We infer from these results that Peptide-25-primed TCRVß11+ T cells may be required for differentiation of Peptide-25-primed TCRVß11 T cells into IFN-
-producing cells in response to Peptide-25.
We then examined the effect of passive administration of anti-Vß11 antibody on the development of IFN-
-producing Th1 cells specific for Peptide-25. Both anti-Vß11 antibody-treated and non-treated groups of C57BL/6 mice were immunized with Peptide-25. Peptide-25-primed cells from each group of mice were stimulated with Peptide-25. In some experiments, anti-Vß11 antibody was added during the in vitro culture. Results revealed that >60% of TCRVß11+ T cells were detected in the cells cultured with Peptide-25 from control groups of mice. In contrast, <1% of TCRVß11+ T cells were detected in the cells from the anti-Vß11 treated mice (Fig. 4A). CD4+ T cell populations expressing TCRVß4, TCRVß6, TCRVß12 and TCRVß14 were expanded; however, no dominant TCRVß usage was observed. Proliferation of and IFN-
production by Peptide-25-primed cells from anti-Vß11-treated mice was
50% less than these of the control group (Fig. 4B and C). When anti-Vß11 antibody was added to the culture of Peptide-25-primed cells of the control mice, IFN-
production was suppressed by 50% of control response (Fig. 4B) whose levels were comparable to those produced by Peptide-25-stimulated cells of Peptide-25-primed and anti-Vß11 treated mice. Taking these results together, the Peptide-25-reactive T cell population expressing TCRVß11 is a major population of IFN-
-producing cells in Peptide-25-immunized C57BL/6 mice, although TCRVß11 T cells are capable of differentiating into IFN-
-producing cells.
Role of IFN-
in the expansion of Peptide-25-reactive CD4+ T cells
There is a body of evidence that IFN-
plays a pivotal role in the polarization of naive T cells towards Th1 cells (50). To examine the role of IFN-
in the development of TCRVß11+ T cells in Peptide-25-primed C57BL/6 mice, we immunized wild-type and IFN-
/ C57BL/6 mice with Peptide-25, and their lymph node cells were cultured with Peptide-25. Results revealed that Peptide-25-primed cells from IFN-
/ mice displayed proliferation and IL-2 production in response to Peptide-25 to a lesser extent than wild-type mice (Fig. 5A and B). As expected, IFN-
production was not observed in cultures of Peptide-25-primed cells from IFN-
/ mice (Fig. 5C). While
41% of CD4+ T cells expressed TCRVß11 in Peptide-25-primed wild-type cells after Peptide-25 stimulation, Peptide-25-primed IFN-
/ cells contained
22% of TCRVß11+ cells. In addition, CD4+ T cell populations expressing TCRVß5, TCRVß6, TCRVß9 and TCRVß12 were increased slightly; however, no dominant TCRVß usage was observed (data not shown). We analyzed the cell cycle number of TCRVß11+ cells by labeling Peptide-25-primed cells with CFSE before culture. FACS analysis revealed that significant proportions of both TCRVß11+ T and TCRVß11 T cells experienced several cell divisions in Peptide-25-primed wild-type cells for 4 days upon Peptide-25 stimulation at two different concentrations (1 and 10 µg/ml). In contrast, Peptide-25-primed IFN-
/ T cells showed less cell divisions than wild-type cells (Fig. 5D).
Role of MyD88 in vivo induction of Th1 cells by Peptide-25
Various cell types and molecules have been proposed to affect Th1 development from naive T cells. As TLR- and MyD88-dependent signaling pathways are in involved in the activation of dendritic cells and Th1 development (29,51), we compared Th1 and Th2 development in Peptide-25-immunized MyD88/ mice with their development in Peptide-25-immunized wild-type mice. Re-stimulation of Peptide-25-primed cells of MyD88/ mice with Peptide-25 induced IFN-
production and expansion of TCRVß11+ T cells to a similar extent to cells of wild-type mice (Fig. 6A). Proportions of IL-4- and IL-5-producing T cells in Peptide-25-primed cells of MyD88/ mice were also similar to those of wild-type mice. TCRVß usage of Peptide-25-primed cells of MyD88/ mice was unaltered from that of wild-type cells (Fig. 6B).
