Apr 28, 1998 - Grand Island, NY) containing 10% FCS, 2 mM L-glutamine,. 100 U/ml of ..... 31 Hickling, J. K., Fenton, C. M., Howland, K., Marsh, S. G. E. and.
International Immunology, Vol. 10, No. 8, pp. 1167–1174
© 1998 Oxford University Press
Possible involvement of autoreactive CD4 T cells in generation of cytotoxic T lymphocytes on in vitro stimulation with H-2 class I-binding tumor antigen peptide Shinichiro Honda1,2, Hisashi Wada1, Akiko Uenaka1 and Eiichi Nakayama1 1Department
of Parasitology and Immunology, Okayama University Medical School, 2-5-1 Shikata-cho, Okayama 700-8558, Japan 2Department of Oncology, Nagasaki University School of Medicine, 1-12-4 Sakamoto-machi, Nagasaki 852-8523, Japan
Keywords: autoreactive CD4 T cells, cytotoxic T lymphocyte, IL-2, I-A, tumor antigen peptide
Abstract We demonstrated that RLY1-specific cytotoxic T lymphocytes (CTL) were generated in spleen cells from the tumor-rejected CB6F1 mice on in vitro stimulation with Ld-binding pRL1a peptide. We showed that CD4 T cells and antigen-presenting cells were necessary for CTL generation. Furthermore, CTL generation was inhibited by the addition of anti-I-Ad mAb, but not anti-I-Ek/d mAb, to the culture. However, no binding of the pRL1a peptide to the I-Ad molecule could be demonstrated. No inhibition by the pRL1a peptide of I-Ad-restricted IL-2 production by ovalbumin (OVA)-specific CD4 T cell hybridoma DO-11.10 was observed on stimulation with the specific OVA peptide. Moreover, no specific proliferative response or IL-2 production was observed with CD4 T cells in spleen cells from the tumor-rejected mice on stimulation with a range of mitomycin C-treated RLY1 cells, or with RLY1 lysate or the pRL1a peptide. Rather, the activation of autoreactive CD4 T cells and the IL-2 production from them were observed. CD4 T cells from CB6F1 mice that had rejected other tumors such as EL4 or MOPC-70A also helped the generation of pRL1a-specific CTL. These findings suggested the involvement of autoreactive CD4 T cells in CTL generation against the pRL1a peptide. Introduction Tumor rejection antigen has been demonstrated on BALB/c radiation-induced leukemia RLY1 cells (1–5). Inocula of 53105 RLY1 cells into BALB/c mice continued to grow and the mice were killed by the tumor in 3–4 weeks, but the same inocula in CB6F1 mice regressed after initial growth of the tumor (1). The inocula at a lower dose (~13105) in BALB/c mice often resulted in tumor regression. Regression of the tumor was blocked by in vivo depletion of CD8 T cells but not CD4 T cells (2,3). Cytotoxic T lymphocytes (CTL) were generated in vitro in spleen cells from BALB/c and CB6F1 mice in which the tumor was growing or had regressed (4). These findings indicated that the tumor rejection antigen recognized by CTL was present on RLY1 tumor cells and that CB6F1 mice were better responders than BALB/c mice. Recently, we identified the tumor rejection antigen peptides on RLY1 cells that were recognized by specific CTL (5).
The antigenic peptides were pRL1a (IPGLPLSL) and pRL1b (SIIPGLPLSL). The peptides were derived from the normally untranslated 59 region of the c-akt gene (6). The long terminal repeat from murine leukemia virus was inserted next to the first coding exon of c-akt and an altered Akt molecule was produced in RLY1 cells. The findings that pRL1a, but not pRL1b, was recovered from affinity-purified Ld molecules on RLY1, and that more efficient sensitization of target cells was observed with pRL1a than pRL1b, suggest that pRL1a could be the final product bound to Ld molecules and that pRL1b is the precursor peptide (7). In this study, we investigated the generation of CTL in spleen cells from RLY1-rejected CB6F1 mice on in vitro stimulation with the pRL1a peptide. We demonstrated that CD4 T cells and antigen-presenting cells (APC) were necessary for CTL generation. IL-2 secreted from CD4 T cells was crucial.
Correspondence to: E. Nakayama Transmitting editor: M. Miyasaka
Received 7 April 1998, accepted 28 April 1998
1168 In vitro CTL generation by tumor antigen peptide Activation of CD4 T cells in the tumor-rejected mice was not against the tumor antigen, but appeared to be against the self in an I-Ad-restricted manner.
Methods
Mice BALB/c (H-2d), C57BL/6 (B6) (H-2b), (BALB/c3B6)F1 (CB6F1) and athymic BALB/c mice were bred in our laboratory animal center.
