International Immunology Advance Access originally published online on September 19, 2007
International Immunology 2007 19(10):1157-1164; doi:10.1093/intimm/dxm080
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T cells specific to hapten–carrier but not to carrier alone assist in the production of anti-hapten and anti-carrier antibodies
1 Division of Structural Immunology
2 Division of Immunobiology, Research Institute for Biological Sciences, Tokyo University of Science, Yamazaki 2669, Noda, Chiba 278-0022, Japan
3 Present address: Department of Biochemistry, Sapporo Medical University School of Medicine, South-1 West-17, Chuo-ku, Sapporo 060-8556, Japan
4 Present address: National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki 305-8566, Japan
5 Present address: Department of Cellular Macromolecule Chemistry, Graduate School of Agriculture, Kyoto Prefectural University, Sakyo-ku, Kyoto 606-8522, Japan
Correspondence to: T. Azuma; E-mail: tazuma{at}rs.noda.tus.ac.jp
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Abstract |
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We examined the immune response of Balb/c mice to antigens prepared by conjugating 2-phenyloxazolone (phOx) to a foreign protein, ovalbumin (OVA), or a self-protein, mouse serum albumin (MSA), in order to study how these chemical modifications would affect immune recognition. We found that anti-OVA antibodies and CD4+ T cells produced by OVA immunization reacted with OVA as well as with phOx–OVA. Anti-phOx antibodies were produced by phOx–OVA immunization and, interestingly, T cells from these mice reacted only with phOx–OVA but not with the intact OVA. These results suggested that the classical model of hapten–carrier immunization, in which B cells specific to hapten are activated with assistance from T cells specific to a carrier protein, might not be a major route for production of anti-hapten antibodies in hapten–carrier immunization. Furthermore, phOx–MSA immunization induced production of anti-phOx antibodies, which could not be accounted for in terms of the assistance of carrier-specific T cells because of the absence of MSA-specific T cells. Therefore, we proposed a new model in which anti-hapten B cells are assisted by T cells specific to the haptenated carrier.
Keywords: antigenicity, B lymphocyte, hapten–carrier immunization, T lymphocyte, TD antigen
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Introduction |
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Haptens are small chemicals which induce a T cell-dependent immune response when conjugated to a protein carrier. Hapten–protein conjugates have been used to study the T cell-dependent immune response, especially antibody affinity maturation because of the ease in measuring the affinity of anti-hapten antibodies (1) and in separating hapten-specific B cells from carrier-specific T cells. Hapten–carrier systems have helped to elucidate the mechanisms involved in the T cell-dependent immune response (2); however, many studies have focused only on the production of anti-hapten antibodies. In addition, the specificity of the T cell population which is activated in the immune response to haptenated protein has not been carefully examined, although that of T cell clones or hybridomas established by hapten–carrier immunization has been investigated extensively (3–13). It is uncertain whether the same population of T cells is induced when animals are immunized with an intact or haptenated protein. In order to apply hapten–carrier as a T cell-dependent antigen model, the immune-competent cells responsible for antibody production must be identified.
According to the ‘classical model’ of hapten–carrier immunization, carrier-specific T cells are thought to provide assistance to hapten-specific B cells which differentiate into antibody-secreting cells. This theory is dependent on the fact that haptenated protein without adjuvant can induce anti-hapten antibody production when T cells specific to carrier are pre-activated by immunization with carrier protein with adjuvant (14), which is referred to as carrier/hapten–carrier immunization. However, it is not clear whether the same pair of T cells and B cells is involved in both carrier/hapten–carrier immunization and the conventional hapten–carrier immunization. Because haptenation may affect the antigenicity of the carrier protein, there would be differences in the repertoire of the provoked B cells and T cells between carrier-immunized and haptenated carrier-immunized animals. One extreme would be immunization with haptenated self-protein. In this case, no Th specific to self-protein would exist. If anti-hapten antibodies were secreted by immunization of haptenated self-protein, this antigen would function in a T cell-independent manner or induce Th specific to the hapten–carrier.
