Engineering N-glycosylation mutations in IL-12 enhances sustained ...

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p40 is important for generating sustained long-term cell-mediated immunity. Thus, the mutant IL-12 could be utilized for the development of DNA vaccines as an ...
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RESEARCH ARTICLE

Engineering N-glycosylation mutations in IL-12 enhances sustained cytotoxic T lymphocyte responses for DNA immunization Sang J. Ha1†, Jun Chang1†, Man K. Song1, You S. Suh1, Hyun T. Jin1, Chu H. Lee1, Gyu H. Nam1, Gildon Choi1, Kwan Y. Choi1, Sung H. Lee2, Won B. Kim2, and Young C. Sung1*

Interleukin-12 (IL-12), consisting of p40 and p35 subunits, produces both p70 heterodimer and free p40. p70 is essential for the induction of T-helper 1 (Th1) and cytotoxic T-cell (CTL) immunity, whereas p40 inhibits p70mediated function. Here, we found that mutations introduced into N-glycosylation sites (N220 of murine p40 and N222 of human p40) reduced secretion of p40 but not p70. Co-immunization of N220 mutant mIL-12 gene with hepatitis C virus (HCV) E2 DNA significantly enhanced long-term E2-specific CD8+ T-cell response and protection against tumor challenge compared with that of wild type. Our results indicate that the ratio of p70 to p40 is important for generating sustained long-term cell-mediated immunity. Thus, the mutant IL-12 could be utilized for the development of DNA vaccines as an adjuvant for the generation of long-term memory T-cell responses.

Interleukin-12 (IL-12) is secreted by monocytes, dendritic cells, and B cells after appropriate stimulation and comprises two separate subunits: a light chain of 35 kDa (p35) and a heavy chain of 40 kDa (p40)1. The p35 subunit alone is not secreted in a free form, while the p40 subunit is secreted as monomer/homodimer of p40 as well as a form of p70 heterodimer2. p70 has a broad range of activities, including proliferation of activated T and natural killer (NK) cells, differentiation of CD8+ T cells, and stimulation of hematopoietic stem cells1. Free p40 blocks the binding of p70 to its receptor and inhibits IL-12-mediated biological activity3,4, suggesting that in vivo production of p40 may be a self-regulatory process that antagonizes the activity of p70. To minimize the in vivo production of p40, a number of studies have focused on the generation of a genetically engineered single-chain fusion5–8. Although Lieschke et al. reported that singlechain IL-12 fusion proteins were found to have similar bioactivity to wild type in vitro and in vivo5, these fusion proteins appear to have lower specific activity than native p70 (refs 6–8). Moreover, there remains a potential disadvantage that the linker sequence used for constructing the fusion protein may generate immunogenic epitopes. Glycosylation can affect folding, conformation, secretion, stability, and biological activity. p35 and p40 subunits of IL-12 have three and four putative N-glycosylation sites, respectively. The N-glycosylation of IL-12 was reported to be a regulatory step that determines its secretion9. This finding led us to hypothesize that mutation of N-glycosylation sites may generate IL-12 derivatives that can alter the secretion of p40. We found that N-glycosylations at Asn222 of human p40 (hp40) and at Asn220 of murine p40 (mp40) were important for the secre-

tion of p40, but not for p70, and the reduced secretion of p40 resulted in an enhanced interferon-γ (IFN-γ) induction. The N-glycosylation mutant (mIL-12N220L) was found to provide important enhancement of long-term E2-specific CTL response in HCV E2 DNA immunization and to induce more efficient protection against tumor challenge than wild type.