Tumors RLY1 is a BALB/c radiation-induced leukemia (1). MOPC70A is a BALB/c plasmacytoma (8). EL4 is a chemically induced leukemia of C57BL origin (9). These tumors were maintained by tissue culture and as ascites form. For immunization of mice, tumor lines maintained by in vivo passage were used. P815 is a DBA/2 methylcholanthrene-induced mastocytoma (10). A20.2J is a BALB/c B cell lymphoma (11) and was obtained from the ATCC (Rockville, MD). These tumors were maintained by tissue culture.
Antigens pRL1a (IPGLPLSL) is the tumor rejection antigen peptide recognized by RLY1-specific CTL and pRL1b (SIIPGLPLSL) is its precursor peptide (5). pRL1b(–) (SIIPGLPL), derived from pRL1b, was used as a control. Chicken ovalbumin (OVA) peptide (323–339) (ISQAVHAAHAEINEAGR) is an I-Ab/d-binding peptide recognized by T cell hybridoma DO-11.10 (12). λ repressor peptide (LEDARRLKAIYEKKK) was described previously (13). These peptides were synthesized by standard solid-phase method using Fmoc chemistry in an 430A peptide synthesizer (Applied Biosystems, Foster City, CA). Cleavage of the peptide from the resin and removal of the side chainprotecting groups were carried out using trifluoroacetic acid. The peptides were purified by reversed-phase HPLC (model 151A; Applied Biosystems) on a preparative C8 column (103100 mm, 20 µm particle size; Applied Biosystems) in 0.1% trifluoroacetic acid with an acetonitrile gradient. Chicken OVA was obtained from Sigma (St Louis, MO). For preparation of the lysate, RLY1 cells were lysed by freezing and thawing 3 times as described (14).
mAb Anti-L3T4 (CD4) mAb, a rat IgG2b antibody produced by hybridoma GK1.5 (15), was provided by Dr F. Fitch (University of Chicago, Chicago, IL). Anti-Lyt-2.2 (CD8) mAb, a mouse IgG2a antibody produced by hybridoma 19/178, was provided by Dr U. Ha¨mmerling (Memorial Sloan-Kettering Cancer Center, New York) (16). Anti-H-2Kd and anti-H-2Dd mAb are mouse IgG2a antibodies produced by hybridomas HB159 and HB102 respectively. Both mAb were obtained from ATCC. Anti-H-2Ld mAb is a mouse IgG2a antibody produced by hybridoma 30-5-7s (17). Anti-I-Ad mAb is a mouse IgG2b antibody produced by hybridoma MK-D6 (18). Anti-I-Ek/d mAb is a mouse IgG2b antibody produced by hybridoma ISCR3 (19). These mAb were provided by Dr N. Shinohara (Kitasato University, Sagamihara, Japan).
In vitro generation of CTL CB6F1 mice were inoculated s.c. in the back with 53105 RLY1 cells. After regression of the tumor, spleen cells (53106) were prepared and cultured with various concentrations of peptide or with mitomycin C (MMC)-treated RLY1 cells (53105) in 24-well tissue culture plates (Falcon 3047; Becton Dickinson, Franklin Lakes, NJ) for 5 days at 37°C in 5% CO2 air. For generation of allogeneic CTL, BALB/c or C57BL/6 (B6) spleen cells were cultured with MMC-treated stimulator spleen cells in 24-well tissue culture plates for 5 days at 37°C in 5% CO2 air. The culture medium was RPMI 1640 (Gibco, Grand Island, NY) containing 10% FCS, 2 mM L-glutamine, 100 U/ml of penicillin, 100 µg/ml of streptomycin and 53 10–5 M 2-mercaptoethanol. MMC treatment was done by incubating cells with MMC at a concentration of 50 µg/ml for 30 min at 37°C. For inhibition of CTL generation, various mAb were added to the 5 day culture.
Cell-mediated cytotoxicity assay A conventional 4 h 51Cr-release assay was performed as described previously (4). Briefly, tumor cells were labeled with 1.85 MBq of Na251CrO4 (New England Nuclear, Boston, MA) for 2 h at 37°C in 5% CO2 air. They were then washed and used as target cells. For peptide pulsing, 10–5 M peptide was added to P815 cells (23106) during labeling. In direct assays, 53103 labeled target cells (100 µl) were incubated with the effector cell suspension (100 µl). In antibody blocking assays, serially diluted antibody (50 µl) was added to the cultures of effector cells (50 µl) and labeled target cells (50 µl). After incubation for 4 h at 37°C in 5% CO2 air, the supernatants (100 µl) were removed and their radioactivity was measured. The percentage of specific lysis was calculated using the following equation: [(a – b)/(c – b)]3100, where a is the radioactivity in the supernatant of target cells mixed with effector cells, b is that in the supernatant of target cells incubated alone and c is that in the supernatant after lysis of target cells with 1% Triton-X.
In vitro elimination of CD4 T cells Spleen cells (53107) were incubated with anti-L3T4 (CD4) mAb (diluted 1:20 with MEM) in a volume of 200 µl for 30 min on ice. Then 750 µl of preselected rabbit serum (diluted 1:12 with MEM) was added as a source of complement and incubated for 30 min at 37°C.