The hapten–carrier system is also of interest as a model of epitope modulations or altered self-proteins since it raises questions as to how chemical modification of proteins affects antigenicity and results in changes to the immune response in vivo. In some situations, T cell clones specific to haptenated self-protein cause chemically induced allergies and autoimmune diseases (15–18), suggesting that these T cells play important roles in these types of immune response. However, the relation of these T cells and carrier-specific T cells in the immune response to haptenated proteins in vivo has not been investigated. In this study, we compared the antigenicity of carrier and haptenated carrier using ovalbumin (OVA) and mouse serum albumin (MSA) as representatives of foreign and self-protein, respectively. These were conjugated with different numbers of 2-phenyloxazolone (phOx) molecules and used as antigens for analyzing the effect of haptenation on the magnitude of the primary immune response in vivo (19, 20). Then, we examined the specificity of antibodies and T cells using these carrier proteins. It is expected that a detailed investigation of the effect of haptenation on protein antigenicity in invoking B-cell and T cell responses in vivo would provide a greater understanding of the immune response to chemically modified proteins.
We found that haptenation of OVA did not result in significant changes in antigenicity because anti-OVA antibodies and T cells specific to OVA were able to recognize phOx–OVA. Interestingly, however, only T cells responding to phOx–OVA, but not to OVA, were activated in phOx–OVA-immunized mice. Similarly, T cells specific to phOx–MSA but not to MSA were activated by immunization with phOx–MSA. These results suggested that haptenation can induce changes in the immunogenicity of proteins. The interactions between B cells and T cells and the contribution of haptenated carrier-specific T cells will be discussed.
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Preparation and characterization of haptenated proteins
OVA and BSA were purchased from Sigma (St Louis, MO, USA). MSA was prepared from ascites or serum of Balb/c mice by ammonium sulfate precipitation and dialyzed against PBS. For preparation of phOx-conjugated proteins, 4-ethoxymethylene-2-phenyl-2-oxazolin-5-one (Aldrich Chem., St Louis, MO, USA) was reacted with carrier proteins at different molecular ratios at room temperature for 2 h and dialyzed against PBS. The concentration of phOx was determined at an absorbance of 348 nm and calculated using a molar absorption coefficient of 3.20 x 104 M–1 cm–1 (19). In order to determine the concentration of phOx-conjugated protein, the absorbance was measured at 280 nm. After subtracting the UV absorbance of phOx at 280 nm, which was determined to be OD348 x 0.221 using phOx-Cap, the protein concentration was calculated using the molar absorption coefficients of 3.21 x 104 M–1 cm–1 for OVA and 4.60 x 104 M–1 cm–1 for BSA (21). The molecular absorption coefficient of MSA was calculated to be 4.69 x 104 M–1 cm–1. The average molar ratio of phOx haptens to each molecule of carrier protein was calculated from the respective concentrations of both and expressed as a subscript (e.g. phOx7–OVA). Gel filtration chromatography was performed using a Superdex 200 column (Amersham Biosciences, Uppsala, Sweden) with buffer containing 10 mM Tris–HCl pH 7.4, 1 mM EDTA and 150 mM NaCl. Isoelectric focusing (IEF) was performed using a Phast System (Amersham Biosciences) according to the manufacturer's protocol.
Mice and immunization
Balb/c mice were purchased from Sankyo Labo Service (Tokyo, Japan) and kept in the animal facility of our institute. The experiments were carried out following the Guidelines for Animal Protocols of our university. Groups of 8- to 10-week old Balb/c mice were immunized with a single intra-peritoneal injection of 100 µg of antigen emulsified in an equal volume of CFA (WAKO, Osaka, Japan).