Results Effects of N-glycosylation on IL-12 secretion. The effect of N-glycosylation on expression, heterodimerization, and secretion of IL-12 was investigated by introducing point mutations at putative N-glycosylation sites of the hp40 gene. We transfected each plasmid into COS-7 cells and then examined expression by western blot (Fig. 1). Two major bands (40 and 37.1 kDa) and two minor bands were visible in both cell lysates and supernatants of wild-type hp40, hp40N125Q, or hp40-N135Q (Fig. 1A and B, lanes 2–4). hp40-N222Q generated four bands that are smaller than native hp40, and hp40N303Q showed different band patterns in which the amount of the 40 kDa protein was dramatically decreased, suggesting that Asn222 and 303 residues of p40 are N-glycosylation sites (Fig. 1A, lanes 5 and 6). These results are consistent with a previous report that Asn222 is N-glycosylated as shown in chemical modification assay10. Interestingly, the intensity of the bands from supernatant of cells transfected with hp40-N135Q and hp40-N222Q, but not that of hp40-N303Q, was significantly reduced (Fig. 1B, lanes 4–6). In a separate pulse–chase experiment, p40 subunit expressed from hp40N222Q construct was comparable to that from wild type but accumulated inside cells, indicating that N222Q could considerably reduce the secretion of p40 (data not shown). To further confirm our

1National

Research Laboratory of DNA Medicine, Department of Life Science, Pohang University of Science & Technology, San 31, Hyoja-Dong, Nam-Ku, Pohang, Kyungbuk, 790-784, Korea. 2ProGen Co. Ltd., 47-5, Sanggal-Ri, Kiheung-Up, Youngin, Kyunggi, 449-905, Korea. *Corresponding author ([email protected]). †These authors contributed equally to this work. http://biotech.nature.com



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RESEARCH ARTICLE Figure 1. Western blot analysis of human p40 and its derivatives with mutations at putative N-glycosylation sites. Cell lysates (A) and supernatants (B) were obtained after transfection of wild-type hp40 or its mutant constructs. The concentration of p40 in each sample was estimated by ELISA. Electrophoresis was carried out on 5 ng of p40 or its derivatives in the samples, followed by immunoblotting using antibody against human p70. The supernatant samples of constructs containing hp40-N135Q, hp40-N222Q, and hp40 (tu), as well as the cell lysate and supernatant samples of mock plasmid (mock), were loaded after normalizing them by SEAP assay rather than quantification of their proteins. (tu), Sample treated with tunicamycin.

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results indicate that Asn135 and 222 residues are critical for the secretion of p40, but not for the heterodimerization and secretion of p70. Furthermore, it is noteworthy that the p40 level in supernatant of N222Q was even lower than that of N135Q (9% vs. 29%). Double and triple mutations further suppressed p40 and p70 production, as compared with single mutations. It is unlikely that the introduced mutations affected the quantification of IL-12 by ELISA, in that amounts of p70 and p40 proteins as determined by ELISA corresponded well with those by SDS–PAGE followed by both silver staining (data not shown) and western blot (Fig. 1). When we investigated the biological activity of wild-type IL-12 and its mutants by comparing IFN-γ induction from activated T cells, hp40-N135Q, hp40-N222L, and hp40-N222Q exhibited the increased IFN-γ induction by 21%, 42%, and 46%, respectively (Table 1). To determine if these mutants produced a higher level of IFN-γ from peripheral blood monocytes (PBMCs) than wild-type IL-12 as a result of the decreased level of p40, we compensated for the differing amounts of p40 protein in the culture supernatant by adding recombinant p40. The reconstitution of