Substitution mutant of Peptide-25, 244D247V, induces Th1 cells in vivo in C3H/HeN mice
In our previous analysis, P1 (at position 244), P4 (at position 247), P6 (at position 249) and P9 (at position 251) sites of Peptide-25 were important for I-Ab binding (26). Peptide-25 was not immunogenic in C3H/HeN mice because APC of C3H/HeN (I-Ak) did not bind to Peptide-25. To develop an immunogenic peptide for Th1 development in C3H/HeN mice, we prepared several mutant peptides by substituting amino acid residues of the I-Ab-binding motif of Peptide-25 to the I-Ak-binding motif. We synthesized substituted mutants (244D247V, 244D or 247V) of Peptide-25 whose P1 and P4 sites were aspartic acid (244D) and valine (247V) respectively. A group of C3H/HeN mice was immunized with 244D247V (Fig. 1), 244D or 247V. Lymph node cells from each group of immunized mice were stimulated with the relevant peptide, and proliferative response and IFN-
secretion were examined. As shown in Fig. 7, stimulation of 244D247V-immunized cells with 244D247V induced a significant proliferative response and remarkable IFN-
production in a dose-dependent manner, but it did not induce either IL-4 or IL-5 (data not shown). All IFN-
-producing T cells were TCRVß11 T cells. The 244D247V-primed cells showed no significant response to 244D or 252R (data not shown). Neither 252R nor 244D was immunogenic (Fig. 7).

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Fig. 7. 244D247V is immunogenic in vivo in C3H/HeN mice. C3H/HeN mice were immunized with 10 µg of 244D247V, 244D or 252R in IFA, and boosted 7 days before the experiment. Lymph node cells from each group of mice were stimulated in vitro with various concentrations of relevant peptide for 4 days. Proliferative response and IFN- secretion in culture supernatants were monitored post-culture according to procedures described in Fig. 3.
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Discussion
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After engagement of the TCR by the appropriate peptideMHC complex, which triggers clonal expansion, Th cells rapidly undergo programmed differentiation. Th subsets are distinguished by their ability to produce distinct cytokine patterns and promote specific immune responses. The IFN-
produced by Th1 cells amplifies Th1 development and inhibits the expansion of Th2 cells (7). Conversely, IL-4 and IL-10 produced by Th2 cells antagonizes the production of IFN-
by Th1 cells (3). Dysregulation of Th cell responses can result in immunopathology in that aberrant Th1 responses can be responsible for organ-specific autoimmunity and exaggerated Th2 responses have been associated with allergic diseases. Thus, understanding the factors and microenvironments that influence the development of naive T cells into different effector subsets has important implications for the development of new strategies to induce beneficial immune responses.
Considerable progress has being made in identifying and analyzing regulatory function to specify cell fate decisions in the lymphoid system during the generation of Th1 versus Th2 cells. While various microenvironments including cytokines, co-stimulatory molecule expression and transcription factors regulate the development of Th1 cells, other mechanisms such as the type of APC, antigen dose and peptide ligand density can also be involved in the differentiation of naive T cells into Th1 and Th2 cells (4,5,52). A number of studies have attempted to define the T cell determinants of various antigens, which are common structures or specific alignments of amino acids within peptides (motifs), required for binding to certain MHC molecules. In contrast to MHC class I-bound peptides, antigenic peptides comprised of 1012 amino acid residues are able to bind MHC class II molecules (53,54). However, the role of protein and peptides in the polarized development of Th1 versus Th2 cells remains elusive.
The major focus of our study is to understand mechanisms underlying preferential induction of Th1 cells by peptides derived from M. tuberculosis. As we reported, Peptide-25 corresponding to amino acid residues 240254 of Ag85B is a major T cell epitope of Ag85B-specific TCRVß11+ Th1 cells (26). Antigen processing of Peptide-25 is required for triggering TCRVß11+ Th1 cells specific for Peptide-25 in an I-Ab-restricted manner (26). Ramachandra et al. found Ag85B241256I-Ab complexes in M. tuberculosis phagosomes that later appeared on the plasma membrane (55). Thus Ag85BMHC class II complexes are formed within and expressed on M. tuberculosis-infected macrophages.