In vivo depletion of CD4 T cells CB6F1 mice were administered i.v. anti-L3T4 (CD4) mAb (diluted 1:8 with MEM) in a volume of 200 µl on days 7 and 3 before removal of the spleen.
Depletion of APC Spleen cells (23108) were incubated for 1 h at 37°C in a 10 ml syringe packed with 0.6 g nylon wool (Wako Pure Chemical Industries, Osaka, Japan). Yields of Thy-11 cells in the eluate were 78.5–90.5%.
Paraformaldehyde fixation Cells were fixed in 0.1% paraformaldehyde in PBS for 30 min on ice and were washed 3 times before use.
In vitro CTL generation by tumor antigen peptide 1169 T cell proliferation assay In vitro CD8-depleted responder spleen cells from RLY1rejected CB6F1 mice were stimulated with MMC-treated RLY1 cells, RLY1 lysate or pRL1a peptide in 96-well plates (Falcon 3072; Becton Dickinson) for 4 days at 37°C in 5% CO2 air. The cells were then harvested and [3H]thymidine uptake was measured. Production of IL-2 Responder cells were cultured with or without antigen for 1– 4 days. For the inhibition assay, cells were cultured in the presence of mAb. The amount of IL-2 in the culture supernatant was measured using the IL-2-dependent cell line NRB (20). Briefly, NRB cells (53103) were incubated with culture supernatant for 24 h and then [3H]thymidine uptake was measured.
Competitive inhibition of the peptide binding to I-Ad molecule Assays were described previously (21). B cell lymphoma A20.2J cells were fixed with 0.1% PFA for 30 min. Various concentrations of competitor peptides were added to the fixed A20.2J (13105) cells and incubated for 30 min, then 10–5 M OVA peptide was added to the 96-well plate (Falcon 3077; Becton Dickinson). T cell hybridoma DO-11.10 cells (13105) were then added and incubated for 24 h at 37°C in 5% CO2 air. The response of DO-11.10 was assessed by measuring the amount of IL-2 secreted. Results
Generation of CTL in spleen cells from RLY1-rejected CB6F1 mice on in vitro stimulation with the pRL1a peptide The inocula of 53105 RLY1 cells into CB6F1 mice grew into tumors and then regressed. Spleen cells (53106) obtained from the tumor-rejected mice were cultured with the pRL1a peptide for 5 days. As shown in Fig. 1, cytotoxicity was generated against RLY1 and pRL1a-pulsed P815, but not against the P815 target, at an optimal concentration of 10–6 M pRL1a. The extent of cytotoxicity generated on stimulation with the pRL1a peptide was generally less than that generated on stimulation with MMC-treated RLY1 cells. Spleen cells cultured with control peptide (SIIPGLPL) or alone showed only marginal cytotoxicity. The cytotoxicity generated on stimulation with the pRL1a peptide was blocked by the addition of anti-Lyt-2.2 (CD8) mAb and anti-Ld mAb (30-57s), but not anti-L3T4 (CD4) mAb (GK1.5), anti-Kd mAb (HB159) or anti-Dd mAb (HB102) to the assay culture (data not shown).
Requirement of CD4 T cells for generation of CTL on in vitro stimulation with pRL1a peptide We investigated the requirement of CD4 T cells for the generation of CTL on in vitro stimulation with the Ld-binding pRL1a peptide. As shown in Fig. 2, the generation of cytotoxicity was diminished by treatment of spleen cells from RLY1-rejected CB6F1 mice with anti-L3T4 (CD4) mAb (GK1.5) and complement before culturing with the pRL1a peptide. The generation of cytotoxicity was also diminished by the treatment of the tumor-rejected CB6F1 mice with anti-L3T4
Fig. 1. Generation of cytotoxicity in spleen cells from RLY1-rejected CB6F1 mice on in vitro stimulation with pRL1a peptide. Spleen cells (53106) were cultured for 5 days with 10–6 M pRL1a peptide (A), 53105 MMC-treated RLY1 cells (B) or 10–6 M control peptide (SIIPGLPL) (C). (D) Spleen cells cultured alone. Cytotoxicity was determined by 4 h 51Cr-release assay with RLY1, P815 pulsed with pRL1a peptide and P815 target cells.
(CD4) mAb (GK1.5) in vivo. On the other hand, the generation of CTL on stimulation with MMC-treated RLY1 cells was not significantly affected by the in vitro or in vivo depletion of CD4 T cells. The requirement of CD4 T cells was observed with a range of peptide concentrations (Fig. 3A). The addition of in vitro CD8-depleted spleen cells from RLY1-rejected mice, but not naive CB6F1 mice, to in vitro CD4-depleted spleen cells from the rejected mice reconstituted the generation of cytotoxicity on in vitro stimulation with the pRL1a peptide (Fig. 3B). Moreover, the addition of rIL-2 (.0.25 U/ml) to the culture of in vitro CD4-depleted spleen cells also reconstituted the generation of cytotoxicity (Fig. 3C). No cytotoxicity against the P815 target was observed with rIL-2 (data not shown).