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data, double and triple mutants for Asn135, 222, and 303 were constructed and analyzed by western blot. As expected, the decrease of protein band intensities in supernatant could be correlated with overlapping mutations at Asn135 and 222 (Fig. 1B, lanes 7–10). Also, the band patterns of hp40-N222,303Q and hp40-N135,222,303Q were similar to those of tunicamycin-treated p40 (Fig. 1A, lanes 9–11). Taken together, our results indicate that Asn222 and 303 are glycosylation sites and play an important role in the secretion of p40. We generated hp35 mutant constructs and co-transfected each mutant with wild-type p40. As judged by ELISA, hp35- Table 1. Heterodimerization, secretion, and bioactivity of IL-12 and its derivativesa N141Q and hp35-N251Q produced similar levels of p70 Transfected constructs Cell lysates Supernatants IFN-γ inductionb p40 p70 p40 p70 compared to wild type (Table 1). However, the NDc ND ND ND 5.2 ± 2.3 mutants, hp35-N127Q and Mock hp35 + hp40 100 ± 8.3 100 ± 12 100 ± 13 100 ± 7.4 100 ± 11 hp35-N127,141Q, exhibited (1,827 ± 152) (98 ± 12) (2,721 ± 354) (251 ± 19) dec-reased levels of p70 hp40 102 ± 11 ND 91 ± 16 ND 8.3 ± 2.1 (71–72% in cell lysates and hp35-N127Q + hp40 99 ± 4.3 72 ± 7.7 98 ± 16 55 ± 14 98 ± 19 102 ± 18 90 ± 12 87 ± 11 95 ± 8.1 91 ± 21 52–55% in supernatants, hp35-N141Q + hp40 97 ± 10 88 ± 8.4 88 ± 12 89 ± 8.9 102 ± 18 respectively), indicating the hp35-N251Q + hp40 88 ± 6.7 71 ± 4.6 103 ± 13 52 ± 11 90 ± 7.9 possible inv-olvement of hp35-N127,141Q + hp40 hp35 ND ND ND ND 3.4 ± 1.4 Asn127 of p35 in the assem- hp35-N125Q + hp35 89 ± 11 97 ± 17 108 ± 17 101 ± 11 108 ± 21 bly and secretion of p70. hp40-N135Q + hp35 92 ± 13 99 ± 4.4 29 ± 4.7 102 ± 14 121 ± 9.1 These results are consistent hp40-N135Q + hp35 [+ hp40]d 97 ± 14 90 ± 16 89 ± 21 8 ± 6.2 93 ± 12 142 ± 24 with the previous report, in hp40-N222L + hp35 88 ± 17 90 ± 14 9 ± 4.3 94 ± 7.5 146 ± 12 that N-glycosylation of p35 hp40-N222Q + hp35 d 103 ± 18 seems to be a key requirement hp40-N222Q + hp35 [+ hp40] hp40-N303Q + hp35 108 ± 12 102 ± 22 104 ± 11 121 ± 19 97 ± 7.8 for the assembling and secre- hp40-N135,222Q + hp35 86 ± 11 88 ± 6.9 4.0 ± 2.1 81 ± 8.4 147 ± 15 tion of p70 (ref. 9). Similar hp40-N135,303Q + hp35 89 ± 7.1 97 ± 11 23 ± 4.5 82 ± 11 103 ± 8.3 results were also obtained for hp40-N222,303Q + hp35 93 ± 7.9 102 ± 24 19 ± 6.7 85 ± 4.8 125 ± 12 85 ± 7.4 65 ± 4.3 2.2 ± 1.1 65 ± 7.4 148 ± 28 other mutants such as hp40-N135,222,303Q + hp35 ND ND ND ND 5.2 ± 2.4 hp35-N127L, hp35-N141S, Mock mIL-12 100 ± 17 100 ± 11 100 ± 22 100 ± 12 100 ± 22 hp35-N251K, hp40-N125L, (56 ± 9.5) (19 ± 2.1) (87 ± 19) (48 ± 5.8) hp40-N222L, and hp40- mIL-12N220L 94 ± 22 96 ± 7.9 2.2 ± 0.5 98 ± 18 172 ± 19 N303G (data not shown). When wild-type p40 or its aThe levels were measured by ELISA and presented relative to the level of wild-type p70 or p40 expression, which is arbitrarily at 100%. The numbers in parentheses represent the absolute concentration (ng/ml) of p70 or p40. The results are the mean mutants were co-transfected set ± s.d. of three independent experiments. with p35, the levels of p40 bThe equal amount of p70 (10 ng) in each mutant supernatant was used in the IFN-γ induction assay. The level of induced IFN-γ and p70 in the cell lysates is presented relative to the level of wild type, which is set at 100%. c were not significantly differ- dND, not detected. Recombinant p40 protein obtained from hp40-transfected COS-7 cells was added to the indicated mutant supernatant to ent between wild type and its equalize p40 concentration to that in wild-type IL-12 supernatant. Each reconstituted supernatant is used for the subsequent mutants (Table 1). The IFN-γ induction assay. 382