In vitro stimulation of lymph node cells from Peptide-25-immunized C57BL/6 mice with Peptide-25 induces massive expansion of both TCRVß11+ and TCRVß11 CD4+ T cells that produce IFN-
(Figs 2A and 3B). As far as we examined, development of IL-4- and IL-5-producing T cells was hardly detectable. Peptide-25 was not immunogenic for C3H/HeN (I-Ak) or BALB/c (I-Ad) mice (data not shown). We have also examined immunogenicity of three different I-Ab-binding peptides (Peptide-9, 50V and OVA265280) in C57BL/6 mice. Results revealed that all peptides were immunogenic and induced significant IFN-
production (Fig. 2A). Among them, only Peptide-25 could induce remarkable development and expansion of TCRVß11+ IFN-
-producing cells. Moreover, Peptide-25 was the most potent immunogen to induce the development of the IFN-
-producing cells. A significant proportion of Peptide-25-reactive IFN-
-producing cells were TCRVß11 T cells. IFN-
-producing cells reactive to Peptide-9, 50V and OVA265280 were TCRVß11 T cells (Fig. 2A). Although passive administration of anti-Vß11 at 1 day before Peptide-25 immunization fully suppressed the development of TCRVß11+ T cells (Fig. 4A), development of IFN-
-producing T cells was inhibited partially, up to 50% of untreated control, by anti-Vß11 mAb administration (Fig. 4B). It was demonstrated that IFN-
derived from CD4+ T cells is sufficient to mediate Th1 cell development (56). We do not know whether IFN-
is produced by cells other than T cells in Peptide-25-immunized mice.
There is a core sequence (P1 to P9) of 9 amino acids in the MHC class II binding motif (53). Using substitution mutants of Peptide-25 and Th1 clones specific for Peptide-25, we demonstrated that the amino acid residues of Peptide-25 at positions 244, 247, 249 and 252 are I-Ab contact residues, and are essential for stimulatory activity (26). Alanine-substituted mutant Y at position 244 of Peptide-25 significantly reduced the stimulatory activity of Peptide-25. Thus, the Y residue at position 244 of Peptide-25 appears to be the P1 site. The amino acid residues of Peptide-25 at positions 244, 247, 249 and 252 are at P1, P4, P6 and P9 sites respectively. Substitution mutant of Peptide-25, 244D247V, at P1 and P4 sites that are capable of binding to I-Ak was immunogenic in C3H/HeN mice for Th1 development, while neither of 244D or 247V was immunogenic (Fig. 7). The cells responding to 244D247V were exclusively TCRVß11 T cells (data not shown). Similarly, a mutant peptide 1431 of Peptide-25 capable of binding to I-Ad was immunogenic in BALB/c mice to induce solely Th1 cell development (data not shown). This further supports the notion that Peptide-25 and its substituted mutants can induce Th1 cells preferentially in various strains of mice.
It has been shown that the immunodominant encephalitogenic T cell epitope of major basic protein (MBP), in mice of the H-2u haplotype (PL/J or B10.PL), is the acetylated N-terminal 9 or 11mer (NAc19 or NAc111) peptide (57). T cell recognition of the epitope is restricted by MHC class II (I-Au) molecule. The MBPNAc111 epitope stimulates CD4+ T cells expressing Vß8.2 TCR that are capable of causing experimental allergic encephalomyelitis (EAE) (5860). The MBPNAc111 epitope has been characterized to have a post-translational modification to the MBP peptide, N-terminal acetylation, which is required for induction of EAE (58). Both TCRVß8+ and TCRVß8 T cell clones were shown to recognize MBPNAc19. The non-acetylated MBP111 peptide is incapable of inducing EAE. Interestingly, not all of the I-Au-restricted T cell clones specific for MBPNAc111 induce EAE with paralysis. A strong positive correlation between levels of lymphotoxin and TNF-
activity, their capacity to induce paralysis, and the clinical hallmarks of EAE has been elucidated (61). No correlation was found between IL-2 or IFN-
production and encephalitogenicity. Although Peptide-25 is derived from Ag85B secreted from M. tuberculosis and is able to induce Th1 development, Peptide-25 by itself is not pathogenic and is actually capable of enhancing anti- tuberculosis immunity. It is not clear whether Peptide-25 is modified in APC during antigen process.
