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International Immunology, Vol. 13, No. 11, 1361-1365, November 2001
© 2001 Japanese Society for Immunology

Immunization with a carbohydrate mimicking peptide augments tumor-specific cellular responses

Behjatolah Monzavi-Karbassi, Gina Cunto-Amesty, Ping Luo, Shahram Shamloo, Magdalena Blaszcyk-Thurin11 and Thomas Kieber-Emmons

Department of Pathology and Laboratory Medicine, University of Pennsylvania, and
1 The Wistar Institute, Philadelphia, PA 19104, USA

Correspondence to: T. Kieber-Emmons


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
The metastatic potential of some tumor cells is associated with the expression of the neolactoseries antigens sialyl-Lewis x (sLex) and sialyl-Lewis a (sLea) as they are ligands for selectins. We have recently shown that peptide mimetics of these antigens can potentiate IgG2a antibodies, which are associated with a Th1-type cellular response. As L-selectin is preferentially expressed on CD4+ Th1 and CD8+ T cell populations, specific induction of these phenotypes could augment a response to L-selectin ligand-expressing tumor cells. Here we demonstrate that immunization with a multiple antigen peptide (MAP) mimetic of sugar constituents of neolactoseries antigens induces a MHC-dependent peptide-specific cellular response that triggers IFN-{gamma} production upon peptide stimulation, correlating with IgG2a induction. Surprisingly, T lymphocytes from peptide-immunized animals were activated in vitro by sLex, also triggering IFN-{gamma} production in a MHC-dependent manner. Stimulation by peptide or carbohydrate resulted in loss of L-selectin on CD4+ T cells confirming a Th1 phenotype. We also observed an enhancement in cytotoxic T lymphocyte (CTL) activity in vitroagainst sLex-expressing Meth A cells using effector cells from Meth A-primed/peptide-boosted animals. CTL activity was inhibited by both anti-MHC class I and anti-L-selectin antibodies. These results further support a role forL-selectin in tumor rejection along with the engagement by the TCR for most likely processedtumor-associated glycopeptides, focusing on peptide mimetics as a means to induce carbohydrate reactive cellular responses.

Keywords: cancer vaccine, carbohydrate, L-selectin, Meth A cells, peptide mimeotope


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Tumor-associated carbohydrate antigens are correlated with metastatic phenotype and poor survival in epithelial malignancies of different origins. The expression of the neolactoseries antigens represented by sialyl-Lewis x (sLex), Lewis x (Lex) and Lewis Y (LeY) are increased significantly on a variety of carcinomas (1–6), with sLex and sialyl-Lewis a (sLea) also reported to be expressed on melanoma cells (7). Early studies indicated a possible relationship between metastatic properties of tumor cells and the expression of these antigens leading to tumor cell dissemination (8–11). An inhibitory effect on the establishment and growth of metastatic colonies of tumor cells expressing these antigens has been noted when anti-sLea or anti-sLex antibody was administered to mice in a pancreatic tumor murine model (12).

A cellular role played by L-selectin expressed on lymphocytes has also been noted in that anti-L-selectin antibody (Mel14) can influence cytotoxic T Lymphocyte (CTL) sensitization and metastatic colony formation (13), and Mel 14 can inhibit CTL activity of effector cells in vitro derived from immunization with selectin-ligand expressing cells (14). These results strongly suggest that blood-borne tumor cells may utilize these antigens with selectin molecules when tumor cells adhere to the endothelia at metastatic sites (1,12,15–17). Over-expression of selectin ligands on tumor cells are also targeted by NK cells (18). Consequently, targeting these antigens may thwart tumor cell dissemination.

