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Oct 13, 1999 - and heterotypic T-cell antigenic site for pigs that is capable of ..... Francis, M. J., G. Z. Hastings, A. D. Syred, B. McGinn, F. Brown, and D. J..
JOURNAL OF VIROLOGY, May 2000, p. 4902–4907 0022-538X/00/$04.00⫹0 Copyright © 2000, American Society for Microbiology. All Rights Reserved.

Vol. 74, No. 10

Interspecies Major Histocompatibility Complex-Restricted Th Cell Epitope on Foot-and-Mouth Disease Virus Capsid Protein VP4 ESTHER BLANCO,1 KENNETH MCCULLOUGH,2* ARTUR SUMMERFIELD,2 JUDE FIORINI,2 ´ S,3 PAUL BARNETT,4 DAVID ANDREU,3 CRISTINA CHIVA,3 EVA BORRA 1,5 AND FRANCISCO SOBRINO Centro de Investigation en Sanidad Animal, INIA, Valdeolmos, 28130 Madrid,1 Departament de Quı´mica Orga `nica, Universitat de Barcelona, 08028 Barcelona,3 and Centro de Biologı´a Molecular “Severo Ochoa” (CSIC-UAM), Cantoblanco, 28049 Madrid,5 Spain; Institute of Virology and Immunoprophylaxis, Mittelha ¨usern, Switzerland2; and BBSRC Institute for Animal Health, Pirbright, Surrey GU24 ONF, England4 Received 13 October 1999/Accepted 16 February 2000

T-cell epitopes within viral polypeptide VP4 of the capsid protein of foot-and-mouth disease virus were analyzed using 15-mer peptides and peripheral blood mononuclear cells (PBMC) from vaccinated outbred pigs. An immunodominant region between VP4 residues 16 and 35 was identified, with peptide residues 20 to 34 (VP4-0) and 21 to 35 (VP4-5) particularly immunostimulatory for PBMC from all of the vaccinated pigs. CD25 upregulation on peptide-stimulated CD4ⴙ CD8ⴙ cells—dominated by Th memory cells in the pig—and inhibition using anti-major histocompatibility complex class II monoclonal antibodies indicated recognition by Th lymphocytes. VP4-0 immunogenicity was retained in a tandem peptide with the VP1 residue 137 to 156 sequential B-cell epitope. This B-cell site also retained immunogenicity, but evidence is presented that specific antibody induction in vitro required both this and the T-cell site. Heterotypic recognition of the residue 20 to 35 region was also noted. Consequently, the VP4 residue 20 to 35 region is a promiscuous, immunodominant and heterotypic T-cell antigenic site for pigs that is capable of providing help for a B-cell epitope when in tandem, thus extending the possible immunogenic repertoire of peptide vaccines. 2-ml dose (this payload has a 50% protective dose [PD50] of 112 in cattle, as defined by the European Pharmacopoeia). The vaccine was formulated as a water-in-oil-in-water emulsion with Montanide ISA 206 (SEPPIC), and the animals were boosted at 4 and 8 weeks with an equivalent dose. At least four different swine leukocyte antigen (SLA) alleles were present in these vaccinated animals (Birte Kristensen, personal communication). With the first litter, three additional littermates were inoculated with phosphate-buffered saline (PBS) and three were inoculated with adjuvant alone as negative controls. The second litter provided two additional littermates for each of the PBS and adjuvant controls. Three additional outbred pigs were immunized intramuscularly with a commercial vaccine (Merial) prepared from a type O-Manissa virus and boosted 4 and 8 weeks later with the same dose. Seroconversion in vaccinated pigs. The serum neutralization test (European Pharmacopoeia) was employed to determine if the generated response was as expected from such a vaccine. The anticipated seroconversion did, indeed, occur in all of the animals between 5 and 7 days postvaccination, peaking at 3 to 4 weeks postvaccination (data not shown). Proliferative response of PBMC against VP4 peptides. PBMC were obtained from the vaccinated pigs (22, 24). Proliferation assays (29) employed 14 overlapping synthetic peptides spanning the entire VP4 sequence (20). The sequence was that of FMDV type C isolate C-S8 (20), which is identical to the C1-Obb isolate (4) employed in the vaccine. These peptides (Table 1) were synthesized by solid-phase methods (21, 25) to ⬎80% purity and checked by amino acid and matrix-assisted laser desorption ionization–time of flight mass spectrum analyses. An additional peptide—VP4-0—represented the VP4 residue 20 to 34 antigenic site described from bovine analyses (40). Dose-dependent in vitro responses of the PBMC were obtained (Fig. 1 shows examples of peptides VP4-3, VP4-4, VP4-5, and VP4-0). A high level of variation