ELISA
In order to determine anti-OVA or anti-phOx antibody production, microtiter plates were coated with OVA or phOx12–BSA (10 µg ml–1) followed by blocking with 0.5% skim milk in PBS. Sera were prepared 2 weeks after immunization and were diluted 1/200 or 1/1000 and reacted with immobilized OVA or phOx12–BSA, respectively. IgG bound to antigen was detected with a peroxidase-conjugated goat anti-mouse IgG polyclonal antibody (Southern Biotechnology Associates, Birmingham, AL, USA) using o-phenylenediamine dihydrochloride as the substrate. For a competitive ELISA, serum from mice immunized with OVA in CFA was diluted 1/200 and reacted overnight with different concentrations of OVA or phOx–OVA. Unbound anti-OVA IgG in the reaction mixture was detected by ELISA using microtiter plates coated with OVA. In order to analyze anti-MSA and anti-phOx antibody production in phOx–MSA immunization, sera were diluted 1/1000 and reacted with immobilized MSA or phOx–MSA and IgG bound to antigen was detected as described above.
Surface plasmon resonance
In order to establish an anti-phOx mAb, spleen cells from phOx11–OVA-immunized mice were fused with SP2/0 myeloma cells using PEG1500 (Roche Diagnostics, Penzberg, Germany). After HAT selection, surviving cells were screened for anti-phOx antibody production by ELISA. An anti-OVA IgG1-producing clone was obtained and sub-cloned by limiting dilution. IgG1 mAb was purified from the culture supernatant of the hybridoma clone using phOx17–BSA conjugated to a Sepharose column. The Biacore biosensor system used for surface plasmon resonance (SPR) measurement was a Biacore 2000 (Biacore AB, Uppsala, Sweden). The anti-phOx mAb was coupled to the sensor chip through rabbit anti-mouse Fc antibodies (Rockland Immunochemicals, Gilbertsville, PA, USA) which were covalently linked to the sensor chip (CM5), and the interaction between mAb and phOx–OVA was analyzed using a Scatchard plot as previously described (21).
T cell proliferation assay
A single-cell suspension of spleen cells of OVA- or phOx–OVA-immunized mice was prepared 2 weeks after immunization. CD4+ T cells were enriched using IMag anti-mouse CD4 particle MSC (BD Biosciences, San Jose, CA, USA) and by passing through a MACS LS column (Miltenyi Biotec, Bergisch Gladbach, Germany). The purity of the CD4+ cells was >95%. As a form of antigen-presenting cell (APC), spleen cells of non-immunized mice were prepared and inactivated by 
-ray irradiation (30 Gy). CD4+ cells (4 x 105) were incubated in 96-well microtiter plates for 72 h in the presence of OVA or phOx–OVA (5 µg ml–1) and APCs (4 x 105 cells) in RPMI 1640 medium containing 10% FCS, 10 mM HEPES, non-essential amino acids, sodium pyruvate and 50 µM 2-mercaptoethanol. During the last 8 h, 0.5 µCi of [3H]-labeled thymidine ([3H]TdR) was added. Cells were harvested and the incorporated radioactivity was counted using a MicroBeta counter (PerkinElmer, Wellesley, MA, USA). For the proliferation assay of cells from phOx–MSA-immunized mice, 5 x 105 of total spleen cells were incubated with MSA, phOx–MSA or phOx–BSA (10 µg ml–1) for 72 h and [3H]TdR was added 10 h before incubation was terminated. Cells were harvested and the incorporated radioactivity was counted.
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Preparation and characterization of antigens
We prepared a series of phOx–OVA conjugates with different valencies and each preparation was assumed to be heterogeneous in terms of the number of phOx molecules per OVA. Since our laboratory has a strong interest in the production of antibodies to epitopes present on the same antigen molecule, the phOx–OVA preparations had to be free of unmodified OVA or aggregated forms. We examined their molecular heterogeneity in terms of their isoelectric points (pIs) and molecular weights using IEF and gel filtration chromatography. Since phOx molecules were conjugated to the
-amino group of lysine residues, this resulted in a pI shift. We found that the pI of OVA changed from 4.6 to 4.3 on conjugation with phOx. No unmodified OVA was observed in the phOx4–OVA and phOx8–OVA preparations, judging from the pI shifts (Fig. 1A). The difference in the pI of phOx4– and phOx8–OVA was not significant, which may have been due to protonation of carboxyl groups below pH 4. Gel filtration profiles indicated, however, that phOx8–OVA was significantly aggregated (Fig. 1B). In order to remove the aggregated forms, we purified the monomers of phOx4– and phOx8–OVA by gel filtration. After purification, the number of phOx haptens per OVA molecule was recalculated to be phOx4– and phOx7–OVA, respectively. When the purified monomeric antigens were analyzed again by gel filtration, almost no aggregated forms were observed (data not shown). We used these monomers as antigens for further study. We also examined the molecular form of phOx–MSA preparations by gel filtration and no aggregated materials were observed (data not shown).