We have recently shown that peptide mimetics of selectin ligands can induce a humoral response that targeted sLex in a murine tumor model facilitating tumor growth inhibition (19). We have gone on to show that immunization with peptide mimetics can elicit carbohydrate-reactive IgG2a antibodies, associated with Th1 (20,21). As QS-21 potentiates a Th1 response (22) and L-selectin is preferentially expressed on CD4+ Th1 cells (23) and on CD8+ T cells (14,24), we were in the first instance interested in determining if peptide mimetic immunization activated lymphocytes that expressed L-selectin. As glycopeptides are also presented by MHC class I that express carbohydrate constituents (25,26) and T cells can recognize carbohydrate moieties on MHC-associated glycopeptides (27), we were also interested in whether mimeotope immunization could augment a CTL response to fibrosarcoma Meth A cells.

We observed that peptide immunization promotes a specific cellular response with IFN-{gamma} production upon activation of splenocytes and T lymphocytes with immunizing peptide or with sLex and its homologue Lex. More importantly, we observed that immunization with peptide mimetic facilitated a specific cellular response to Meth A cells. Peptide boosting of mice primed with sLex-expressing Meth A cells enhanced the Meth A directed in vitro CTL activity. This activity was blocked not only by anti-MHC class I but also by anti-L-selectin antibody. This latter observation parallels other studies describing a role for L-selectin in T cell-mediated rejection of cells that express its ligands, such as sLex (14). The possibility of boosting lymphocyte subsets that cross-react with tumor cells that express L-selectin ligands opens new perspectives in designing cancer vaccines for further reducing micrometastases.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Immunization of animals
Peptides were synthesized as multiple antigen peptides (MAP; Research Genetics, Huntsville, AL) made by Fmoc synthesis on polylysine groups, resulting in the presentation of eight peptide clusters. Multivalent sLex, Lex and LeY were obtained from GlycoTech (Rockville, MD). Each BALB/c mouse received i.p. injections with 100 µg of MAP, or 50 µg of carbohydrates, and 20 µg of the adjuvant QS-21 (Aquila Biopharmaceuticals, Framingham, MA), both re-suspended in 100 µl of PBS as we described earlier (19). QS-21 was used in all peptide and carbohydrate immunization as adjuvant. As controls we used naive mice, animals injected with QS-21 (20 µg per mouse) or with multivalent sLex and LeY (50 µg of carbohydrates and 20 µg of the adjuvant QS-21 per mouse) antigens.

sLex-expressing Meth A cells were selected by sorting of positive cells with anti-sLex FH-6 antibody from an original Meth A cell line and were 100% sLex+ upon prolonged culture. Meth A cells were repeatedly passed in vitro to decrease their tumorgenicity. Mice were immunized i.p. with 3x105 of these cells. Non-tumor bearing mice were used for analysis or for further experiments.

Antibodies
All antibodies were purchased from PharMingen (San Diego, CA). Anti mouse I-Ad (AMS 32.1), H-2Kd (SF1-1.1) and CD1d (1B1), and their IgG2a and IgG2b isotype controls were first dialyzed in PBS buffer and then used in the proliferation cultures. For fluorescent staining of the splenocytes we used biotinylated rat anti-mouse anti-CD62L (anti-L-selectin Mel-14) and its isotype control IgG2a; anti-CD4–FITC (L3T4) and its isotype control IgG2b–FITC.

Flow cytometry
Fresh or in vitro stimulated splenocytes were blocked before staining with PBS containing 1% BSA and 1% rat serum for 10 min at 4°C. Cells were subsequently stained with rat anti-mouse mAb labeled with biotin, FITC or phycoerythrin. Appropriate rat isotype controls were used to set up background fluorescence. Streptavidin–FITC or avidin– phycoerythrin (Sigma, St Louis, MO) were used after biotin-labeled antibodies, as required. Acquisition of data was performed immediately after staining by using the FACScan analyzer and analysis performed by CellQuest software (both from Becton Dickinson Immunocytometry Systems, Mansfield, MA).

In vitro proliferation assays
Spleens were aseptically removed from each group. Splenocytes were harvested from spleens, and isolated as the responder cells by lysis of erythrocytes and consequent washing several times with fresh media. Prepared responder cells were used for detection of cell proliferation, as described (28,29). Briefly, cells (2.5x105/well) were cultured in flat-bottom 96-well plates and incubated with MAP, carbohydrates, ovalbumin (OVA) or media only. After 3 days of incubation, 1 µCi of [3H]thymidine was added to each well and cells were incubated for an additional 16–18 h. Cultures were then harvested and radioactive emission counted on a Betaplate liquid scintillation counter (EG & G Wallac, Turku, Finland).