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RESEARCH ARTICLE Figure 2. Comparison of receptor binding and IFN-γ induction between wild-type and N222L mutant p70 on Con A blast cells. Con A blast cells were examined for the ability to bind wild-type or mutant p70 proteins of the indicated concentration (A). The cells were stained with anti-p70 and the relative percentage of stained cells was represented. Similar results were obtained in an additional experiment. Con A blast cells were also used for the IFN-γ induction assay (B). The cells were incubated with wild-type or mutant p70 protein of different concentrations for 16 h and the levels of induced IFN-γ were estimated by ELISA. Data are represented as the average ± s.d. in triplicate readings and similar results were found in three independent experiments.

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p40 decreased the IFN-γ induction levels in N135Q and N222Q mutants from 121% and 146% to 97% and 103%, respectively (Table 1). In addition, we partially purified wild-type and N222L mutant of p70 proteins using recombinant adenovirus expression system, and then compared the receptor-binding affinity of wild-type and mutant p70 for high-affinity IL-12 receptors expressed on concanavalin A (Con A)-activated human PBMCs (Fig. 2A). Binding affinity of both wild-type and mutant p70 as measured by flow cytometry was very simi- B lar and dose-dependent. IFN-γ induction by equal amounts of each protein was comparable (Fig. 2B). These results point toward the decrease of p40 secretion as the main reason for the enhancement of IFN-γ induction by these mutants, rather than changed binding affinity or receptor interaction dynamics. Enhancement of Th1 immune response by mIL-12N220L. Our results demonstrated that Asn222 mutant of hp40 could reduce the secretion of p40 and induce a higher level of IFN-γ production than wild type in vitro. Hence, we became interested in determining its effect in vivo using a DNA vaccination model in mice. To locate a murine IL-12 mutation equivalent to the human Asn222 mutation, we aligned mp40 cDNA with hp40 cDNA, and found that Asn220 of mp40 is located in a very similar sequence context to Asn222 of hp40. Asn220 residue of mp40 was found to be N-glycosylated, as judged by western blot (data not shown). In addition, a murine IL-12 mutant containing mp40-N220L in single-construct mIL-12N220L showed similar characteristics to the human Asn222 mutant, with regard to the secretion of p40 and p70 as well as IFN-γ induction ability (Table 1). After mice were injected twice with mock or HCV E2 DNA as a marker antigen in the presence and absence of mIL-12 or mIL12N220L DNA, the in vivo adjuvant effect of mIL-12N220L was compared with wild-type mIL-12. The single DNA immunization