1170 In vitro CTL generation by tumor antigen peptide
Fig. 2. Effect of in vitro (A and B) and in vivo (C and D) depletion of CD4 T cells on CTL generation. For (A) and (B), spleen cells from RLY1-rejected CB6F1 mice were treated with anti-L3T4 (CD4) mAb, or anti-Lyt-2.1 mAb (control) plus complement, or complement alone before use as responders. For (C) and (D), spleen cells from the tumor-rejected CB6F1 mice administered anti-L3T4 (CD4) mAb, antiLyt-2.1 mAb (control) or MEM in vivo on days –7 and –3 were used as responder cells. Cells (53106) were cultured with 10–6 M pRL1a (A and C) or MMC-treated RLY1 (53105) cells (B and D) for 5 days. Cytotoxicity was determined by 4 h 51Cr-release assay with RLY1 target cells.
Requirement of APC for generation of CTL in spleen cells from RLY1-rejected CB6F1 mice on in vitro stimulation with pRL1a peptide To investigate the requirement of APC for in vitro generation of CTL, spleen cells from RLY1-rejected CB6F1 mice were passed through a nylon wool column and then stimulated with the pRL1a peptide in vitro. As shown in Fig. 3(D), no cytotoxicity was generated. Cytotoxicity was generated by the addition of MMC-treated, but not PFA-treated, peritoneal exudate cells (PEC) from naive CB6F1 mice to nylon woolpassed spleen cells. The results were essentially similar on in vitro stimulation with MMC-treated RLY1 cells (data not shown).
Inhibition of CTL generation by anti-H-2 class II mAb The inhibition of CTL generation by anti-H-2 class II mAb was then investigated. Purified anti-I-Ad mAb (MK-D6) and anti-I-
Fig. 3. (A) Spleen cells from RLY1-rejected mice that were untreated (open symbols) or treated with anti-L3T4 (CD4) mAb plus complement (closed symbols) were cultured with various concentrations of pRL1a peptide for 5 days. (B) Spleen cells from RLY1-rejected mice treated with anti-L3T4 (CD4) mAb plus complement were mixed with spleen cells from RLY1-rejected or naive CB6F1 mice treated with anti-Lyt2.2 (CD8) mAb plus complement and cultured with 10–6 M pRL1a for 5 days. (C) Various concentrations of rIL-2 were added to spleen cells from tumor rejected mice treated with anti-L3T4 (CD4) mAb plus complement and cultured with 10–6 M pRL1a for 5 days. In (D), RLY1-rejected CB6F1 spleen cells passed through a nylon wool column were mixed with MMC- or PFA-treated PEC from naive CB6F1 mice, and cultured with 10–6 M pRL1a for 5 days. Cytotoxicity was determined by 4 h 51Cr-release assay with RLY1 target cells.
Ek/d mAb (ISCR3) were dialyzed with PBS, and added to the culture of spleen cells from RLY1-rejected CB6F1 mice and then stimulated with the pRL1a peptide for 5 days. The titers (half maximal staining) of purified I-Ad mAb (MK-D6) and of the anti-I-Ek/d mAb (ISCR3) used were both 1:100,000 as determined by flow cytometry. As shown in Fig. 4, anti-I-Ad mAb (MK-D6), but not anti-I-Ek/d mAb (ISCR3), inhibited CTL generation on stimulation with the pRL1a peptide. No significant inhibition was observed with CTL generation on stimulation with RLY1 cells, or with allogeneic CTL generation.
In vitro CTL generation by tumor antigen peptide 1171
Fig. 5. Competitive inhibition of I-Ad-restricted peptide recognition. Competitor λ repressor or pRL1a peptide was added to PFA (0.1%)fixed A20.2J cells (13105) at the indicated concentrations in 96-well plates and incubated for 30 min before 10–5 M OVA peptide was added. T cell hybridoma DO-11.10 cells (13105) were then added and incubated for 24 h. The IL-2 content of the culture supernatant was measured using IL-2-dependent cell line NRB as described (20).
Proliferation of CD4 T cells and IL-2 production from them in spleen cells from RLY1-rejected CB6F1 mice against self, but not tumor antigen
Fig. 4. Inhibition of CTL generation by anti-H-2 class II mAb. Purified anti-I-Ad mAb (MK-D6), anti-I-Ek/d mAb (ISCR3) or anti-Lyt-2.1 mAb (control) was added to the culture of spleen cells from RLY1-rejected CB6F1 mice at the indicated concentrations and stimulated with 10–6 M pRL1a peptide (A) or MMC-treated RLY1 cells (B). (C) mAb were added to the control culture of BALB/c spleen cells stimulated with MMC-treated B6 spleen cells. Similarly, in (D), mAb were added to the culture of B6 spleen cells stimulated with MMC-treated BALB/c spleen cells. Five days after culture, cytotoxicity was determined by 4 h 51Cr-release assay with RLY1 (A, B and D) or EL4 (C) target cells.