Foot-and-mouth disease virus (FMDV) is the causative agent of a highly contagious disease affecting cloven-hoofed animals that is capable of periodic reintroduction into areas such as Europe, where routine vaccination has been terminated (18). FMDV belongs to the Aphthovirus genus of the family Picornaviridae (27). The virus particle contains a positive-strand RNA molecule within a nonglycosylated icosahedral capsid composed of four viral polypeptides, VP1 to VP4 (2, 31). Vaccination traditionally uses inactivated whole-virus vaccines, the objective being induction of the specific antibody central to protective immune defenses (23). Although recombinant VP1 and peptides containing the VP1 (residues 137 to 156) continuous B-cell epitope or carboxy terminus have been tested (6, 13), these conferred lower protection than wholevirus vaccines (7, 39), primarily due to the absence of T-cell epitopes (11, 15). Of the T-helper (Th)-cell epitopes identified on FMDV proteins (9, 10, 15, 16, 29, 35, 40), those conserved among different FMDV strains and recognized by different major histocompatibility complex (MHC) allelic forms would be preferred for vaccine application (31). In this respect, the VP4 structural protein (32) is highly conserved among FMDV serotypes and other picornaviruses (4) and possesses an MHC-promiscuous T-cell site for cattle—with respect to four MHC class II alleles (40). The present study therefore sought to identify T-cell epitopes on VP4 recognized by peripheral blood mononuclear cells (PBMC) from vaccinated pigs. Outbred White Landrace pigs from two litters, 3 to 6 month old, were immunized intramuscularly with an inactivated-virus vaccine made with FMDV strain C1 Oberbayern (C1 Obb) at 2.86 ␮g of 146S antigen per * Corresponding author. Mailing address: Institute of Virology and Immunoprophylaxis, Sensemattstrasse 293, 3147 Mittelha¨usern, Switzerland. Phone: 41-31-8489361. Fax: 41-31-8489222. E-mail: kenneth [email protected]. 4902

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TABLE 1. Overlapping synthetic 15-mer peptides derived from the sequence of structural protein VP4 Peptide

Sequence

Residues

1 2 3 4 0 5 6 7 8 9 13 14 BTa

GAGQSSPATGS SPATGSQNQSGNTGS SQNQSGNTGSIINNY GNTGSIINNYYMQQY SIINNYYMQQYQNSM IINNYYMQQYQNSMD YMQQYQNSMDTQLGD QNSMDTQLGDNAISG TQLGDNAISGGSNEG NAISGGSNEGSTDTT NTQNNDWFSKLASSA DWFSKLASSAFSGLF TASARGDLAHLTTTHARHLPSIINNYYMQQYQNSM

1–11 6–20 11–25 16–30 20–34 21–35 26–40 31–45 36–50 41–55 61–75 66–80 VP1 137–156 ⫹ VP4 20–34

a

This tandem peptide contained VP1 residues 137 to 156 colinearly synthesized with those of peptide VP4-0.

was noted between PBMC from different animals, in terms of both the kinetics of the response and the recognition of individual peptides. For example, at 14 days postvaccination (shown in Fig. 1), PBMC from pig 314 responded strongly, whereas cells from animal 316 were less responsive (Fig. 1, solid diamond compared with open circle). The quality of these proliferations can be ascertained in the context of the responses generated using PBMC from the negative controls— littermates of the vaccinated animals which had received PBS or adjuvant alone instead of the vaccine. These control animals

FIG. 1. Effect of peptide dose on the proliferative response of PBMC obtained from C1-Obb-vaccinated pigs at 10 days (pigs 112 and 113) or 14 days (pigs 313 to 316) postimmunization (booster vaccination). VP4-3 residues 11 to 25 (A), VP4-4 residues 16 to 30 (B), VP4-0 residues 20 to 34 (C), and VP4-5 residues 21 to 35 (D) are shown as examples, for which the background proliferation of each animal’s PBMC has been subtracted (now corresponds to 0 cpm on the y axis). For comparison, the arrow shows the maximum number of counts per minute obtained with stimulation of PBMC from negative control (nonimmune) pigs.