Proliferation assay was also performed using CellTiter 96 Aqueous One Solution (Promega, Madison, WI) based on the manufacturer's instructions. Cell culture was exactly performed as above and at the third day of incubation the provided solution was added to each well. Plates were incubated for an additional 1–2 h in a humidified, 5% CO2 incubator at 37°C. Absorbency was measured immediately at 490 nm using a 96-well plate reader (Spectra Fluor; Tecan, Triangle Park, NC) as a measure for cell proliferation.

For detection of cell proliferation in T lymphocyte-enriched populations, a single-cell suspension of isolated splenocytes was depleted of B cells by positive selection after incubation with pan-B (B220) magnetic beads (Dynal, Oslo, Norway), at 4°C, for 30 min. To separate adherent from non-adherent cells, the B cell-depleted population was resuspended in RPMI media with 20% FBS and incubated for two consecutive adherence periods of 1 h each, at 37°C in humidified atmosphere with 5% CO2. T lymphocytes were recovered and washed 3 times with RPMI/10% FBS and used in cell proliferation assays with or without mitomycin C (MMC)-treated antigen-presenting cells (APC). Adherent cells were recovered separately, treated with MMC (100 µg/ml in RPMI supplemented with 10% FBS, for 40 min at 37°C) and used as APC in proliferation of purified T lymphocytes at 10% ratio. Purified T cell populations, checked by FACS analysis, were assessed as >95% CD3+, with no CD19+ cells, and used in cell proliferation assays as explained above. Medium used for the proliferation assays was RPMI 1640 (Life Technologies, Rockville, MD) supplemented with 5% heat-inactivated FCS, L-glutamine, penicillin (100 IU/ml) and streptomycin (100 µg/ml).

Cytokine production
Supernatants were collected from the co-cultures of splenocytes or purified T lymphocytes 72 h after in vitro stimulation with carbohydrate, peptides and media. The concentrations of IL-4, IL-10 and IFN-{gamma} were measured by quantitative capture ELISA (PharMingen), according to the manufacturer's instructions.

CTL assay
Cytotoxic activity was measured by a 5 (P815 cells)- or 16 (Meth A cells)-h 51Cr-release assay, as described elsewhere (30,31). Briefly, effector cells were stimulated for 5 days in the presence of stimulator cells and 10% RAT-T-STIM without concanavalin A (Becton Dickinson Labware, Bedford, MA). Peptide-pulsed murine mastocytoma P815 cells were used as stimulators (10:1 effector:stimulator ratio) and targets in 5-h assay, as described before (30,32). Meth A cells were treated with MMC and used as stimulator in 300:1 effector:stimulator ratio. For MMC treatment, 1x107 cells were treated in 1 ml of RPMI media supplemented with 10% FBS, with 100 µg MMC at 37°C for 40 min. Cells were washed 3 times after treatment.

Untreated Meth A cells were used as target cells. All target cells were labeled with 100 µCi/ml Na251CrO4 and mixed with effector cells at E:T ratios ranging from 50:1 to 3:1. Maximum and minimum (spontaneous) releases were determined by lysis of target cells in 5% Triton X-100 and medium respectively. Lysis was calculated as [(experimental – spontaneous)/(maximum – spontaneous)]x100. An assay was not considered valid if the values for the `spontaneous release' count were >20% (for 5-h assay) or 30% (for 16-h assay) of the `maximum release'. To calculate specific lysis of targets, the percent lysis of non-specific targets (P815 cells) was subtracted from the percent lysis of specific targets (peptide-pulsed P815 cells).