No inhibition by pRL1a peptide of I-Ad-restricted OVA-specific T cell recognition To examine the binding of the pRL1a peptide to the I-Ad molecule, we investigated the inhibition by the pRL1a peptide of IL-2-secretion by the I-Ad-restricted OVA-specific CD4 T cell hybridoma DO-11.10 on stimulation with the appropriate peptide. Whereas peptide derived from the λ repressor gene, which has been shown to bind to the I-Ad molecule, clearly inhibited IL-2 secretion by DO-11.10, no inhibition by the pRL1a peptide was observed (Fig. 5).
We investigated the proliferation of CD4 T cells and IL-2 production from them in spleen cells from RLY1-rejected CB6F1 mice. As shown in Fig. 6, no specific proliferative response was observed with differing amounts of in vitro CD8depleted spleen cells on a range of MMC-treated RLY1 cells, RLY1 lysate or the pRL1a peptide. Rather, significant proliferation was observed with the responder spleen cells cultured alone. The response was abrogated by in vitro depletion of APC and restored by adding PEC from either the tumor-rejected or naive CB6F1 mice to them (Fig. 6D). As shown in Fig. 7, a significant amount of IL-2 was detected in the culture supernatant of in vitro CD8- but not CD4depleted spleen cells from RLY1-rejected CB6F1 mice. IL-2 production was abrogated by passing the spleen cells through a nylon wool column and restored by adding PEC from either the tumor-rejected or untreated CB6F1 mice to them. IL-2 production by in vitro CD8-depleted spleen cells was blocked by the addition of purified anti-I-Ad mAb (MK-D6), but not anti-I-Ek/d mAb (ISCR3) to the culture (data not shown).
Generation of CTL against pRL1a peptide in in vitro CD4-depleted spleen cells from RLY1-rejected CB6F1 mice by adding CD8-depleted spleen cells from CB6F1 mice that had rejected other tumors We further investigated whether the autoreactive proliferation of CD4 T cells similarly took place in mice that had rejected
1172 In vitro CTL generation by tumor antigen peptide
Fig. 7. IL-2 production. Spleen cells (33106) from RLY1-rejected (A) or naive (B) CB6F1 mice treated with anti-L3T4 (CD4) mAb (GK1.5) or anti-Lyt-2.2 (CD8) mAb plus complement, passed through a nylon wool column or passed through a nylon wool column and mixed with 53105 PEC from naive CB6F1 mice, were cultured in 24-well plates. Four days after culture, the IL-2 content of the supernatant was measured using the IL-2-dependent cell line NRB as described (20).
Fig. 6. Proliferative response of CD4 T cells. 53105 spleen cells from RLY1-rejected CB6F1 mice treated with anti-Lyt-2.2 (CD8) mAb plus complement were cultured with MMC-treated RLY1 cells (A), RLY1 lysate (B) or pRL1a peptide (C) at the indicated dosages in 96-well plates. (D) Spleen cells (53105) from the RLY1-rejected mice were passed through a nylon wool column and mixed with 13105 PEC from RLY1-rejected or naive CB6F1 mice. Four days after culture, cells were harvested and [3H]thymidine uptake was measured. Each bar indicates the mean of triplicate cultures.
other tumors and could also help generation of pRL1a-specific CTL. As shown in Fig. 8, the addition of in vitro CD8-depleted spleen cells from CB6F1 mice that had rejected EL4 or MOPC70A to the culture of in vitro CD4-depleted spleen cells from RLY1-rejected CB6F1 mice generated CTL on stimulation with the pRL1a peptide. Discussion In this study, we demonstrated that RLY1-specific CTL were generated in spleen cells from the tumor-rejected CB6F1 mice on in vitro stimulation with the pRL1a peptide. We showed that CD4 T cells and APC were necessary for in vitro CTL generation. The addition of rIL-2 to the culture of in vitro CD4-depleted spleen cells from tumor-rejected CB6F1 mice reconstituted CTL generation, suggesting that the IL-2 produced by CD4 T cells is crucial. Furthermore, generation of CTL was blocked by the addition of anti-I-Ad mAb, but not anti-I-Ek/d mAb, to the culture. These findings may suggest that the Ld-binding octamer pRL1a peptide also binds to the
I-Ad molecule on APC and stimulates CD4 T cells to produce IL-2. However, no such binding could be demonstrated by competitive inhibition of I-Ad-restricted recognition of the OVA peptide by CD4 T cell hybridoma DO-11.10. Moreover, no specific proliferative response or IL-2 production was observed with CD4 T cells in spleen cells from the tumorrejected mice on stimulation with a range of MMC-treated RLY1 cells, RLY1 lysate or the pRL1a peptide. Rather, the activation of autoreactive CD4 T cells and the IL-2 production by them were observed in spleen cells from the tumor-rejected CB6F1 mice, which appeared to be responsible for CTL generation. The finding that CD4 T cells from CB6F1 mice that had rejected other tumors such as EL4 or MOPC-70A also helped the generation of pRL1a-specific CTL confirmed the involvement of autoreactive CD4 T cells in CTL generation against the pRL1a peptide. It was reported that murine thymocytes and splenic T cells showed a proliferative response against syngeneic APC (22– 25). A