Engineering Th determinants for efficient priming of humoral and cytotoxic T cell responses
Ascensión López-Díaz de Cerio1,
Juan José Lasarte1,
Noelia Casares1,
Pablo Sarobe1,
Marta Ruiz1,
Jesús Prieto1 and
Francisco Borrás-Cuesta1
1 Universidad de Navarra, Division de Hepatología y Terapia Génica, Facultad de Medicina. Irunlarrea 1, 31008 Pamplona, Spain
Correspondence to: F. Borrás-Cuesta; E-mail: fborras{at}unav.es
Transmitting editor: T. Watanabe
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Abstract
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To engineer Th determinants (THd) to prime help for humoral or cytotoxic T cell responses, we modified ovalbumin [OVA323337] and myoglobin [MYO106118] eliciting Th1 and Th0 cytokine profiles respectively. Residues along the sequence of both THd were replaced with amino acids representative of different families. Replacements at positions P1 and P5 pointing to the TCR in both THd afforded higher levels of IFN-
and IL-4 production. Peptides eliciting different proportions of IFN-
and IL-4 were co-immunized with a peptide hapten or a T cytotoxic determinant (TCd) respectively. OVA323337- and MYO106118-derived peptides afforded the best THd for the induction of cytotoxic T lymphocyte (CTL) and anti-hapten antibodies respectively. IFN-
and IL-4, primed by MYO106118-derived peptides, correlated significantly with antibody production against the hapten (P < 0.05 for IFN-
and P < 0.05 for IL-4). Interestingly, two peptides derived from OVA323337, 323G and 327G, which induced the clearest Th2 cytokine profiles, were not the most efficient to prime cell help for the induction of anti-hapten antibodies. For CTL induction, OVA323337-derived peptides, inducing a Th1-like profile, required a lower dose (5 nmol) than Th0 peptides (50 nmol). The dose of 50 nmol was detrimental for Th1-like peptides. Interestingly, IFN-
primed by the THd correlated significantly with that induced by the TCd (P < 0.01).
Keywords: antibody, antigen, cytotoxicity, epitope, peptide, Th1, Th2, TCR
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Introduction
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Th lymphocytes (HTL) play a central role in the induction of antibodies (1) as well as cytotoxic T lymphocytes (CTL) (2,3). HTL are usually classified according to the cytokine pattern they induce: Th1 (IFN-
, tumor necrosis factor-ß and IL-2), Th2 (IL-4, IL-5, IL-6 and IL-13) and Th0 (a mixture of cytokines from Th1 and Th2) (4,5). It has been described that a Th1 cytokine profile is associated with CTL responses, whereas a Th2 profile is associated with humoral (antibody) responses (2,68). HTL liberate cytokines following the recognition by the TCR of the complex between the peptide and the MHC class II molecules (MHC-II) presented at the surface of antigen-presenting cells (APC). Peptides presented by MHC-II are referred to as Th determinants (THd). Several factors such as dose of antigen (911), co-stimulatory molecules on APC (1215), cytokine environment (16) and nature of the pathogen (17,18) may have a profound effect on T cell differentiation to produce Th1 or Th2 cytokine profiles. Other factors that affect this differentiation are the strength of interaction between the THd and the MHC-II (1921), and the strength of recognition of the THdMHC-II complex by the TCR (2224). Based on this knowledge, several groups have carried out modifications in the sequence of THd in order to modify their interaction with either the MHC-II (19,21) or the TCR (2224) and reported that strong interactions favor Th1 cytokine profiles. We speculated that systematic replacement of amino acids along the sequence of two well-characterized peptides [both recognized by I-Ad class II molecules (25,26)] by amino acids representative of different families might offer an insight on how changes in the interaction between peptide, MHC-II and TCR of HTL would alter the pattern of cytokines following the recognition of the peptideMHC-II complex by the TCR. Thus, we selected K/E, M/Y and G/A as residues representative of hydrophilic (positively and negatively charged), hydrophobic (aliphatic and aromatic) and small residues respectively.