Statistical analysis
Data were analyzed by using Student's t-test. Values of P < 0.05 were considered statistically significant. All experiments were performed at least 3 times.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Peptide immunization primes for a specific cellular response
To facilitate a cellular response, we had designed peptide 106 (Table 1Go) with the potential to bind to H2-Kd and I-Ad. MHC binding prediction calculations identifies an H2-Kd and I-Ad binding motif centered on the RYDIYWRYDI sequence of the 106 peptide (Table 1Go) (33). Furthermore, the WRYDI sequence tract of the 106 peptide also displays similarity to a peptide sequence tract (DIYRW) identified in a peptide that binds to CD1 (34), which may play a role in activating NK T cells.


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  		Table 1. Peptides used in this study
 
To analyze the cellular response upon peptide 106 immunization, BALB/c mice were immunized with the mimeotope and the proliferative response of isolated splenocytes to the peptides 106, 711 (Table 1Go) as MAP forms, as well as OVA (as an additional control) was determined. As expected, in vivo primed splenocytes from 106 peptide-immunized animals were specifically activated by peptide 106, and not control peptides, in a statistically significant concentration-dependent manner (Fig. 1Go). Peptide 106 did not stimulate splenocytes from naive animals or from animals immunized with QS-21 alone (data not shown). These results indicate that immunization with peptide 106 mediates a peptide-specific cellular response.



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  		Fig. 1. Proliferative response of splenocytes from peptide 106-immunized mice. (A) Mice (four per group) were immunized 3 times with peptide 106 formulated in QS-21 every other week. Seven days after the third injection, spleens were collected and splenocytes were incubated with two concentrations (10 and 2 µg/ml) of the indicated MAP or OVA for 72 h. A 3H-incorporation assay was performed as described in Methods. Results represent the mean value ± SEM based on four independent experiments with triplicate samplings. *P < 0.05; **P < 0.025.

 
Anti-MHC class II and I inhibit proliferative response
A standard cell proliferation was performed with purified T lymphocytes alone and after addition of MMC-inactivated APC (Fig. 2AGo). The results indicate that proliferation is APC dependent. Proliferation of the T lymphocytes was not observed in the absence of APC. Proliferation was inhibited by the addition of specific anti-I-Ad anti-class II and to a lesser extent with anti-class I antibodies (Fig. 2BGo). The addition of anti-CD1 antibody inhibited proliferation to almost the same extent as addition of anti-class I antibody. Therefore, the 106 peptide might be presented in multiple ways to T cells; however, MHC class II-dependent presentation appears to be the major pathway (60% inhibition by anti-class II antibody).



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  		Fig. 2. Cell proliferation is APC dependent, and is blocked by anti-MHC class II and I antibodies. Mice were immunized with 106 peptide as explained in the legend to Fig. 1Go and splenocytes were collected 7 days after the last immunization. (A) Proliferation assay was performed on splenocytes and purified T lymphocytes (with or without addition of inactivated APC) with a peptide concentration of 10 µg/ml. (B) Anti-I-Ad, -H-2Kd and -CD1d, and IgG2a and IgG2b isotypes were used for blocking of proliferation of splenocytes. Isotype controls acted very similarly, so just one of those is presented. (C) Splenocytes were stimulated using peptides 106, 711 and OVA (each 10 µg/ml), and IFN- {gamma} levels were quantified. All results are expressed as the mean ± SEM of four independent experiments with triplicate samplings. *P < 0.05; **P < 0.01.

 
For analysis of cytokine profiles, plates for proliferation were doubled and the supernatant of duplicated plates were harvested after 72 h of in vitro activation. IFN-{gamma} production was found to be peptide specific (Fig. 2CGo), with no detectable presence of IL-4 or IL-10 (data not shown). The sensitivity for IL-4 and IL-10 detection was 7.8 and 31.3 pg/ml respectively. Taken together, these results demonstrate that 106-MAP-peptide immunization along with QS-21 potentially activates lymphocyte populations with a predominant Th1 phenotype.