could not induce any E2-specific antibody responses, but the booster injection seroconverted 50–60% of the E2 DNA-immunized mice (Fig. 3, GII–IV). Co-administration of mIL-12N220L (GIV) or mIL-12 (GIII) showed 2 or 3 times higher total IgG level than that of E2 DNA (GII), and the IgG2a levels of GIII and GIV were 3 and 10 times higher than those of GII mice, respectively (Fig. 3A, C). As a negative control, control DNA-immuTable 2. Frequency and kinetics of HCV-E2-specific CD8+ T cells nized mice (GI) showed no E2-specific IgG response. It is No. of Weeks Frequency of HCV-E2-specific CD8+ T cells worth noting that IgG2a level immun- after final Intracellular IFN-γ staining (%)b Limiting dilution assay (No.)c was significantly enhanced by ization immunization co-injection of mILa I II III IV I II III IV Group 12N220L, compared with that 2 0 0.08 0.32 0.42 0.58 7.1 36.1 39.0 47.1 of mIL-12 (P < 0.001) 3 0.05 0.34 0.48 0.72 8.4 39.0 48.8 66.7 (Fig. 3B, C), indicating that 6 0.06 0.29 0.39 0.73 6.9 34.8 46.8 64.9 mIL-12N220L augments the 10 0.07 0.21 0.20 0.68 7.2 18.2 19.8 64.3 14 0.07 0.09 0.09 0.57 8.1 9.6 8.8 45.7 induction of Th1 immune 1 4 0.06 0.33 0.42 0.55 7.5 33.2 41.0 48.7 responses. We also observed 8 0.08 0.26 0.27 0.51 8.4 11.3 12.5 48.2 that there was significantly 14 0.06 0.10 0.07 0.42 7.1 6.8 8.1 44.7 enhanced and sustained level aThe expression plasmids of each group are as follows: GI, pTV2; GII, pTV2-E2 + pTV2; GIII, pTV2-E2 + pTV2-mIL-12; GIV, of IFN-γ produced from pTV2-E2 + pTV2-mIL-12N220L. splenocytes of mice co-immubThe percentage of IFN-γ-producing CD8+ T cells among live CD8+ T cells was calculated by plotting of live CD8+ T cells with nized with mIL-12N220L, CD8 and IFN-γ. Data are presented as the average value obtained with two mice per group in two independent experiments. cThe frequency of precursor CTLs per 107 spleen cells was calculated by regression analysis of the number of negative wells compared with mIL-12 (P < at each dilution of responder cells. The numbers represent the average value of two independent experiments. 0.001) (data not shown). http://biotech.nature.com



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RESEARCH ARTICLE Figure 3. Kinetics of E2-specific antibody responses in DNA immunization. Each group of mice (n = 20) was immunized twice at 0 and 4 weeks with control plasmid, pTV2 (GI, open circles), pTV2-E2 (GII, filled circles), pTV2E2 + pTV2-mIL-12 (GIII, open squares), and pTV2-E2 + pTV2-mIL-12N220L (GIV, filled squares), respectively. The mice were bled 0, 3, 6, and 10 weeks after booster immunization, and then sera collected at the indicated times were pooled for determination of E2-specific total IgG (A), IgG1 (B), and IgG2a (C) levels. Data are represented as the average of three individual assays ± s.d. Seroconversion rates of GII, GIII, and GIV after booster immunization are 56, 50, and 60%, respectively. Sera obtained from the seroconverted mice were also tested for determination of total IgG, IgG1, and IgG2a levels and showed results similar to those obtained from pooled sera of all immunized mice. *P < 0.001.

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lar IFN-γ staining and the limiting dilution assay, it appears that GIV mice exhibited the highest frequency of E2-specific CD8+ T cells and GIII mice showed an intermediate frequency initially (four and three weeks after primary and booster immunization, respectively). These results indicate that co-administration of mIL12N220L augments E2-specific memory CD8+ T-cell response induced by E2 DNA immunization at early time points, which seems to be inconsistent with the result of CTL assay. This discrepancy may be explained by a previous report that the chromium release assay is not as quantitatively sensitive as intracellular IFN-γ staining and LDA (ref. 11). Interestingly, GII and GIII mice showed a decreased frequency of E2-specific CD8+ T cells at later times, converging to background level at 14 weeks following booster or primary immunization (Table 2). In contrast, GIV mice maintained the frequency of E2-specific CD8+ T cells at a high level even at 14 weeks after primary or booster immunization, demonstrating that mIL12N220L co-immunization can induce prolonged memory CD8+ T-cell immunity in E2 DNA immunization. Improved protection of immunized mice against tumor challenge. To test whether sustained long-term CTL immunity measured in vitro is correlated with in vivo protection against tumor challenge, the immunized mice were challenged with modified CT26 tumor cells expressing E2 at 12 weeks after booster immunization. When we examined tumor growth and survival rate of the immunized mice up to 66 days after tumor challenge with a lethal dose (Fig. 4G, H), GII mice, but not GI mice, seemed to have delayed tumor growth and showed a 20% survival rate. GIII mice exhibited further delayed tumor growth compared with GII mice, and had a 30% survival rate. In GIV mice, tumor growth was significantly delayed and 90% of the immunized mice survived, implying that the enhanced E2-specific CTL response induced by mIL12N220L co-delivery might promote protection against tumor challenge. These results demonstrated that the increased biological activity of mutant IL-12 proved to be effective on antitumor immunity in an animal model.