The crystal structure of the complex between the I-Ad class II molecule and the THd OVA323337, which encompasses residues 323337 from ovalbumin (27), has identified those residues from OVA323337 that point towards the cleft of the I-Ad molecule and those pointing to the TCR of the HTL (28). By analogy to this structure, the same group assigned the relative orientation of amino acid residues from another THd, MYO106118, which encompasses amino acids 106118 from sperm whale myoglobin (29). The knowledge of these two structures offered us the possibility of assigning the effect of amino acid replacements as mainly due to peptideMHC-II or to peptideTCR interactions. Moreover, since OVA323337 and MYO106118 induce Th1 and Th0 cytokine profiles, and are able to prime T cell help for CTL and antibody induction respectively (30,31), we thought that both peptides might be good leading compounds to engineer different types of THd for the induction of humoral and CTL immune responses.
As shown by our group, antibodies against a non-immunogenic B cell epitope can be elicited by co-immunization of this epitope and a THd (31,32). Similarly, CTL responses can be induced by co-immunization of a T cytotoxic determinant (TCd) and a THd (3336). THd eliciting Th1 cytokine profiles are more efficient than those eliciting Th0 profiles (30). As co-immunizations are simple to carry out, it was thought that both strategies offered a convenient way of testing the relative efficacy of a large panel of THd to prime both humoral and cytotoxic T cell responses. The results of these experiments are discussed.
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Methods
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Peptides
Peptides were synthesized by the solid-phase method of Merrifield (37) using a manual multiple solid-phase peptide synthesizer as described in (38). At the end of the synthesis, peptides were cleaved, deprotected and washed 6 times with diethyl ether. They were lyophilized and analyzed by HPLC. The purity of peptides was >80% as judged by HPLC. Two well-characterized THd were synthesized: FISEAIIHVLHSR (MYO106118) corresponding to residues 106118 from sperm whale MYO (26) and ISQAVHAAHAEINEA (OVA323337) encompassing residues 323337 from OVA (27). Both peptides were used as leading compounds to engineer different types of THd. Peptide MSYSWTGALVTPCAAE (P17), which encompasses residues 24222437 from hepatitis C virus NS5 protein and which is not immunogenic per se (39), was used as a TCd in experiments to induce CTL responses. Peptide hapten NNTRKRIRIQRGPGR (gp120301315) encompassing residues 301315 from the principal hypervariable neutralizing domain of HIV type 1 glycoprotein 120 (31) was used in experiments on antibody induction.
Mice
Female BALB/c mice (46 weeks old) were purchased from IFFA Credo (Barcelona, Spain). They were hosted in appropriate animal care facilities and handled following institutional guidelines.
Immunization and measurement of cytokine production
Groups of five mice were immunized by s.c. injection at the base of the tail and footpads with 50 nmol peptide emulsified in incomplete Freunds adjuvant (IFA). Ten days later, animals were killed and lymph nodes removed. Lymphocytes were then plated on 96-well plates at 8 x 105 cells/well with culture medium (RPMI 1640 supplemented with 10% FCS, 2 mM glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin and 5 x 105 M 2-mercaptoethanol) alone or with 30 µM peptide in a final volume of 0.25 ml. Supernatants (50 µl) were removed 24 h later to measure IL-2 production, and 48 h later to measure IFN-
and IL-4. IL-2 was measured using the CTL-L2 bioassay as previously described (30). IFN-
production was measured by ELISA (PharMingen, San Diego, CA) according to the manufacturers instructions. IL-4 was measured using the CT4S cell line (kindly provided by Drs W. E. Paul and C. Watson, National Institutes of Health, Bethesda, MD) as described (30).
Induction and titration of anti-gp120301315 antibodies
Groups of five mice were immunized by i.p. injection of 200 µl of a 1:1 emulsion of complete Freunds adjuvant and saline solution containing a mixture of 60 µg (33 nmol) of the peptide hapten gp120301315 and 50 nmol THd peptide. A group of mice was bled from the retro-orbital plexus at day 30, and another group was boosted with the same dose of gp120301315 and THd peptide in IFA on day 30 and 45, and bled on day 60 after the first injection. Anti-gp120301315 antibodies were titrated by ELISA as follows. Microtiter wells (Maxisorp; Nunc, Roskilde, Denmark) were coated with gp120301315 by overnight incubation at 4°C with 50 µl peptide solution (20 µg/ml in 0.1 M sodium carbonate buffer). The wells were then washed 3 times with a solution of PBS containing 0.1% Tween 20 (PBST). To block non-specific antibody binding, the wells were incubated at room temperature for 1 h with the above buffer, but containing 1% powdered milk (PBSMT). After removing the PBSMT, 50 µl different serum dilutions was added and incubated at 37°C for 1 h. Wells were washed 3 times with PBST and incubated at 37°C for 1 h with a 1/500 solution of goat anti-mouse IgG biotinylated whole antibody (Amersham, Uppsala, Sweden) in PBSMT. After washing 3 times with PBST, the wells were incubated with 1/500 dilution of horseradish peroxidasestreptavidin (Amersham) at 37°C for 1 h, washed 3 times with PBST, and the color reaction started by adding 100 µl of a solution prepared by mixing 10 ml 0.6% acetic acid (pH 4.7), 7.5 µl 33% (w/v) hydrogen peroxide and 100 µl 40 mM water solution of 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (Sigma, St Louis MO). Plates were read at 405 nm in a Titertek Multiscan MKII (Flow, dabsystems, Finland) after 1 h.