were handled and bled identically to the vaccinated pigs. For clarity, only the maximum response by these nonimmune cells is shown in Fig. 1 (arrow). It was subsequently determined that the characterization of pigs 314, 316, 112, and 113 as poor responders or nonresponders was incorrect. If the PBMC were prepared at 35 days postvaccination, certain peptides were found to be immunostimulatory. This effect is demonstrated in Fig. 2A. Optimum responses for PBMC from pigs 313 and 315 were found at 14 days postvaccination, whereas PBMC from pigs 314 and 316 did not show optimum responses until 35 days postvaccination (these are shown in Fig. 2A). Figure 2A also shows that the responses against the peptides were specific. Of particular use in this sense were the poorly immunogenic VP4-14 peptide and the VP4-3 peptide which was nonstimulatory for PBMC from pigs 314 and 316. The low level of stimulation obtained with these peptides demonstrated the relevance of the levels of stimulation obtained with the other peptides. Such comparisons were considered to be more pertinent than the use of a peptide bearing no resemblance to the VP4 sequence. Spectrum of VP4 recognition by different vaccinated pigs. Most of the peptide sequences tended to be restricted in terms of recognition only by PBMC from particular individual animals. Despite this variation between animals, certain peptides were consistently recognized by PBMC from all of the pigs vaccinated. VP4-5, VP4-0, and to some extent VP4-4 provided consistent interanimal recognition by PBMC (Fig. 2A), dependent on the time postvaccination: the highest responses were found at 14 days after booster vaccination for pigs 313 and 315 and at 35 days for pigs 314 and 316. A similar situation was noted with vaccinated pigs 112 and 113 (data not shown). In contrast to the significant stimulation induced by VP4-5 and VP4-0 in PBMC from the vaccinated pigs, there was a lack of stimulation with PBMC from the negative control littermates inoculated with PBS or adjuvant alone: the highest stimulation index (SI) obtained with these negative control PBMC was always ⬍2 (data not shown). These antipeptide responses coincided with that against whole-virus antigen (Fig. 2A). When the antipeptide response was relatively poor, as with the PBMC from pigs 314 and 316 at 14 days, the antivirus response was also weak. With stronger antipeptide responses, as with PBMC from animals 314 and 316 at 35 days postvaccination, the antivirus response was also higher. Importance of the VP4 residue 20 to 35 T-cell antigenic site in porcine immune responses against FMDV. Considering the

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FIG. 2. (A) Proliferative responses of PBMC obtained at 14 days (pigs 313 and 315) and 35 days (pigs 314 and 316) against optimum stimulatory concentrations of VP4 peptides 0 to 14 (4 ␮g/ml). The animals were vaccinated twice with monovalent FMDV serotype C1-Obb vaccine in an oil adjuvant. Data are expressed as SIs (counts per minute in the presence of peptide divided by counts per minute in medium alone), and each bar represents the mean of triplicate cultures. The level obtained with medium alone was always ⱕ2,000 cpm. (B) Relative positions on VP4 of the sequences of VP4-4, -5, and -0.

above results, analyses focused on the nested peptides within the region between residues 16 and 35. Although significant responses against peptide VP4-0 were found with PBMC from all of the animals analyzed, the highest levels of stimulation were obtained with VP4-4 for animal 315 PBMC and with VP4-5 for PBMC from animals 313, 314, and 316 (Fig. 2A). Peptide VP4-5 also induced the highest levels of proliferation in PBMC from the vaccinated pigs in the other experiment— animals 112 and 113 (data not shown). Peptides VP4-4 and VP4-5 share 10 and 14 amino acids, respectively, with peptide VP4-0 (Fig. 2B), demonstrating that the VP4 residue 22 to 30 region contains at least one particularly potent T-cell determinant. It is possible that the three peptides possessed the same immunogenic motif, while their different flanking sequences could modify lymphocyte activation (41) or the susceptibility of the T-cell epitopes to proteases in antigen processing (12). Alternatively, 15-mer peptides may bind directly to MHC molecules rather than be processed through the endocytic pathway (36). Thus, the differences in the responses induced by VP4-0 and VP4-5 may reflect variations in affinity for interaction with MHC molecules or the T-cell receptor of the lymphocytes they activate.