similar observation was also made on human T cells from peripheral blood lymphocytes (26). These syngeneic mixed lymphocyte responses appeared to be the basis of IL-2 production by CD4 T cells required for CTL generation. The response involved the MHC class II antigen and was independent of FCS (24,25). In this study, for immunization of mice, we used tumor cell lines maintained by in vivo passage as ascites form, suggesting that the involvement of FCS was unlikely. The proliferation of CD4 T cells and the IL-2 production by them were also observed with those from unsensitized mice. However, the extent of production from the tumor-rejected CB6F1 mice was 2- to 5-fold greater than that from naive CB6F1 mice. CD4 T cells in the tumor-rejected CB6F1 mice must somehow be activated against the self antigen and show efficient proliferation and IL-2 production, thus explaining the requirement of in vivo sensitization with the tumor itself. It is unknown why autoreactive CD4 T cells
In vitro CTL generation by tumor antigen peptide 1173
Fig. 8. Generation of CTL on in vitro stimulation with pRL1a peptide by the help of autoreactive CD4 T cells. Spleen cells from RLY1rejected CB6F1 mice treated with anti-L3T4 (CD4) mAb plus complement were mixed with spleen cells from EL4- or MOPC-70Arejected, or naive CB6F1 mice treated with anti-Lyt-2.2 (CD8) mAb plus complement, and were cultured with 10–6 M pRL1a for 5 days. Cytotoxicity was determined by 4 h 51Cr-release assay with RLY1 target cells.
and failure to activate CD4 T cells due to lack of the helper epitope. For binding to the class II antigen, peptide length was shown to be critical (31,32) and an octamer or a nonamer peptide does not generally bind to class II antigens. Thus, for vaccination with viral antigen peptides, long peptides (15– 20mer) were used which potentially included class II-binding helper epitopes for CD4 activation (33,34). Recently, however, there were some reports which showed CTL induction by class I-binding short peptides derived from viruses or bacteria which appeared to be strongly immunogenic (35–37). This suggested that the CD4 requirement was inversely related to the antigenic strength of the peptide used. The requirement of CD4 T cells for in vitro CTL generation in spleen cells from mice without in vivo sensitization on stimulation with MHC class I-binding peptide has been shown previously (38). Endogenous p2Ca peptide derived from oxoglutarate dehydrogenase is an octamer peptide which binds to Ld molecules and is recognized by allogeneic Ldspecific 2C CTL line (39). We and others showed that the p2Ca peptide is capable of generating CTL in in vitro primary cultures of spleen cells (38,40). CD4 T cells and APC were both required for generation of CTL. Furthermore, anti-I-Ad mAb, but not anti-I-Ed mAb, blocked CTL induction. It could be possible that the peptide was bound to H-2 class II molecules. However, direct proof of the binding of the peptide to class II molecule or that of any specific proliferative response of CD4 T cells or IL-2 production from them is lacking. Rather, it could be possible that activation of autoreactive CD4 T cells occurred in spleen cells from naive mice (see above) providing enough help for CD8 T cells to react against such a strongly antigenic peptide. Cytokines such as IL-1 secreted from macrophages which endocytosed the high concentration (~10–6 M) of peptide used for in vitro stimulation would facilitate the activation of CD4 T cells.
Acknowledgements were activated in tumor rejection in this study. Activation of autoreactive CD4 T cells could be due to direct activation by the self antigen overexpressed on APC, or bystander activation (27,28) during rejection of large tumor mass. Previously, Kuribayashi et al. (29) established a CTLL-D4 cell line comprising Lyt-112–3– and Lyt-162131 populations from RLY1rejected CB6F1 spleen cells by in vitro stimulation with tumor cells without exogenously added IL-2. Adoptive transfer of CTLL-D4 was effective for tumor rejection only in the presence of the Lyt-112–3– population of which proliferative response was shown to be against the self, but not the tumor antigen (30). These findings were consistent with our findings. The finding that CTL were generated in CD4-depleted, RLY1rejected CB6F1 spleen cells on stimulation with MMC-treated RLY1 cells (as shown in Fig. 2) indicated that strongly antigenic tumor cells were capable of activating CD8 precursor CTL even without help from CD4 T cells. RLY1 did not produce IL-2 in culture. Thus, autoreactive CD4 T cells optimized the generation of CD8 CTL. In vitro CTL induction following vaccination with class I-binding short epitope peptides alone was generally difficult. This is probably due to the low immunogenicity of the peptide
We thank Ms M. Isobe for excellent technical assistance and Ms J. Mizuuchi for preparation of the manuscript. This work was supported in part by a Grant-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Science, Sports and Culture of Japan.