Immunization and measurement of CTL responses
Groups of two mice were immunized by s.c. injection at the base of the tail and footpads with a mixture of 60 µg (36 nmol) TCd P17 and 0.5, 5 or 50 nmol THd emulsified in IFA. Animals were killed 10 days after immunization and their lymph nodes removed. Cells (8 x 105/well) were re-stimulated in vitro in 96-well plates using 30 µM THd or 24 µM TCd in 250 µl/well culture medium. CTL activity was estimated by a conventional 51Cr-release assay (30) and by measuring IFN-
production following stimulation of 48-h cultures with P17 as described above.
Statistical analysis
Correlation between variables was carried out using a Pearson test for parametric samples using the SPSS program version 6.1 for Windows package.
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Results
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Effect of single amino acid replacements in the sequence of OVA323337 and MYO106118 on IFN-
and IL-4 production
To develop THd peptides eliciting different cytokine profiles, we selected as leading peptides OVA323337 and MYO106118, for which we knew the relative orientation of their residues to the I-Ad class II molecule and the TCR (28), and carried out single amino acid substitutions in their sequence. Positions of the modified residues are indicated as P3 to P13 according to the nomenclature of Scott et al. (28) who aligned both peptides with respect to their interactions with MHC and TCR molecules, P1 being the main anchor residue of both peptides to I-Ad molecules. Residues P1, P5 and P8 point to the TCR in both peptides whereas residues P4 and P9 point to the MHC-II. The orientation of positions P3, P6, P7, P10, P11, P12 and P13 is not clearly defined. All these positions were systematically replaced by K, E, M, Y, G and A, representative of different families of amino acids; hydrophilic positively or negatively charged (K and E) hydrophobic, aliphatic or aromatic (M and Y) and small (G and A). This afforded 114 peptides that were used to immunize mice in order to study the capacity of these peptides to prime IFN-
, IL-4 and IL-2 production. Figures 1 and 2 show the results of these experiments for OVA323337-modified and MYO106118- modified peptides respectively. At first glance it is clear that many substitutions in both peptides have a profound effect on cytokine production. In general, OVA323337-modified peptides elicited higher levels of IFN-
and lower levels of IL-2 than MYO106118-modified peptides. In both groups, the most dramatic changes in IFN-
and IL-4 production occurred at positions P1 and P5, which point to the TCR. These changes do not go hand-in-hand with changes in IL-2 production, suggesting that modifications affect cytokine profiles rather than cell proliferation. In some cases the levels of IFN-
and IL-4 were more than an order of magnitude higher than the level induced by the parent wild-type peptide. In OVA323337-modified peptides, replacements at positions P1 and P5 with K, E, M and Y enhanced IFN-
and IL-4 production (with the exception of 328K which only induced IFN-
). Modifications with G and A maintained or slightly decreased IFN-
, but increased IL-4 production (with the exception of 328G, which did not induce either of these cytokines). In contrast, modifications in MYO106118 at positions P1 and P5, with K, E, M, Y, G or A, enhanced IFN-
and IL-4 production. Moreover, modified peptides 108G (P1) and 113A (P5) induced the highest levels of IFN-
in these positions. Amino acid replacements in positions other than P1 and P5 affected mainly IFN-
production both in OVA323337 and MYO106118 modified peptides. However, these changes were less important than those observed in P1 and P5.

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Fig. 1. Induction of IFN- and IL-4 (A) and IL-2 (B) following immunization with modified peptides from OVA323337. Mice were immunized with 50 nmol THd and cytokines measured at day 10 (see Methods). The amino acids of the wild-type peptides are indicated using the single letter code as well as the number they occupy in their corresponding protein sequence. P3 to P13 are used to designate positions of residues after alignment of OVA323337 with MYO106118 following the nomenclature given in (28). P1, P5 and P8 (and probably P12, see Discussion) are probably the main residues pointing to the TCR, whereas P1 (not studied), P4 and P9 are those pointing to the MHC. The corresponding wild-type peptide is indicated with an asterisk, whereas amino acid replacements are indicated with a letter above each bar.