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The production of anti-carrier and anti-hapten antibodies
In order to study the relationship between hapten valence and the primary immune response in vivo, we immunized Balb/c mice with 100 µg of unmodified OVA, phOx4–OVA or phOx7–OVA emulsified in 100 µl of CFA. Two weeks after immunization, serum samples were collected and production of anti-phOx and anti-OVA antibodies was analyzed by ELISA. As shown in Fig. 2(A), anti-phOx antibody was produced in phOx–OVA-immunized mice and there was no significant difference in the amount of anti-phOx antibody between mice immunized with phOx4–OVA and those immunized with phOx7–OVA. On the other hand, anti-OVA antibody was detected in all mice, but the amount decreased with an increase in the number of phOx haptens on the OVA molecule (Fig. 2B). When phOx11–OVA was used as the immunizing antigen, only marginal production of anti-OVA antibodies was observed (data not shown). These results indicated that the magnitude of the immune response to carrier protein decreased with an increase in the number of haptens. Next, we compared the specificities of B cells and T cells responding in mice immunized with OVA or phOx–OVA.
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Interaction between phOx–OVA and anti-OVA antibodies
One possible explanation for the decrease in anti-OVA antibody production is that there was a change in antigenicity, i.e. all epitopes recognized by anti-OVA B cells could have been altered by haptenation. In order to test this possibility, we analyzed the interaction between phOx–OVA and anti-OVA antibodies by a competitive ELISA. In this assay, we used anti-OVA antisera obtained from OVA-immunized mice because they contained a set of antibodies recognizing different epitopes of OVA. As shown in Fig. 3, OVA inhibited the binding of anti-OVA antibodies to OVA immobilized on plastic plates in a dose-dependent manner. When phOx4–OVA or phOx7–OVA was used as the competitor, it inhibited the interaction between anti-OVA antibodies and OVA as efficiently as did unmodified OVA. Therefore, most of the OVA epitopes recognized by anti-OVA antibodies, and possibly by anti-OVA B cell receptors (BCRs), did not change when conjugated with up to seven molecules of phOx, so that anti-OVA B cells were able to recognize phOx–OVA as well as they recognized the unmodified OVA. This suggested that the decrease in anti-OVA antibody production in phOx–OVA-immunized mice compared with OVA-immunized mice was not due to a change in the antigenicity of OVA in provoking a B cell response.
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Avidity of phOx4– and phOx7–OVA in interaction with a primary mAb
No significant difference in the production of anti-phOx antibodies was observed between phOx4–OVA and phOx7–OVA, although the production of anti-OVA antibodies decreased with an increase in hapten valence (Fig. 2). We next examined whether anti-phOx antibodies could distinguish between phOx4–OVA and phOx7–OVA. We prepared an anti-phOx IgG1-producing hybridoma cell line using spleen cells obtained from a mouse 7 days after immunization with phOx11–OVA. This mAb was considered to be a primary antibody because no somatic hypermutations were identified in the cDNA sequence of the Ig heavy chain (data not shown). The interaction of this mAb and phOx4–OVA or phOx7–OVA was measured by SPR and the results were subjected to Scatchard analysis (Fig. 4). The apparently straight lines of the plots provided the avidity (Ka) of the interaction, which was 2.6 x 108 M–1 for phOx4–OVA and 3.9 x 108 M–1 for phOx7–OVA. Therefore, anti-phOx antibody and possibly anti-phOx BCR were able to recognize the difference in the hapten valence from the avidity of the interaction. However, it is likely that this difference in avidity was not reflected in the production of anti-phOx antibody because there was no difference in the anti-phOx antibody production of phOx4–OVA- and phOx7–OVA-immunized mice.