Abbreviations B6 CB6F1 CTL PFA MMC OVA PEC
C57BL/6 (BALB/c3C57BL/6)F1 cytotoxic T lymphocyte paraformaldehyde mitomycin ovalbumin peritoneal exudate cells
References 1 Sato, H., Boyse, E. A., Aoki, T., Iritani, C. and Old, L. J. 1973. Leukemia-associated transplantation antigens related to murine leukemia virus. The X.1 system: immune response controlled by a locus linked to H-2. J. Exp. Med. 138:593. 2 Nakayama, E. and Uenaka, A. 1985. Effect of in vivo administration of Lyt antibodies. Lyt phenotype of T cells in lymphoid tissues and blocking of tumor rejection. J. Exp. Med. 161:345.
1174 In vitro CTL generation by tumor antigen peptide 3 Udono, H., Mieno, M., Shiku, H. and Nakayama, E. 1989. The roles of CD81 and CD41 cells in tumor rejection. Jpn. J. Cancer Res. 80:649. 4 Nakayama, E., Shiku, H., Takahashi, T., Oettgen, H. F. and Old, L. J. 1979. Definition of a unique cell surface antigen of mouse leukemia RLY1 by cell-mediated cytotoxicity. Proc. Natl Acad. Sci. USA 76:3486. 5 Uenaka, A., Ono, T., Akisawa, T., Wada, H., Yasuda, T. and Nakayama, E. 1994. Identification of a unique antigen peptide pRL1 on BALB/c RLY1 leukemia recognized by cytotoxic T lymphocytes and its relation to the Akt oncogene. J. Exp. Med. 180:1599. 6 Wada, H., Matsuo, M., Uenaka, A., Shimbara, N., Shimizu, K. and Nakayama, E. 1995. Rejection antigen peptides on BALB/c RLY1 leukemia recognized by cytotoxic T lymphocytes: derivation from the normally untranslated 59 region of the c-Akt proto-oncogene activated by long terminal repeat. Cancer Res. 55:4780. 7 Ono, T., Uenaka, A. and Nakayama, E. 1996. Production of murine leukemia RLY1 rejection antigen peptide pRL1a by proteolysis of natural precursor pRL1b. Jpn. J. Cancer Res. 87:1165. 8 Potter, M. 1967. The plasma cell tumors and myeloma protein of mice. Methods Cancer Res. 2:106. 9 Gorer, P. A. 1950. Studies in antibody response of mice to tumour inoculation. Br. J. Cancer 4:372 10 Dunn, T. B. and Potter, M. 1957. A transplantable mast-cell neoplasm in the mouse. J. Natl Cancer Inst. 18:587. 11 Kim, K. J., Kanellopoulos-Langevin, C., Merwin, R. M., Sachs, D. H. and Asofsky, R. 1979. Establishment and characterization of BALB/c lymphoma lines with B cell properties. J. Immunol. 122:549. 12 White, J., Haskins, K., Marrack, P. and Kappler, J. 1983. Use of I region-restricted, antigen-specific T cell hybridomas to produce idiotypically specific anti-receptor antibodies. J. Immunol. 130:1033. 13 Guillet, J. G., Lai, M. Z., Briner, T. J., Buus, S., Sette, A., Grey, H. M., Smith, J. A. and Gefter, M. L. 1987. Immunological self, nonself discrimination. Science 235:865. 14 Monach, P. A., Meredith, S. C., Siegel, C. T. and Schreiber, H. 1995. A unique tumor antigen produced by a single amino acid substitution. Immunity 2:45. 15 Dialynas, D. P., Quan, Z. S., Wall, K. A., Pierres, A., Quintans, J., Loken, M. R., Pierres, M. and Fitch, F. W. 1983. Characterization of the murine T cell surface molecule, designated L3T4, identified by monoclonal antibody GK1.5: similarity of L3T4 to the human Leu-3/T4 molecule. J. Immunol. 131:2445. 16 Nakayama, E. 1982. Blocking of effector cell cytotoxicity and T-cell proliferation by Lyt antisera. Immunol. Rev. 68:117. 17 Ozato, K., Hansen, T. H. and Sachs, D. H. 1980. Monoclonal antibodies to mouse MHC antigens. II. Antibodies to the H-2Ld antigen, the products of a third polymorphic locus of the mouse major histocompatibility complex. J. Immunol. 125:2473. 18 Kappler, J. W., Skidmore, B., White, J. and Marrack, P. 1981. Antigen-inducible, H-2-restricted, interleukin-2-producing T cell hybridomas. Lack of independent antigen and H-2 recognition. J. Exp. Med. 153:1198. 19 Watanabe, M., Suzuki, T., Taniguchi, M. and Shinohara, N. 1983. Monoclonal anti-Ia murine alloantibodies crossreactive with the Ia-homologues of other mammalian species including humans. Transplantation 36:712. 20 Matsutake, T., Sasaki, T., Ogino, T., Kaku, M. and Nakayama, E. 1995. Prevention of mortality by in vivo depletion of αβ T cells in murine lethal listeriosis and involvement of γδ T cells in bacterial elimination. Immunobiology 193:71. 21 Marrack, P., Ignatowicz, L., Kappler, J. W., Boymel, J. and Freed,