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Induction of prolonged CD8+ T-cell responses by mIL12N220L. To investigate whether mIL-12N220L has an effect on the induction of long-term CTL response, CTL activity was determined using splenocytes from the DNA-immunized mice. Splenocytes from mice immunized with E2 DNA with or without IL-12 (GII-GIV) exhibited strong E2-specific CTL activity after booster immunization (Fig. 4A). The IgG response to E2 was undetectable at this time point, indicating that the threshold for the induction of CTL response might be lower than that of IgG response. Co-delivery of mIL-12N220L did not show a significant difference in the level of CTLs compared to that of wild type at 0 and 3 weeks following booster immunization (Fig. 4A, B); however, the difference became apparent at later time points (6–14 weeks, Fig. 4C–E). The results indicate that the co-delivery of mIL12N220L could induce sustained CTL activity, which was E2-specific as indicated by the lack of any specific lysis against parental target cells (Fig. 4F). To further confirm our results, the frequency and kinetics of E2specific CD8+ T cells were determined by both intracellular IFN-γ staining and limiting dilution assay (LDA). As expected, mock DNA-immunized mice (GI) did not exhibit a significant number of E2-specific CD8+ T cells. In contrast, GII–IV significantly increased the frequency of E2-specific CD8+ T cells at four weeks following primary immunization (Table 2). Given the results of the intracellu384

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Discussion Immunization with E2 DNA alone showed detectable CTL response to E2 for up to 10 weeks following the last immunization. Co-administration of mutant mIL-12-N220L increased Th1 and particularly CTL responses better than that of wild-type mIL12. In addition, intratumoral (i.t.) injection of recombinant defective adenovirus expressing mIL-12N220L (rAd/mIL12N220L) was shown to induce sustained Th1 and CTL immunity to tumor-associated antigen to a greater degree than that of wildtype mIL-12-expressing adenovirus. When the cured mice were rechallenged with the same tumor cells, protection against tumors was more efficient in rAd/mIL-12N220L-injected mice than rAd/mIL-12-injected mice (our unpublished data). These results suggest that the effect of mIL-12N220L is independent of the coexpressed antigens and experimental system. It is worthwhile to note that there were no significant differences in the levels of mIL•

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Figure 4. Effect of mIL-12N220L on E2-specific CTL responses and protection against challenge with modified tumors. CTL activity was determined by measuring specific lysis of CT26-hghE2t with splenocytes obtained from three mice at 0 (A), 3 (B), 6 (C), 10 (D), and 14 (E) weeks after booster immunization. As a control, CT26-neo cells were used as target cells in the assay for splenocytes of GIV mice obtained at 14 weeks after booster immunization (F). Data are represented as the mean percentage of specific lysis ± s.d. for the effector:target ratios listed in triplicate cultures. This result was reproduced in three independent experiments. The DNA-immunized mice (10 mice per group) were injected subcutaneously with 106 CT26-hghE2t tumor cells at 12 weeks after booster immunization. The mean local tumor growth was determined by measuring the volume of tumors every 3 days (G). Data are represented as the mean tumor volume ± s.e.m. The viability of individual mice was monitored for 66 days after tumor challenge (H). Symbols and constructs for GI through GIV are as in Figure 3. *P < 0.015; **P < 0.0001.