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Fig. 2. Induction of IFN- , and IL-4 (A) and IL-2 (B) following immunization with modified peptides from MYO106118. For nomenclature and conditions, see the legend to Fig. 1.
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T cell help for antibody induction
As discussed earlier in the Introduction, one of the aims of the present study was to develop THd peptides capable of priming T cell help for efficient antibody induction. For this purpose we selected a wide range of peptides derived from MYO106118 as well as from OVA323337, inducing different ratios of IFN-
and IL-4. Thus, we tested 108G, 108M, 111Y, 112Y, 112G, 113K, 113M and 115M, and also 323K, 329E, 329Y, 330M, 330Y and 336G, as well as wild-type peptides MYO106118 and OVA323337 with respect to their ability to elicit Th responses for the induction of antibodies against peptide gp120301315. This peptide, from the principal neutralizing domain of HIV type 1 gp120 envelope protein, is not immunogenic per se (31). Each of the THd peptides was co-immunized with gp120301315 as previously described. Anti-gp120301315 antibodies were measured at day 30 and 60 in two different groups of mice. As shown in Table 1, anti-gp120301315 antibody titers induced with the help of MYO106118-derived peptides were much higher than those induced with OVA323337-derived peptides. Indeed, whereas MYO106118-derived peptides primed antibody induction at day 30, OVA323337-modified peptides did not. Moreover, a difference of over one order of magnitude in the antibody titers against gp120301315 was observed at day 60 when using the best peptide from each series: 111Y and 336G respectively. We studied the correlation between IFN-
or IL-4 production by the THd peptides and the anti-gp120301315 titers induced. It was found that in the case of MYO106118-derived peptides, both cytokines correlated significantly with anti-gp120301315 production at day 30 (R = 0.77, P < 0.05 for IFN-
and R = 0.78, P < 0.05 for IL-4) (Fig. 3A and B respectively), but no correlation was found at day 60 after three immunizations. In the case of OVA323337-derived peptides, no correlation was found between anti-gp120301315 and IFN-
or IL-4 production at day 60. No correlation could be tested at day 30 due to the absence of anti-gp120301315 antibodies at this time. A similar test carried out to study the correlation between IFN-
and IL-4 production showed no correlation between both cytokines.
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Table 1. Induction of antibodies against peptide hapten gp120301315 after immunization with gp120301315 in conjunction with different THd peptides inducing various levels of IFN- and IL-4
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Fig. 3. Correlation between cytokine production and anti- gp120301315 antibody titers induced at day 30 following co-immunization of THd derived from MYO106118 and gp120301315. (A) Correlation with IFN- . (B) Correlation with IL-4. The values used are those from Table 1.
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T cell help for CTL induction
To test how different cytokine profiles affected CTL induction, we used peptides derived from OVA323337, known to be an efficient THd for CTL induction (30,40,41). We selected peptides inducing high levels of IFN-
(330M, 331G and 336G), peptides inducing IL-4 and high levels of IFN-
(323K, 327E and 328M), as well as OVA323337 to immunize mice at three different doses of THd (0.5, 5 and 50 nmol) in conjunction with 60 µg (36 nmol) of P17, a TCd which is unable to induce a CTL response per se (42). We tested different doses of THd in order to get an insight on how the dose and the cytokine profile might affect the efficacy of CTL induction. Lymph node cells from all groups of immunized mice were extracted as described in Methods and used to measure CTL activity against P17-pulsed target cells. Since percentages of CTL lysis were found to be too low (data not shown), we decided to measure IFN-
production as an alternative measure of induced CTL activity via the T cell help from THd peptides. It was thought that this was a valid approach because immunization with P17 alone is unable to induce IFN-
production (data not shown). Thus, following in vitro stimulation with the corresponding THd peptide or with the CTL epitope P17 (42), we measured the level of IFN-
production. In three peptides (OVA323337, 323K and 328M) the highest levels of IFN-
induced, both against the THd itself and the TCd, were detected in those animals immunized with the higher dose of THd (50 nmol). By contrast, in peptides 331G, 336G and 330M, the higher levels were detected at the intermediate dose of immunization (5 nmol) (Fig. 4A). Peptide 327E induced IFN-
against itself, but not against the TCd. As shown in Fig. 4(B), when the level of IFN-
produced after stimulation with the THd was plotted against the level of IFN-
produced by the TCd peptide P17, a highly significant correlation was found between both parameters (R = 0.842, P < 0.01).