22 23
24 25 26
27 28
29 30 31 32 33 34 35
36
37
38
39
40
J. H. 1993. Comparison of peptides bound to spleen and thymus class II. J. Exp. Med. 178:2173. Glimcher, L. H., Longo, D. L., Green, I. and Schwartz, R. H. 1981. Murine syngeneic mixed lymphocyte response. I. Target antigens are self Ia molecules. J. Exp. Med. 154:1652. Smith, J. B. and Pasternak, R. D. 1978. Syngeneic mixed lymphocyte reaction in mice: strain distribution, kinetics, participating cells, and absence in NZB mice. J. Immunol. 121:1889. Lattime, E. C., Gillis, S., Pecoraro, G. and Stutman, O. 1982. Iadependent interleukin 2 production in sygeneic cellular interactions. J. Immunol. 128:480. Nussezweig, M. C. and Steinman, R. M. 1980. Contribution of dendritic cells to stimulation of the murine syngeneic mixed leukocyte reaction. J. Exp. Med. 151:1196. Kuntz, M. M., Innes, J. B. and Weksler, M. E. 1976. Lymphocyte transformation induced by autologous cells. IV. Human Tlymphocyte proliferation induced by autologous or allogenic nonT lymphocytes. J. Exp. Med. 143:1042. Rocken, M., Urban, J. F. and Shevach, E. M. 1992. Infection breaks T-cell tolerance. Nature 359:79. Ehl, S., Hombach, J., Aichele, P., Hengartner, H, and Zinkernagel, R. M. 1997. Bystander activation of cytotoxic T cells: studies on the mechanism and evaluation of in vivo significance in a transgenic mouse model. J. Exp. Med. 185:1241. Kuribayashi, K., Keyaki, A., Sakaguchi, S. and Masuda, T. 1985. Effector mechanisms of syngeneic anti-tumor responses in mice. Immunology 56:127. Keyaki, A., Kuribayashi, K., Sakaguchi, S., Masuda, T., Yamashita, J., Handa, H. and Nakayama, E. 1985. Effector mechanisms of syngeneic anti-tumor responses in mice. Immunology 56:141. Hickling, J. K., Fenton, C. M., Howland, K., Marsh, S. G. E. and Rothbard, J. B. 1990. Peptides recognized by class I restricted T cells also bind to MHC class II molecules. Int. Immunol. 2:435. O’Sullivan, D., Sidney, J., Del Guercio, M. F., Colon, S. M. and Sette, A. 1991. Truncation analysis of several DR binding epitopes. J. Immunol. 146:1240. Fayolle, C., Deriaud, E. and Leclerc, C. 1991. In vivo induction of cytotoxic T cell response by a free synthetic peptide requires CD41 T cell help. J. Immunol. 147:4069. Gao, X. M., Zheng, B., Liew, F. Y., Brett, S. and Tite, J. 1991. Priming of influenza virus-specific cytotoxic T lymphocytes in vivo by short synthetic peptides. J. Immunol. 147:3268. Dyal, R., Vasovic, L. V., Molano, A. and Nokolic-Zugic, J. 1995. CD4-independent in vivo priming of murine CTL by optimal MHC class I-restricted peptides derived from intracellular pathogens. Int. Immunol. 7:1205. Fayolle, C., Abdel-Motal, U. M., Berg, L., Deriaud, E., Jondal, M. and Leclerc, C. 1996. Induction of cytotoxic T-cell response by optimal-length peptides does not require CD41 T cell help. Immunology 89:41. Sauzet, J. P., Gras-Masse, H., Guillet, J. G. and Gomard, E. 1996. Influence of strong CD4 epitopes on long-term virus-specific cytotoxic T cell responses induced in vivo with peptides. Int. Immunol. 8:457. Wada, H., Ono, T., Uenaka, A., Monden, M. and Nakayama, E. 1995. Requirement of CD41 T cells and antigen-presenting cells for primary in vitro generation of CD81 cytotoxic T cells against Ld-binding self-peptide p2Ca. Immunology 84:633. Udaka, K., Tsomides, T. J., Walden, P., Fukusen, N. and Eisen, H. N. 1993. A ubiquitous protein is the source of naturally occurring peptides that are recognized by a CD81 T-cell clone. Proc. Natl Acad. Sci. USA 90:11272. Tjoa, B. A. and Kranz, D. M. 1994. Generation of cytotoxic T-lymphocytes to a self-peptide/class I complex: a model for peptide-mediated tumor rejection. Cancer Res. 54:204.