Activation down-modulates L-selectin
We examined L-selectin expression on CD4+ T cells. L-selectin is preferentially expressed on Th1 over Th2 cells (23). The activation of T cells through the TCR results in the differential regulation of L-selectin (24). Using FACS analysis, we studied the expression of L-selectin on CD4+ T cells from naive and peptide-immunized animals before and after in vitro stimulation with peptide 106 to assess activation (Fig. 3Go). In keeping with other studies, a population of cells from peptide 106-immunized mice remained clearly L-selectin+ after in vitro stimulation (24). The demonstrated presence and loss of L-selectin on the surface of CD4+ T cells occurring upon stimulation to the 106 peptide further confirms a Th1 nature for these cells. It was observed that sLex could activate these cells as effectively as peptide 106 as assessed by L-selectin loss (Fig. 3Go).



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  		Fig. 3. Analysis of L-selectin expression on the surface of CD4+ lymphocytes obtained from naive and 106/QS21-immunized animals. Staining was performed with phycoerythrin-conjugated anti-mouse L-selectin (Mel-14) and anti-CD4–FITC (L3T4) mAb before and after 3 days of in vitro stimulation. For stimulation either 106 or sLex (10 µg/ml) were used. Analysis of Mel-14 binding was performed on gated CD4 + lymphocytes. Mean fluorescence values for L-selectin expression on CD4+ cells are shown in the top right corner of the histograms. Naive, cells from naive mice before proliferation. 106 immunized, cells from 106-peptide immunized mice before proliferation. Naive/106 and 106/106, cells from naive and 106 immunized mice stimulated in vitro for 3 days with 106 peptide. Naïve/sLex and 106/sLex, cells from naive and 106 immunized mice stimulated in vitro for 3 days with carbohydrate sLex.

 
Carbohydrate stimulates proliferation of 106 peptide-primed splenocytes
In separate experiments, isolated splenocytes were incubated with sLex, Lex and LeY (Fig 4AGo). sLex and Lex, but not LeY stimulated a proliferative response with splenocytes from 106-immunized animals. Splenocytes from mice immunized with the carbohydrate antigens sLex or LeY did not proliferate upon incubation with peptide 106, LeY, sLex or Lex (Fig. 4B and CGo). This result suggests that priming with the peptide mimeotope leads to a specific carbohydrate cross-reactive cellular response.



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  		Fig. 4. Cell proliferative response to carbohydrates in splenocytes from 106 peptide (A)-, sLex (B)- and LeY (C)-immunized mice. Mice (four per group) were immunized 3 times at a 2-week interval. Seven days after the last immunization spleens were removed, and splenocytes collected and prepared as explained in Methods. Cells were incubated with peptide and carbohydrates for 3 days. Proliferation was measured at the third day after incubation. For recording the proliferation response the number of viable cells was detected using the CellTiter 96 Aqueous One Solution cell proliferation assay (Promega). The absorbance in the presence of culture medium is subtracted as background proliferation. All results present the mean value ± SD of triplicate samplings. Data are representative of three independent experiments with pooled cells from four mice. *P < 0.05; **P < 0.025; ***P < 0.01.

 
MHC class II molecule is involved in proliferation of peptide-primed T lymphocytes by carbohydrate
sLex-stimulated proliferation of peptide-primed T lymphocytes was inhibited by the I-Ad-specific anti-MHC class II antibody, suggesting a role played by the class II molecule in the cross-reactivity between the carbohydrate antigen and peptide-primed responder cells and APC (Fig. 5AGo). The specificity of the antibody for I-Ad has been assessed by others, indicating specific MHC class II inhibition of proliferation (35–37). IFN-{gamma} production for sLex activation was detected (Fig. 5BGo). As in the peptide case, we could not detect any IL-4 and IL-10 production after activation. This production was specific for peptide-immunized mice compared with T lymphocytes derived from mice immunized with QS-21 alone or from naive animals.