J. VIROL.

The relative roles played by flanking sequences and direct binding to MHC molecules must await further analyses on shorter peptide sequences or the use of T-cell clones. Nevertheless, the residue 20 to 35 region of VP4 clearly contained at least one potent T-cell determinant. Whether this is a single or multiple motif is currently under study with shorter peptide sequences. It was interesting that the VP4-0 residue 20 to 34 and VP4-5 residue 21 to 35 sequences overlapped residues 19 to 20 and 25 to 35 of VP4, which were predicted to be amphipathic using the AMPHI algorithm (19). In contrast, the peptide sequence of weakly immunogenic and restricted (interanimal) VP4-13 residues 61 to 75 overlapped an amphipathic segment predicted by the SOHHA algorithm (14) and the AMPHI and MOTIF programs (30). The relevance of the VP4 epitopes in the context of the virus can be seen by comparing the immunogenicity of the VP4 peptides with that of similar overlapping peptides derived from VP1. In agreement with Rodrı´guez et al. (29), no single VP1 peptide consistently and efficiently stimulated PBMC from all of the vaccinated pigs, even when these PBMC were obtained at different times postvaccination (data not shown). Furthermore, none of the VP1 peptides were as potent for restimulation as VP4-5, as shown in Fig. 2A. Antigenicity of VP4-0 residues 20 to 34 in tandem with the sequential B-cell site at VP1 residues 137 to 156. If such antigenic VP4 T-cell epitope peptides were to have value in vaccine formulations, they should remain immunostimulatory when in combination with B-cell epitopes. A tandem peptide of VP4-0 residues 20 to 34 colinearly synthesized with the VP1 residue 137 to 156 sequential B-cell site still induced lymphoproliferation in vitro (Fig. 3A). This demonstrated the retained availability of the T-cell antigenic site. PBMC from one animal also displayed some proliferation upon stimulation with VP1 residues 137 to 156 alone, but less efficiently compared with the responses against the tandem peptide and VP4-0. It is not clear why the B-cell epitope peptide induced this low level of stimulation, but it should be emphasized that this was not observed with all of the animals and that the level was rather low. One possibility is that the peptide could, on occasion, stimulate the B lymphocytes to proliferate, perhaps with the assistance of T lymphocytes which had already been stimulated in vivo before isolation of the PBMC. Certainly, the B-cell epitope-containing peptide was antigenic. Whether alone or in tandem with the T-cell epitope peptide, it reacted with monoclonal antibodies (MAb) raised against the whole virus in immunoblotting analyses (data not shown). It was also necessary to know if the T-cell epitopes within VP4-0 and VP4-5 were functionally active. That is, whether they could stimulate T cells to provide the necessary immunological help for antigen-stimulated B lymphocytes, resulting in specific-antibody production. Collen et al. (9) had demonstrated that a VP1-derived T-cell epitope peptide was immunogenic in cattle when used in tandem with a residue 140 to 160 B-cell epitope peptide. In the present analyses, a T-cell– B-cell tandem consisting of VP4-0 residues 20 to 34 and VP1 residues 137 to 156 induced the production in vitro of antiFMDV neutralizing activity by PBMC from vaccinated pigs over a 2-week period (Table 2). This activity was serotype specific and was therefore considered to be due to the antivirus antibody. The B-cell epitope peptide alone was inefficient at inducing such an antibody, and the T-cell peptide alone provided only background interference with the virus, as obtained with supernatants from unstimulated PBMC. Consequently, the ability of the VP4-0 peptide to stimulate lymphocyte proliferation could be translated into functional immunological

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TABLE 2. Induction of anti-FMDV neutralizing antibodies by in vitro restimulation of immune porcine PBMC with a T-cell–B-cell tandem peptide Stimulating peptide