Considerable progress has being made in identifying and analyzing regulatory functions that specify cell fate decisions at various nodal points in the lymphoid system, during the generation of Th1 versus Th2 cells. It remains unclear why Peptide-25 can induce potent Th1 response preferentially with particular TCR usage. There are several possibilities to account for our observations. First, Pepetide-25 might activate dendritic cells to enhance expression of co-stimulatory molecules or transcription factors such as T-bet that lead to prompt production of IFN-
, IL-12 or an unidentified master cytokine for Th1 development (62,63). Expansion of not only TCRVß11+ T cells, but also TCRVß11 T cells, was observed when Peptide-25-immunized cells of wild-type mice were stimulated with Peptide-25. We examined the contribution of IFN-
in the development of Peptide-25-reactive T cells using IFN-
/ mice. In IFN-
/ mice, both TCRVß11+ T cell and TCRVß11 T cell populations were significantly decreased (Fig. 5D). Taken together, IFN-
production is necessary for full development of TCRVß11+ and TCRVß11 T cells in response to Peptide-25. Reduced IL-2 production in Peptide-25-primed IFN-
/ cells compared with wild-type cells (Fig. 5A and 5B) may account for the reduced proportions of Peptide-25-reactive TCRVß11+ and TCRVß11 T cells in IFN-
/ mice. Second, the avidity of Peptide-25 for its specific TCR may be strong enough to induce Th1 development. It is quite intriguing that >40% of CD4+ T cells express a predominant TCRß11 gene. TCRVß11+ cells are necessary for full activation and expansion of Th1 development in response to Peptide-25 (Figs 3 and 4). Third, Peptide-25-reactive T cells or their products indirectly stimulate CD4+ T cells that enhance Th1 cell development. Other possibilities are not exclusive.
In our preliminary data, co-immunization of OVA with Peptide-25 in C57BL/6 mice induced the augmented generation of OVA-specific cytotoxic T cells compared with immunization of OVA alone (unpublished observations). These results suggest that Peptide-25 enhances the development of a concomitant Th1 response by unknown mechanisms. Taking all the results together, Peptide-25 has unique features represented by expansion of Th1 cells with a high proportion of TCRVß11+ T cells and oligoclonal response as well. To gain further insight in to the site of action and mechanisms on induction of preferential Th1 development by Peptide-25, transgenic mice expressing TCR that specifically recognize Peptide-25 together with I-Ab would provide a useful model. We recently succeeded in generating such TCR-Tg mice and found that T cells of the TCR-Tg mice could differentiate only to Th1 in response to Peptide-25 within 3 days in primary culture in the absence of IL-12 and IL-18 (unpublished observation).
Recently, TLR have been shown to regulate Th1 responses in addition to innate immune responses in response to relevant stimuli (6467). Although most TLR recognize carbohydrate or lipid of bacteria, some TLR, e.g. TLR5, recognize protein antigen such as flagellin (68). Stimulation of lymph node cells from Peptide-25-immunized MyD88/ mice with Peptide-25 showed similar patterns of IFN-
production to those observed in wild-type mice (Fig. 6). These results indicate that at least the MyD88-dependent pathway is not involved in major steps of Peptide-25-induced Th1 development.
In conclusion, this study demonstrates that immunization of C57BL/6 mice with Peptide-25 leads to the preferential induction of development of TCRVß11+ Th1 and oligoclonal response. The IFN-
production is required for full development and activation of TCRVß11+ T cells and TCRVß11 T cells in response to Peptide-25. Cytokines other than IFN-
may also be involved in Peptide-25-induced Th1 development from naive T cells. These experimental systems will provide a useful tool for delineating the regulatory mechanisms of antigenic peptides for Th1-dominant immune response.
 |
Acknowledgements
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We thank Drs T. Yokochi, Y. Yoshikai, H. Nakauchi, W. E. Paul, O. Kanagawa and M. Howard for providing mice and mAb, and Dr S. Takaki and Dr Yuji Kikuchi for their valuable suggestions throughout this study. We are indebted to Drs S. Taki and Yoko Kikuchi for their critical review of this manuscript. This work was supported in part by a Research Grant from the Human Frontier Science Program (K. T.), by Special Coordination Funds for Promoting Science and Technology, and by Grant-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Science, Sports and Culture, in Japan.
 |
Abbreviations
|
---|
APCantigen-presenting cell
EAEexperimental allergic encephalomyelitis
IFAincomplete Freunds adjuvant
MPBmyelin basic protein
OVAovalbumin
TNFtumor necrosis factor
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