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  		Fig. 5. (A) Anti-MHC class II antibody blocks activation of T cells by sLex. Purified T lymphocytes from 106 peptide/QS21-immunized (performed as explained above) animals were used in a proliferation assay with or without sLex (10 µg/ml) in the presence of MMC-treated APC. Incubation was done in the presence or absence (No Ab) of anti-MHC class II antibody (10 µg/ml). Isotype control was used at 10 µg/ml. (B) IFN-{gamma} production in the supernatant of purified T lymphocytes stimulated with sLex (10 µg/ml) for 3 days in the presence of MMC-treated APC. Supernatant from stimulated T lymphocytes was collected and IFN-{gamma} was measured. All results are presented as the mean value ± SEM based on four independent experiments with duplicate samplings. Medium, proliferation in culture medium used as background. *P < 0.05; **P < 0.025; ***P < 0.01; ****P < 0.005. ns, not significant.

 
Peptide boost of Meth A cell-primed animals enhances CTL activity against Meth A cells
In vitro stimulated effector cells from peptide 106-injected animals showed high specific lysis against peptide-pulsed P815 cells, confirming the peptide's ability to function as a MHC class I target (Fig. 6AGo). As expected, cytotoxicity was reduced in experiments in which CD8+ cells were depleted. When Meth A cells were used as stimulators and targets, we observed moderate cell killing (Fig. 6BGo). In contrast, no significant cytotoxicity was detected on the P815 cells, used as a negative control. P815 cells did not express sLex, as assessed by FACS using the mAb FH-6 (data not shown) nor did serum to peptide 106 bind to P815 cells, also assessed by FACS (data not shown). We did not detect any significant increase in lysis of peptide pulsed Meth A cells (not shown). In vitro stimulated splenocytes from naive or QS-21 immunized mice did not show significant cytotoxicity on Meth A cells.



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  		Fig. 6. Induction of cross-reactive CTL response to Meth A cells following immunization with peptide 106. Mice (four per group) were immunized with peptide 106 at 3 times at a 2-week interval and 7 days after the last immunization, spleens were collected and isolated effector cells were re-stimulated with either peptide pulsed P815 cells or Meth A cells in vitro for 5 days. CTL was performed using 106 peptide-pulsed P815 (A) or Meth A (non-pulsed) (B) cells as stimulators and targets. {blacklozenge}, Bulk effector cells from 106-immunized mice; •, CD8+ T cell depleted effector cells from 106-immunized mice; {blacktriangleup}, Bulk effector cells from naive mice. To calculate specific lysis of targets (as shown in A), the percent lysis of non-specific targets (P815 cells) was subtracted from the percent lysis of specific targets (peptide-pulsed P815 cells). Cytotoxicity against P815 cells as control target (open symbols) is shown in (B). All error bars (SD) were calculated based on triplicates. All data are representative of three independent experiments using pooled cells from four mice. Statistically significant compare with cytotoxicity levels of naive effector cells at *P < 0.05 and **P < 0.025 respectively.

 
To examine the hypothesis that peptide immunization could augment cellular responses to Meth A-cells, we studied the proliferative effects of peptides and Meth A cells in groups of mice immunized with Meth A cells only or primed with Meth A cells and boosted with peptide. The results are summarized in Fig. 7Go(A). Immunization with Meth A cells stimulated a proliferative response to the cells, as expected. Boosting of Meth A cell-immunized mice with peptide 106 increased the proliferative response to peptide 106 and to Meth A cells. As the control peptide for proliferation we used a peptide referred to as peptide 109 (Table 1Go). We did not detect cross-reactivity between peptide 109 and Meth A cells, but this peptide showed cross-reactivity in cellular response with 106 peptide (not shown; attributed to the sequence homology between the peptides). We investigated the role of Meth A and P815 cells in stimulation of splenocytes from mice immunized with peptide 106, sLex and OVA as control. Only splenocytes from 106 peptide immunization showed a proliferative response in co-culture with MMC-treated Meth A cells (data not shown), we did not detect significant proliferation using P815 cells as stimulator (data not shown).