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Recognition of phOx–OVA by T cells induced by immunization with unmodified OVA
We analyzed the specificity of Th activated in immunized mice. First, we examined whether T cells from mice immunized with OVA responded to unmodified OVA and phOx–OVA. Two weeks after immunization with OVA, CD4+ Th were purified by MACS. These cells were mixed with
-irradiated spleen cells of non-immunized mice which acted as APCs. After incubation for 3 days with antigen, proliferation of these cells was analyzed by [3H]TdR. CD4+ T cells of OVA-immunized mice showed a proliferative response to OVA, indicating that anti-OVA T cells were stimulated in these mice (Fig. 5A). When CD8+ cytotoxic T cells were used, no proliferation was observed (data not shown). Interestingly, these OVA-stimulated Th could respond to phOx–OVA as well (Fig. 5A). It suggested that the antigenicity of OVA to T cells did not changed by haptenation and that the repertoire of OVA antigen peptides presented on APCs to OVA-specific T cells was not altered.
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Antigen specificity of T cells induced by immunization with phOx7–OVA
We analyzed T cells in phOx–OVA-immunized mice. CD4+ T cells obtained from phOx7–OVA-immunized mice were purified and a proliferation assay was performed. We found that these cells could respond to phOx4–OVA and phOx7–OVA equally well, but surprisingly, did not show a proliferative response to unmodified OVA (Fig. 5B). This suggested the presence of T cells that recognize only phOx–OVA and not the unmodified OVA. In addition, in phOx–OVA-immunized mice, only T cells specific to phOx–OVA were activated while the activation of T cells specific to unmodified OVA were not observed, even though phOx4–OVA and phOx7–OVA were recognized by T cells of OVA-immunized mice (Fig. 5A). These T cells reacted neither to phOx-conjugated chicken gamma globulin nor to (4-hydroxy-3-nitrophenyl) acetyl-conjugated OVA, indicating that both the hapten group and OVA sequence were important for recognition by these cells (data not shown).
Immune response to haptenated self-protein
In order to confirm the existence of T cells recognizing haptenated protein and to determine their contribution to the hapten–carrier immune response, we analyzed the response to a haptenated self-protein, MSA. If T cells recognized only the unmodified peptide of the carrier protein, they would not have provided assistance to anti-hapten B cells because MSA-specific T cells were tolerized in these mice. Therefore, anti-hapten antibody production was not expected. When mice were immunized with phOx4–MSA, only marginal production of anti-phOx antibody was observed. However, when MSA was conjugated to more than eight phOx molecules, anti-phOx antibody production was clearly observed and the increase in hapten valence resulted in greater antibody production (Fig. 6A). Antibodies to MSA could not be detected by ELISA. This suggested that haptenation of self-protein may have changed the antigenicity of MSA to T cells and that haptenated MSA-specific T cells were activated and provided help to anti-phOx B cells. This hypothesis was examined by a T cell proliferation assay. Two weeks after immunization with phOx16–MSA, the response of splenic T cells to phOx-conjugated MSA was determined. As shown in Fig. 6(B), cells from phOx16–MSA-immunized mice could not respond to unmodified MSA. When phOx4–MSA or phOx8–MSA was used as antigen, only marginal proliferation was observed. However, when the amount of conjugated phOx was >10 molecules, there was a clear proliferative response to phOx–MSA but not to phOx–BSA. Therefore, it was suggested that when mice were immunized with phOx–MSA, haptenated carrier-specific T cells were activated and provided help to hapten-specific B cells.