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Discussion
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Effect of single amino acid replacements in the sequence of OVA323337 and MYO106118 on IFN-
and IL-4 production
Previous work from other groups has shown that single amino acid substitutions in peptides recognized by MHC molecules may alter TCR recognition. Thus, Kersh et al. (43) reported that a substitution of an anchor residue, E73, by D73 in the sequence of THd peptide, although it did not affect binding to I-Ek, affected the orientation of the peptide to the TCR molecule, even 10 Å away from the substitution. This replacement strongly reduced the T cell response to the peptide presented by the I-Ek molecule. Similarly, a single amino acid substitution (Q
E) in the sequence of a TCd from vesicular stomatitis virus (44) greatly altered the T cell response triggered after recognition of the complex TCdMHC-I by TCR. Other groups have reported that changes in the sequence of a THd that enhance or decrease binding to MHC-II or TCR molecules may lead to THd inducing Th1 or Th2 cytokine patterns respectively (19,2124). With the aim of learning rules to modulate the production of IFN-
and IL-4 by THd, we studied the effect on IFN-
and IL-4 production after replacing residues along the sequence of two well-characterized THd by amino acids from different families: hydrophilic positively or negatively charged (K and E), hydrophobic aliphatic or aromatic (M and Y) and small (G and A). It was hoped that this strategy might lead to the discovery of THd for the efficient and specialized induction of humoral or cytotoxic cell responses. As shown in Figs 1(A) and 2(A), the most pronounced effects on IFN-
and IL-4 production were observed following replacement of residues at positions P1 and P5, which are reported to point to the TCR (28) both in OVA323337 and MYO106118. Since these changes do not go in the same direction as changes in IL-2 production, it is likely that modifications affect cytokine profiles rather than cell proliferation. It could be interpreted that the enhanced IFN-
and IL-4 production at these positions is related to the selection of best-fit TCR from the existing large diversity of the TCR repertoire (45), for a given peptideMHC-II complex. However, if the substituted residue interacts mainly with the MHC-II, no best-fit accommodation may take place, rendering the possibility of greatly enhanced IFN-
and/or IL-4 production less likely, unless the residue has an important impact in binding to MHC-II. Since we did not measure binding of peptides to I-Ad molecules, we cannot tell if changes in TCR positions may have also affected binding to I-Ad and consequently if changes in cytokine production are due to changes in TCR or MHC-II interactions. However, from the work of Sette et al. (46), we know that substitution 328E binds less well to I-Ad than OVA323337, and, in spite of this reduced binding, the peptide greatly enhances IFN-
and IL-4 production (Fig. 1), suggesting a compensation due to the selection of a population of best-fit TCR.
From Fig. 1 it is clear that substitutions at P1 and P5 in OVA323337 by amino acids K, E, M and Y afforded peptides inducing higher levels of IFN-
than substitutions by G and A. Interestingly, according to the work of Singh and Thornton (47), the first group of amino acids has a higher propensity of interaction in proteins than the second group. By contrast, replacement by G or A in MYO106118 had an equal or greater effect than K, E, M or Y. We postulate that enhanced IFN-
production following replacements by the small size residues G and A in MYO106118 may allow an increased flexibility of the TCR to interact with the I-Ad molecule. This might be related to non-bulky residues A or G not keeping the TCR as far away from the MHC-II as a bulky residue protruding from the I-Ad molecule might. This interpretation is in line with the observation that replacement of V by A at position 5 in the sequence of AH1 (a peptide presented by H-2Ld) enhances the stability of the MHCpeptideTCR complex without altering the binding to H-2Ld (48).