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  		Fig. 7. (A) 106 peptide boosts cellular responses in the Meth A cell-primed animals. Mice were immunized with Meth A cells, rested for 1 month, and then were boosted with the 106 peptide and compared with Meth A cell primed/boosted (at the same interval) only. In each immunization regimen, splenocytes were collected 7 days after boost, and proliferative response was measured using peptides 106, 109 and Meth A cells as antigens. Meth A cells were treated with MMC and then used in the assay. Background c.p.m. is [3H]thymidine incorporation with used medium only. (B) Anti-Meth A cells CTL. Mice were primed and boosted with Meth A cells (circle) or primed with Meth A cells and boosted with 106 peptide (square), as explained above. Cytotoxicity was measured against Meth A (closed symbols) or P815 cells (open symbols). Splenocytes were stimulated in vitro with Meth A cells as described in Methods. All bars show SD based on three replications. All data are representative of three independent experiments using pooled cells from four mice. *P < 0.05; **P < 0.025. In (B) asterisks compare the levels of cytotoxicity of effector cells from Meth A/106-immunized animals with the cytotoxicity levels of the same cells detected against P815 cells as targets.

 
We observed a cytotoxic enhancement effect upon immunization with peptide 106 using effector cells from Meth A-immunized mice compared with those from Meth A-primed/peptide-boosted mice (Fig. 7BGo). The level of CTL was consistent with those observed in similar studies (31,32). A clear increase in lysis was observed in the cell-primed/peptide-boosted animals compared to mice immunized with Meth A cells only. We did not detect any cytotoxicity using re-stimulated effectors from either immunization groups targeting P815 cells. Levels of cyotoxicity against Meth A cells were compared with those levels against P815 cells, statistically significant cytotoxicity against Meth A cells was detected only with effectors from peptide boosted animals. Representative results from 50:1 E:T ratio of a separate experiment are summarized in Table 2Go. Cytotoxicity was dependent on both CD4+ and CD8+ cells as assessed by respective depletion of the CD4+ and CD8+ subsets. Meth A cell-mediated lysis was found to be inhibited by both anti-MHC class I and L-selectin antibodies, but not by anti-class II antibody as Meth A cells do not express MHC class II molecules.


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  		Table 2. Percentage of specific lysis (± SD)a on Meth Ab cells as targets at 50:1 E:T ratio
 
These results further suggest that immunization with peptide or Meth A cells activates CD4+ and CD8+ T cells that affect cytotoxicity. Meth A cells are known to activate both T cell subsets that are involved in cytotoxicity targeting Meth A cells (31,32). However, in the case of peptide mimetic boost of Meth A cell-immunized animals, MHC class I-restricted/CD8+-dependent T cells showed a predominant role in the CTL response. Our results further indicate that L-selectin may participate in the lysis process as described previously (14). With naive mice and QS-21-immunized mice, as before, we did not detect any significant lysis.


   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
We had previously demonstrated that immunization with a multivalent, repetitive peptide mimetic of sLex (peptide 106) induced an anti-sLex cross-reactive antibody response to Meth A tumor cells (19). Antibody responses to sLex can mediate complement-dependent killing of sLex-expressing tumor cells inhibiting the establishment and growth of metastatic colonies as observed in several animal models (12,19,38).

Meth A tumor growth inhibition upon immunization with attenuated Meth A cells is reported to be dependent on CD4+ and CD8+ T cell responses to undefined glycoproteins (31,32,39–41). Commitment of lymphocytes to the Th1 and to CD8+ T cell phenotypes as characterized by the expression of IFN-{gamma}, may be critically involved in tumor rejection (42,43). However, the expression of L-selectin on these cell types may also lend to CTL activity (14). IFN-{gamma} and lymphocytes work together to find and eliminate tumor cells (44). As the adjuvant QS-21 promotes the Th1 phenotype, which expresses L-selectin, and peptide 106 can induce a Th1-associated humoral response, we examined if immunization with peptide 106 along with QS-21 could augment cellular responses to sLex-expressing Meth A tumor cells.