TBc

B T

No. of days poststimulation of immune PBMC in vitroa

Mean % reduction of FMDV PFUb ⫾ SD

4 7 13

49 ⫾ 7 71 ⫾ 11 62 ⫾ 9

7 7

16 ⫾ 5 8⫾2

a We obtained 106 PBMC from FMDV-immune pigs, and placed them into culture as described in the text. The cells were stimulated with the appropriate peptide (TB, B, or T) at 4 ␮g/ml. Cells cultured in the absence of a peptide served as negative controls. Culture supernatants were obtained at 4, 7, and 13 days poststimulation, and the anti-FMDV neutralizing activity was measured. b Anti-FMDV neutralizing activity was measured as described by Rodrı´guez et al. (29). Briefly, 60 to 80 PFU of FMDV type C isolate C-S8 was incubated for 45 min with 125 ␮l of the PBMC culture supernatants. The mixtures were then used to infect preformed BHK-21 cell monolayers and overlaid with 1.3% (wt/ vol) low-melting-point agarose. Infection was allowed to progress for 18 h at 37°C. The monolayers were then fixed and stained with 0.5% (wt/vol) crystal violet in 10% (vol/vol) aqueous acetic acid solution and the PFU were counted. Inhibition of the virus was scored as a percentage of the difference between the number of PFU in negative control supernatants and the number of PFU in peptide-stimulated supernatants divided by the number of PFU in the control supernatants. The specificity of this inhibition was confirmed when no PFU reduction was obtained with a type O virus (O1 Kaufbeuren). c The stimulating peptides for the in vitro PBMC cultures were TB (VP4-0 residues 20 to 34 plus VP1 residues 137 to 156), B (VP1 residues 137 to 156), and T (VP4-0 residues 20 to 34).

FIG. 3. (A) Comparative lymphoproliferation of PBMC obtained from pigs 314 and 316 at 35 days post-booster vaccination, stimulated in vitro with the VP4-0 (residues 20 to 34) T-cell epitope peptide, the sequential VP1 (residues 137 to 156) B-cell epitope peptide, or a T-cell–B-cell tandem of these two peptides (4 ␮g/ml). The levels obtained with medium alone were 350 (pig 314) and 750 (pig 316) cpm (⫾10%). (B) Heterologous peptide-induced lymphoproliferation. Shown is an analysis of the capacity of VP4-4 and VP4-5, in comparison with whole C-S8 FMDV virion antigen, to stimulate in vitro lymphoproliferation of PBMC obtained from pigs vaccinated with the heterologous FMDV O-Manissa vaccine. The levels obtained with medium alone were 900 (pig 12), 3,500 (pig 13), and 950 (pig 14) cpm (⫾10%). In both panels A and B, data are expressed as SIs determined as described in the legend to Fig. 2.

help when the stimulatory peptide was in tandem with a B-cell epitope-containing peptide. Heterotypic recognition of VP4-4 and VP4-5. The VP4 region from residue 16 to residue 30 is totally conserved among FMDV serotypes A, O, and C. Consequently, VP4-4 and VP4-5 were tested for heterotypic recognition through stimulation of PBMC obtained at 30 days postvaccination from pigs immunized with a monovalent type O vaccine (O-Manissa), the cells being obtained at 30 days after the last vaccination. Two sets of the PBMC proliferated significantly in response to the peptides (Fig. 3B). PBMC from pig 12 responded slightly, but only to VP4-5. This level of stimulation was not as high as that obtained with the homologous virus antigen nor that obtained with the whole heterologous virus antigen (Fig. 3B), contrasting with the homologous lymphoproliferation assay results. It is not clear why there should be this bias toward

homotypic lymphoproliferation, unless antigen processing was a more critical element with respect to heterologous recognition. MHC (SLA) restriction of the anti-VP4 peptide lymphoproliferative response. Anti-porcine SLA class I (MAb 74-11-10 [28]) and class II (MAb MSA-3 [17]) were used to block proliferation (8) in response to VP4-0 or VP4-5. A total of 15 ␮l of the appropriate MAb (1 mg/ml) was added per well at the beginning of culture, and an additional 15 ␮l per well was added after 24 h of incubation. The anti-SLA class II MAb inhibited VP4-0-induced lymphoproliferation by more than 80% (Fig. 4A), and a lower level of inhibition (40%) was found with the anti-SLA class I MAb. The level of inhibition of VP4-5-induced proliferation was also higher with the anti-SLA class II MAb than with the anti-SLA class I MAb (data not shown). As negative controls, MAb against the ␥␦ T-cell receptor or the panmyeloid SWC3 marker had no effect on peptide-induced lymphoproliferation (data not shown). This demonstrated that the anti-SLA blocking was specific, the peptide-induced lymphoproliferation being primarily SLA class II dependent, although a certain degree of SLA class I involvement was present. This suggested that Th lymphocytes dominated the response, but lymphocytes of the cytotoxic T (Tc)cell subpopulation were also active (anti-SLA class I antibodies do not inhibit memory Th lymphocyte proliferation [Armin Saalmu ¨ller, personal communication]). Characterization of T-cell subpopulations involved in antiVP4 peptide lymphoproliferative response. The results had not yet demonstrated that T cells were activated. Consequently, analyses turned to the responding T-cell subpopulations, identified by their upregulation of CD25 (1, 3). PBMC obtained at 6 days postvaccination were stimulated in vitro with VP4-0 or the T-cell–B-cell tandem. Control cultures were cells stimulated with medium alone. Triple labeling was for flow cytometry using MAb against CD25, CD4, and CD8 (1, 22, 37). The