In the present work we show that some replacements at P12 enhanced IFN-
and IL-4 production in OVA323337-modified peptides. This, according to our hypothesis, suggests that P12 is likely to interact with some population of best-fit TCR. By contrast, substitutions at P8, which is described as a position pointing to the TCR, had little or no effect on cytokine production, both in MYO106118 and OVA323337, suggesting that P8 might not be that important in TCR recognition. It is interesting to note that, contrary to what was found for substitutions at positions P1 and P5, which affected both IFN-
and IL-4 production, substitutions in other positions affected mainly IFN-
, with little or no effect on IL-4 production. This is particularly evident for substitutions at L115, which corresponds to P7 in MYO106118. It is tempting to speculate that changes leading to a moderate enhancement in interaction increase IFN-
production, whereas strong changes (like the ones observed for the main positions pointing to the TCR) lead to an enhancement of both IFN-
and IL-4 production. This does not agree with the interpretation by Ise et al. (49) that IL-4 is induced before IFN-
when increasing the peptide concentration, but it is in line with the results from Rogers et al. (50) who reported that over the short term of differentiation, IFN-
-producing cells are induced by lower levels of stimulation than IL-4-producing cells, although optimal induction of both cytokines was seen with the same high level of stimulation. However, since the work of Ise et al. (49) and Rogers et al. (50) was carried out using transgenic T cells bearing a single TCR, their results may not be strictly comparable with ours. Indeed, we report here the average effect obtained in vivo under conditions that resemble vaccination (or what may happen after infection with a virus), where a wide polyclonal population of TCR molecules recognizes the THd.
Based on the crystal structure of OVA323339 bound to I-Ad, Scott et al. (28) assigned positions P4 (V327) and P9 (A332) as pointing to the MHC and P1 (I323) and P5 (H328) to the TCR. In the same paper, these authors postulate also that this peptide may bind to I-Ad molecules in two other registers. The possibility of more registers of binding of this peptide to I-Ad molecules has also been put forward by Roberson et al. (51). However, since crystallization leads to a single crystal structure of the peptideMHC complex, and the TCR positions P1 and P5 assigned by Scott et al. as the main TCR positions coincide with those having the greatest effect on cytokine production in our work, we suggest that anchoring of OVA323337 to I-Ad may take place in such a way that positions P1 and P5 are favored to interact with the TCR.
In conclusion, our data suggests that substitutions in the sequence of THd with amino acids having high or a low propensity of interaction in proteins may substantially alter IFN-
and/or IL-4 production. However, in most cases, these changes seem to have greater effect when they correspond to residues pointing to the TCR. This information may be very valuable to engineer peptides eliciting different cytokine profiles.
T cell help for antibody induction
From Table 1 it is clear that to prime T cell help for anti-gp120301315 antibody induction, MYO106118-derived peptides were much more efficient than those derived from OVA323337. The best antibody titers at day 60 were obtained when using THd eliciting both IFN-
and IL-4, i.e. peptides eliciting Th0 cytokine profiles. However, THd inducing only IFN-
(112Y, 329E and OVA323337) were also able to prime T cell help for the induction of anti-gp120301315 antibodies. These results indicate that both IFN-
and IL-4 production are relevant in antibody induction. Moreover, among all modified peptides from MYO106118 and OVA323337, 323G and 327G were those that induced a cytokine profile that was closer to a Th2 pattern (Fig. 1). However, none of these two peptides was the best to provide T cell help for the induction of anti-gp120301315 antibodies, even within those peptides derived from OVA323337. This result suggests that a Th2 pattern is not necessarily the best to induce antibodies. It is not clear to us why MYO106118-derived peptides were much more efficient than OVA323337-derived peptides to provide T cell help for antibody induction, especially when considering that peptide 330M that induces similar levels of IFN-
and IL-4 than peptide 111Y leads to greatly different anti-gp120301315 antibody titers, 5300 and 242,000 respectively. It could be postulated that cytokines from the Th1 and Th2 subsets, other than IFN-
and IL-4, may be of relevance. However, it may also be possible that those steps leading to the presentation of THd by MHC-II may also be of relevance. For instance, the relative ability of THd peptides to enter the APC or bind directly to the MHC-II at the surface of APC. Moreover, since B lymphocytes behave as APC, it may be possible that if the B lymphocytes that recognize the hapten (in our case gp120301315) simultaneously bind the THd, these lymphocytes might expand more readily via the cytokine stimuli induced in situ after recognition of the THdMHC-II complex (presented by the B lymphocyte) by the TCR from a HTL, than if the cytokine stimuli is elicited when the THdMHC-II complex is presented by an APC other than the B lymphocyte itself.