The only difference between mIL-12 and mIL-12N220L is a single amino acid change, which leads to a normal level of p70 secretion and little p40 secretion. It remains to be determined how a relatively high ratio of p70 to p40 generated by co-administration of mIL12N220L induces a sustained long-term CTL response. First, it is likely that p70 is one of the determinants for long-term memory T-cell generation and/or maintenance through increasing the lifespan of T cells. p70 was shown to prevent Fas-mediated apoptosis of antigen-specific T cells12. In addition, we observed that p70 could enhance survival of CD8+ T cells as well as CD4+ T cells from passive cell death and activation-induced cell death (our unpublished data), indicating that p70 directly enhances survival of CD8+ T cells. Second, p70 may directly or indirectly act on the maintenance of memory T cells through expansion of memory T cells. Recent evidence indicates that p70 directly acts on T cells to enhance homeostatic expansion, leading to amplification of competent cells with memory function13. Furthermore, it has been shown that p70 indirectly stimulates selective proliferation of memory CD8+ T cells in vivo by an IFN-γ-dependent pathway14. IFN-γ induced by p70 stimulates the expression of IL-15 (ref. 15), which is a positive regulator of memory CD8+ T-cell turnover in vivo and in vitro15,16. Consistent with these data, our results demonstrated that immunization of mIL-12N220L initially produced more frequent antigen-specific CD8+ T cells than wild type IL-12 and maintained the cells to the later time point (Table 2), suggesting that p70 is involved in both expansion and maintenance of memory T cells. In this regard, p40, which has been known to compete with p70 through binding to the β1 chain of the IL-12 receptor complex3, might inhibit p70-mediated memory Th1 and CTL responses. In immune responses, the stimulatory effects of one process are frequently counterbalanced by the inhibitory effects of another. Ku et al. reported that division of CD8+ memory T cells requires IL-15 and is markedly increased by inhibition of IL-2 and therefore, the frequency of CD8+ memory T cells in vivo is controlled by a balance between IL-15 and IL-2 (ref. 16). Taken together, our data demonstrated that, as with the immunological balance between IL15 and IL-2, the ratio of p70 and p40 has an analogous role in sustaining CTLs as well as Th1 cells.

Experimental protocol Plasmids and mutagenesis. The cDNAs of hp35 and hp40 were cloned using RT-PCR with sequence-specific primers. Each amplified cDNA was subcloned into the universal vector, pSK (Stratagene, La Jolla, CA). For the expression in eukaryotic cells, hp40 and hp35 cDNAs were separately inserted into pCI-neo (pCIN-hp40 and pCIN-hp35). Mouse IL-12 expression cassette from pCIN-mIL-12 (ref. 17) was inserted into pTV2 vector18 to construct pTV2-mIL-12.

12p70 in sera of the immunized mice (13,669 ng vs. 14,047 ng and 186 ng vs. 169 ng at one and two days after i.t. injection of each recombinant adenovirus, respectively), indicating a similar in vivo half-life between wild-type mIL-12 and mIL-12N220L (our unpublished data). http://biotech.nature.com



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RESEARCH ARTICLE from PBMCs by cultivation with 5 µg/ml of Con A and 5 ng/ml of IL-2 for three days. The expression of IL-12Rβ1 and β2 was determined with anti-IL12Rβ1, anti-IL-12Rβ2, or their isotype-matched monoclonal antibodies (mAbs; PharMingen, San Diego, CA) using FACSCalibur (Becton Dickinson, San Jose, CA). For detection of p70 binding to its receptors, cells were incubated with partially purified wild-type or mutant p70 proteins, and anti-p70 mAbs (R&D Systems). Stained cell populations were analyzed using FACSCalibur (Becton Dickinson), and the percentage of p70-binding cells was calculated by relatively gating the range of IL-12-binding cells using isotype-matched antibodies for anti-p70 mAbs.

For the substitution of asparagine residues in putative N-glycosylation sites of hp40 and hp35 to glutamine residues, missense mutations were introduced by PCR using pCIN-hp40 or pCIN-hp35 as a template as described19. Double- and triple-glutamine mutants were constructed using single- or double-glutamine mutants as PCR templates. hp40-N222L and mp40-N220L were generated similarly. For the expression of mIL-12N220L, mp40 in pCIN-mIL-12 was replaced with mp40-N220L. The expression cassette of pCIN-mIL-12N220L was inserted into pTV2 vector to generate pTV2-mIL-12N220L. The pTV2-E2 plasmid was described18. The list of oligonucleotides used for PCR can be found as Supplementary Table 1 in the Web Extras page of Nature Biotechnology Online.