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would be present. Similar profiles were obtained with the tandem peptide and VP4-0. In conclusion, the present work indicates that the region including residues 20 to 35 in FMDV VP4 constitutes a promising candidate for induction of T-cell responses, with Th lymphocytes dominating therein. Lymphocytes from vaccinated pigs can recognize an MHC-promiscuous, immunodominant T-cell epitope within this region of VP4. The immunogenic peptides derived from this region possess a degree of heterotypic lymphostimulatory ability but with a clear homotypic bias. Recognition of the sequence by immune lymphocytes is retained when it is present in a tandem peptide linked to an immunodominant FMDV B-cell site. The results propose that peptides from, or covering, the VP4 region including residues 20 to 35 would be candidates for inclusion in the formulation of synthetic peptide vaccines. We thank V. Ley (CISA-INIA), J. Dominguez (CISA-INIA), and E. Hensen (University of Utrecht) for fruitful discussions, as well as C. Sanchez, Heidi Gerber, Sarah Cox, Annette Arriens, Rene´ Schaffner, Marie-Paule Farkas, and Daniel Brechbu ¨hl for valuable help with the in vitro and animal experimentation. We also thank M. Lombard for providing the type O foot-and-mouth disease virus vaccine. This work was funded in part by the European Union (contract FAIR-PL97-3665). In addition, work done at CISA-INIA and CBMSO was supported by CICYT, Spain (grant BIO96-0400-C02-01), and by the Fundacio ´n Ramo ´n Areces. Work done at the IVI was supported by the Swiss National Science Foundation (grant 31-40887.94), the Federal Office of Science and Education (grant 97.0422), and the Federal Veterinary Office. Work done at the University of Barcelona was supported by DGESIC (grant PB97-0973). REFERENCES

FIG. 4. (A) SLA dependency of peptide-induced specific lymphoproliferation. Anti-SLA class I (white bars) and anti-SLA class II (grey bars) MAb blocking of lymphoproliferation is shown. Each bar represents the number of counts per minute obtained with VP4-0 (residues 20 to 34) in the presence or absence (black bars) of the MAb. Above the bars are the percentages of inhibition obtained with the MAb. (B to E) Identification of the T-cell subpopulations responding to stimulation with the tandem T-cell–B-cell peptide. Interleukin-2 receptor (CD25) was measured on cells gated as CD4⫺ CD8⫹ (B), CD4⫹ CD8⫹ (C), CD4⫺ CD8⫺ (D), and CD4⫹ CD8⫺ (E). The dark grey histograms show CD25 expression in cultures stimulated with the T-cell–B-cell tandem peptide. The light grey histograms show the labeling on lymphocyte subpopulations in control unstimulated cultures.

MAb were anti-CD4 (clone PT90A; Veterinary Medical Research and Development [VMRD]), anti-CD8 (clone PT81B; VMRD), and anti-CD25 (clone K231.3B2; kindly provided by Armin Saalmu ¨ller, BFAV, Tu ¨bingen, Germany) (3). Through this, CD25 expression could be identified on CD4⫹ CD8⫺ (dominated by naive Th cells), CD4⫺ CD8⫹ (Tc cells), CD4⫹ CD8⫹ (dominated by memory Th cells in the pig) (26, 33, 38, 42), and CD4⫺ CD8⫺ (containing ␥␦ T lymphocytes, B lymphocytes, and monocytes) cells (5, 34). Compared with cells from the control cultures (Fig. 4B to E, light grey plots), the expression of CD25 (Fig. 4B to E, dark plots) on CD4⫺ CD8⫹ Tc cells increased in only a minority of cells following peptide stimulation (Fig. 4B). A similar increase was noted with the CD4⫺ CD8⫺ and the CD4⫹ CD8⫺ naive Th subpopulations (Fig. 4D and E). It was the CD4⫹ CD8⫹ subpopulation which most significantly upregulated CD25 (Fig. 4C). Although memory Th cells would dominate these CD4⫹ CD8⫹ lymphocytes, stimulated CD4⫹ CD8⫺ naive Th cells (upregulate CD8)

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