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In this study, we examined the effect of haptenation on the antigenicity as well as immunogenicity of OVA and MSA by analyzing the specificity of B cells and T cells responding in the immunization with phOx–OVA and phOx–MSA. When mice were immunized with the haptenated foreign protein, phOx7–OVA, both anti-OVA and anti-phOx antibodies were produced, indicating that B cells were able to recognize the phOx hapten as well as OVA epitopes on phOx–OVA molecules (Fig. 2) and that T cells activated by phOx–OVA provided assistance to these B cells. On the other hand, since mice that had been pre-immunized with OVA plus adjuvant were able to produce anti-phOx antibodies after immunization with phOx–OVA in saline (data not shown), the T cells specific to OVA was presumed to act as Th in carrier/hapten–carrier immunization. In fact, T cells prepared by immunization with OVA were able to react with both phOx–OVA and intact OVA (Fig. 5A). If these OVA-specific T cells had been activated in phOx–OVA-immunized mice, they would have assisted anti-phOx B cells, as predicted in the classical model. However, CD4+ T cells from phOx–OVA-immunized mice responded to phOx–OVA but not to intact OVA (Fig. 5B), indicating that T cells specific to the carrier alone did not exist. Therefore, the production of anti-phOx antibodies could not be explained by the classical model. Instead, there were phOx–OVA-specific T cells which assisted B cells in phOx–OVA immunization. We proposed a new model in which B cells specific to hapten are activated with assistance from T cells specific to haptenated carrier. By immunization with phOx–MSA, only anti-phOx antibodies and not anti-MSA antibodies were produced (Fig. 6A), and the T cells obtained were found to be stimulated by phOx–MSA but not by MSA (Fig. 6B). These results showed that MSA and phOx–MSA were recognized as totally different antigens, i.e. there was no immune response to MSA due to self-tolerance but antibodies to phOx–MSA were secreted because the complex was recognized as a foreign antigen. Similar to the finding obtained with phOx–OVA immunization, the production of anti-phOx antibodies was assisted by phOx–MSA-specific T cells. Based on this similarity in T cell specificity arising from hapten–carrier immunization, we considered that our new model would be applicable to haptenated proteins in general. The new model suggested a facultative role for T cells in the recognition of chemically modified proteins. They do not respond to each structural element separately, such as distinguishing between hapten and carrier but rather recognize the integrated structure of the antigen molecule. T cells specific to the carrier, i.e. the intact protein, would not be essential for an immune response to the hapten–carrier.
The antigenic determinants of T cells specific to haptenated proteins have been investigated using T cell clones or hybridomas. The epitopes of CD8+ T cell clones specific to trinitrophenyl (TNP) peptide were analyzed in detail and found to react with various TNP-conjugated peptides possessing different sequences (6, 9, 10, 13). Ni2+-specific T cells were isolated from patients with nickel contact dermatitis in which Ni2+ interacts with a peptide on MHC and alters its antigenicity (18). Type II collagen in joint cartilage was post-translationally modified by hydroxylation and glycosylation, and T cells specific to this modified protein were shown to be important in the development of arthritis (15). Antigen recognition of T cell clones from patients allergic to penicillin was also independent of peptide sequence (17). Carrier sequence dependency was observed in a T cell hybridoma specific to phosphorylcholine-conjugated hen egg lysozyme, which weakly recognized unmodified carrier (12). Immunization with moth cytochrome C peptide containing nitrotyrosine-induced T cells specific to modified peptide (22). Maleylation of self-protein modulated immunogenicity and anti-self-T cells were induced (23, 24). Although the relative contribution of peptide sequences and peptide-conjugated hapten seemed to vary in a T cell-dependent manner, the haptenated amino acid residue was found to contribute largely to the binding free energy in interactions with TCR, i.e. the haptenated residue would be major determinant for the interactions. Our results showing that T cells specific to phOx–OVA or phOx–MSA had little cross-reactivity with unmodified OVA (Fig. 5B) or MSA (Fig. 6B) also indicated the large contribution of hapten to the binding energy. In this case, phOx-modified OVA peptides must be presented on APCs. Nalefski and Rao (11) showed that a peptide derived from OVA conjugated with p-azobenzenearsonate, Ars-OVA33–49, competed with OVA323–339, which is known to be the most highly studied OVA antigenic peptide (25) that binds to I-Ad, although unhaptenated OVA33–49 was incapable of I-Ad binding. Since OVA33–49 contains a lysine residue, it may act as an epitope for T cells specific to phOx–OVA. Among the OVA peptides presented with I-Ad, OVA273–288 (26) was expected to be conjugated with phOx because it has two lysine residues. There may also be other OVA epitopes containing lysine residues. Although we have little information regarding the amino acid sequences of phOx–OVA peptides recognized by T cells, the fact that there was no significant difference in stimulation with phOx4–OVA as compared with phOx7–OVA suggested that the lysine residues with higher reactivity with phOx would contribute largely to the antigenicity of the epitopes.