DNA immunization and antibody responses. Female BALB/c mice were purchased from Japan SLC (Shizuoka, Japan). Six- to eight-week-old mice were intramuscularly immunized with 200 µg of plasmid. Antibody responses were monitored at the different time points by ELISA using E2 protein as described18.

Transfection, ELISA, and western blot analysis. Transfection into COS-7 cells was carried out by electroporation in the presence of 20 µg of specimen DNA and 2 µg of secreted alkaline phosphatase (pNEB-SEAP) (New England Biolabs, Inc., Beverly, MA) as an internal control. After 24 h, media were replaced with a minimal volume of serum-free medium. Tunicamycin (Sigma, St. Louis, MO), 1 µg/ml, was added at this time. After incubation for another 24 h, supernatants and cells were harvested. Supernatants were used for SEAP assay to normalize the efficiency of transfection. Cells were pelleted by centrifugation, and resuspended in 200 µl of lysis buffer (Promega, Madison, WI). The levels of p70 and p40 were measured by ELISA (R&D systems, Minneapolis, MN). For western blot analysis, supernatant and cell lysates were electrophoresed through SDS–PAGE (10%) and immunoblotted using polyclonal antibody against human IL-12 (R&D Systems).

Analysis of CD8+ T-cell responses. CTL assay was performed as described18. The frequency of CTLs was measured using LDA (ref. 11) using CT26-hghE2t cells as stimulator and target cells. For determination of CD8+ antigen-specific T-lymphocyte frequency, intracellular cytokine assessment was used as described11, with slight modifications. Briefly, splenocytes (2 × 107) obtained at the indicated week after the booster immunization were stimulated with CT26hghE2t cells (1 × 106) for 40 h. Then 4 µl of GolgiStop (PharMingen) were added and the cells were incubated for another 6 h. CD8+ T cells were purified from the stimulated splenocytes using anti-CD8 microbeads (Miltenyi Biotec, Inc., Auburn, CA), and then used in intracellular IFN-γ staining assay. Stained cells were analyzed by FACSCalibur (Becton Dickinson).

IFN-γ induction assay. Human PBMCs were isolated by density gradient centrifugation from fresh blood and incubated with culture supernatants containing 10 ng/ml of human p70 or its mutant derivatives for 16 h. For the mouse IFN-γ detection, 105 splenocytes from BALB/c mice were incubated with culture supernatants containing 10 ng/ml of mouse p70 or its mutant derivatives for 24 h. The amounts of induced human and mouse IFN-γ were estimated by human and mouse IFN-γ ELISA (R&D Systems), respectively.

Measurement of tumor growth. The local tumor growth was determined by measuring the diameter and volume of tumors with calipers every 3 days. Note: Supplementary information is available on the Nature Biotechnology website.

Receptor-binding assay. Recombinant adenovirus expressing wild-type or N222L mutant human IL-12 was produced according to Quantumn applications manual (Quantumn Biotechnologies, Montreal, PQ, Canada). Recombinant viruses were used to infect 5 × 108 293 cells in serum free media II (CHO-S-SFM II) for the maintenance of CHO cells for two days. For the purification of wild-type and mutant forms of hp70, the obtained culture media were applied to DEAE-Sepharose, hydroxyapatite, and Q-Sepharose columns sequentially as described20. The methods used for the detection of IL-12R and IL-12 binding cells by flow cytometry have been described21. Binding assays were conducted using Con A blast cells, which were prepared

Acknowledgments The authors acknowledge Sang-Chun Lee and Su-Il Park for devoted animal care. We are grateful to Hye-Ryun Kim and Se-Hwan Yang for faithful reading and comments. This work was supported by National Research Laboratory grant from Korea Institute of Science and Technology Evaluation and Planning (2000-N-NL-01-C-202) and grants from POSCO (2000Y013), Superior Research Center supported by Korea Science and Engineering Foundation, and ProGen Co. Ltd. Received 20 July 2001; accepted 24 January 2001

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