To attempt to interpret the effect of different THd in priming antibody production, we correlated the levels of IFN-
and IL-4 induced by these THd, with anti-gp120301315 antibody titers induced at day 60. It was found that neither of these two cytokines correlated significantly with antibody production. Since anti-gp120301315 antibody titers at day 60 are obtained following three immunizations (day 0, 30 and 45) with gp120301315 and THd, whereas the cytokine profiles for all THd were obtained after a single immunization with THd, it occurred to us that in correlation studies the antibody titers at day 30 might better reflect the T cell help provided by the THd. For this reason, we immunized a new group of mice and measured antibody titers at day 30. When IFN-
and IL-4 production by the group of MYO106118-modified peptides was correlated with antibody production at day 30 (Fig. 3A and Fig. 3B), a significant correlation was found for both cytokines (R = 0.77, P < 0.05 for IFN-
and R = 0.78, P < 0.05 for IL-4). These results are in agreement with the published observation that Th1 and Th2 CD4+ T cells provide help for B cell clonal expansion and antibody synthesis in a similar manner in vivo (52). No correlation was found between IFN-
and IL-4 primed by THd used in our experiments, suggesting that in our system both cytokines are independent variables. For OVA323337-modified peptides, cytokines could not be correlated with antibody titers at day 30 because these titers were too low or nil. Although we do not have a clear-cut interpretation of why there is a correlation at day 30, but not at day 60, we believe that a possible interpretation might be that changes in cytokine profiles and concomitant antibody titers may take place after repeated immunizations with THd.
Irrespective of the detailed mechanism by which THd provide help for antibody induction, it is clear that single amino acid replacements in their sequence may have profound effects on their ability to induce antibodies. With respect to MYO106118, some of these peptides have shown an enhanced capacity to provide T cell help for antibody induction, a result that may have useful applications in the production of mAb against non-immunogenic peptides or haptens in general.
T cell help for CTL induction
To study how IFN-
and IL-4 production affected CTL induction, we immunized mice with P17 in combination with different THd peptides derived from OVA323337. We selected OVA323337-derived peptides instead of MYO106118-derived peptides because we had shown that OVA323337 is more efficient than MYO106118 in priming T cell help for CTL induction (30). We selected peptides inducing high levels of IFN-
, but different IFN-
:IL-4 ratios (Fig. 1A). As shown in Fig. 4, peptides having a strong Th1 character (331G, 336G and 330M) induced the strongest CTL response at the intermediate dose of immunization (5 nmol), whereas the wild-type peptide OVA323337 which elicits a weaker Th1 cytokine response pattern, and peptides 323K and 328M, which produce high levels of IFN-
, but also IL-4 (Th0 cytokine pattern), were more active at 50 nmol. Peptide 327E, which also elicited a Th0 cytokine profile, provided very little help to the P17 peptide at the three concentrations tested. When the levels of IFN-
induced by each THd peptide were correlated with the corresponding level of IFN-
induced by P17, a high correlation (P < 0.01) was observed.
In conclusion, the strongest CTL inductions were obtained after immunization with a TCd and a THd eliciting Th1 cytokine profiles (Fig. 4). The dose of THd peptide was also found to be of great relevance for efficient CTL induction. This last result is in agreement with that reported by Alexander et al. (53) where a dose of 5 nmol THd PADRE was more efficient than 100 nmol to prime T cell help for CTL induction. These findings have obvious implications when developing vaccination protocols. As previously reported by us (30,33,41), exogenous THd peptides may prove to be very valuable to induce CTL responses in cancer, as well as in viral chronic infections. Finally, although more speculative, the influence of amino acid residues pointing to the TCR may be of value in predicting immunodominant THd from antigens. Similar reasoning might obviously be applied in the understanding of the immunogenicity of peptides presented by MHC-I.
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Acknowledgements
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This work has been supported by grants from CICYT (SAF2000-0059 and SAF97/0223). It was also supported in part by a grant from Instituto de Salud Carlos III (C03/02). A. L.-D. de C. was a recipient of a scholarship from the Ministerio de Educación y Ciencia (AP 9672673897). N. C. was a recipient of a scholarship from Gobierno Vasco.
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Abbreviations
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APCantigen-presenting cell
CTLcytotoxic T lymphocytes
HTLTh lymphocytes
IFAincomplete Freunds adjuvant
MHC-IIMHC class II molecules
MYOmyoglobin
OVAovalbumin
PBSTPBS containing 0.1% Tween 20
PBSMTPBS containing 0.1% Tween 20 + 1% powdered milk
TCdT cytotoxic determinant
THdTh determinant
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References
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