The preferential production of T cells specific to phOx–OVA but not to intact OVA following immunization with former can be explained in terms of the higher avidity of T cells specific to phOx–OVA, since Franco et al. (27) showed that CD8+ T cells specific to haptenated peptides possess higher binding avidity than those specific to unhaptenated peptides. These authors showed that CD8+ T cells specific to TNP–Sendai virus nucleoprotein 324–332 had a 100-fold higher avidity than those specific to unhaptenated carrier peptide. The T cells specific to phOx–OVA would overwhelm those specific to OVA in competing in binding to APCs and would appear 
100-fold more frequently than the latter if phOx conjugation increased avidity to CD4+ T cells as well. On the other hand, CD4+ T cells of OVA-immunized mice responded to both phOx–OVA and OVA (Fig. 5A). In the processing of phOx–OVA in APCs, OVA peptides with and without phOx would be generated, since the OVA molecule contains 20 lysine residues (28) and only seven of them were conjugated to phOx. Therefore, the discrepancy between the results of Fig. 5(A and B) could be explained by the fact that only a small number of peptide–MHC complexes was required for T cell activation (29, 30). APCs were capable of presenting enough OVA peptides from phOx–OVA to induce anti-OVA T-cell proliferation in the absence of T cells specific to phOx–OVA in vitro.
We observed a decreased production of anti-OVA antibodies after immunization with phOx–OVA which corresponded to a rise in the phOx valence (Fig. 2B). At first, we thought that the decrease in production reflected a change in the antigenicity of OVA resulting from conjugation with phOx. However, anti-OVA antibodies produced by OVA immunization could recognize OVA and phOx–OVA equally (Fig. 3), indicating that the antigenicity of OVA recognized by B cells did not change with haptenation. Therefore, the reduced production of anti-OVA antibodies could not be accounted for, since B cells specific to OVA were able to receive assistance from T cells specific to phOX–OVA, even in the absence of T cells specific to OVA. However, one explanation which appeals to us is that B cells specific to phOx may be superior in terms of antigen binding and presentation. Interactions between anti-hapten BCR and hapten groups are multivalent, while anti-carrier BCR binds antigen in a monovalent fashion. Due to the high avidity of multivalent interactions with hapten groups, B cells specific to phOx would be superior to those specific to OVA in antigen competition. In fact, SPR measurements showed that the avidity of a germ line anti-phOx mAb to phOx–OVA was in the order of 108 M–1 (Fig. 3), and this would be sufficiently higher than the affinity of naive B cells specific to OVA. Therefore, B cells specific to phOx would win the antigen competition and receive more activation signals than those specific to OVA, especially in the case of a high hapten valence (31).
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Ministry of Education, Science, Sports and Culture of Japan, (12470080); Tokyo University of Science.
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Abbreviations |
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| APC, antigen-presenting cell |
| BCR, B cell receptor |
| [3H]TdR, [3H]-labeled thymidine |
| IEF, isoelectric focusing |
| MSA, mouse serum albumin |
| OVA, ovalbumin |
| phOx, 2-phenyloxazolone |
| pI, isoelectric point |
| SPR, surface plasmon resonance |
| TNP, trinitrophenyl |
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Notes |
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Transmitting editor: K. Okumura
Received 29 July 2006, accepted 19 June 2007.
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