Immunization with the mimeotope led to peptide-specific cell proliferation that was concentration dependent (Fig. 1Go). As expected, cell proliferation was primarily MHC class II dependent as determined by inhibition with respective anti-MHC antibody (Fig. 2Go). Cell stimulation with peptide 106 triggered IFN-{gamma} release, suggesting that peptide immunization with QS-21 as expected polarized the Th1 subset (Fig. 2CGo). Consistent with previous studies we observed L-selectin loss on CD4+ T lymphocytes upon peptide stimulation, but also saw this loss upon stimulation with sLex (Fig. 3Go). The proliferative response was peptide, sLex and Lex specific since LeY did not exhibit any cellular responses nor did splenocytes from sLex- and LeY-immunized animals responded to peptide 106, sLex or Lex antigens (Fig. 4Go).

We observed that sLex activated T lymphocytes from 106 immunized animals to proliferate and secrete IFN-{gamma} (Fig. 5Go). Cell activation by sLex was inhibited by anti-MHC class II antibody addition, suggesting a possible role for this molecule in the presentation process (Fig. 5BGo). In vivo stimulated effector cells from peptide 106 immunization displayed cytotoxicity directed toward peptide 106-pulsed MHC class I+ class II– P815 target cells, further verifying a role for CD8+ T cell reactivity with peptide 106 (Fig. 6Go). While Meth A cell priming and boosting can lead to CTL activity against Meth A cells, peptide boosting increased the level of cytotoxicity against Meth A cells to a statistically significant level as compared with cytotoxicity against P815 cells as control target (Fig. 7Go and Table 2Go), indicating a cross-reactive nature between peptide and tumor specific CTL responses.

CTL activity was inhibited by the addition of either anti-class I or anti-L-selectin antibody (Table 2Go). However, we could not block CTL activity by the sLex-reactive antibody FH-6 (data not shown). It is possible that FH-6 binds to a subtype of sLex carbohydrate epitopes that do not always function as ligands for L-selectin just like it defines a subset that does not bind to E-selectin (2). This is also similar to that found for the antibody MECA 79 which binds to a subset of sulfated sLex different than that of L-selectin (45). L-selectin is known to bind to a variety of carbohydrates expressed on glycoproteins (46,47). It is possible that L-selectin functions as an auxiliary molecule (48) and by itself is not sufficient to mediate CTL killing, but requires engagement of antigen-specific TCR (14). NK cells, on the other hand, also express L-selectin and other lectin-type molecules, and NK cells appear to mediate cytolysis of tumor cells that express high levels of sLex (18). However, direct evidence that fuscosylated selectin ligands play a role in tumor rejection is still lacking.

The activation of cross-reactive T cells has been described in many systems. What is the specificity of the CD8+ T cells targeting Meth A? Meth A cell-primed T cells maybe glycopeptide/glycoprotein specific (32), which are cross-reactive with peptide 106, as glycopeptides are known to activate T cells that recognize the carbohydrate moiety on MHC-associated glycopeptides (49–55). Direct interaction of the TCR with the carbohydrate (27,56) is dependent on the chemical structure of the glycan as well as its position within a peptide. Glycopeptides with GlcNAc residues known to associate with class I (25) also induce CTL responses (26). T cells primed to glycopeptides carrying more complex saccharide antigens sometimes show a complicated pattern of cross-responses to glycopeptides with simpler glycan moieties (51). It argues that the presentation of carbohydrate antigens on Meth A should be more thoroughly characterized in terms of the structure of glycopeptides, glycoshingolipids, etc., which is beyond the scope of the present paper.

Proliferation by sLex and Lex is tougher to reconcile. Carbohydrate antigens have been proposed to associate with MHC directly (57); however, this type of association has not yet been confirmed in our system. It is possible that proliferation is associated with some yet undefined superantigen-like association as we observed up-regulation of CD15s on sLex activated T cells (data not shown). Superantigens but not mitogens are capable of inducing up-regulation of E-selectin ligands on human T lymphocytes (58). Likewise, bacterial polysaccharides and mimicking peptides with a distinct charge motif involving Lys and Asp residues have been shown to activate T lymphocytes and that this activity confers immunity to a distinct pathologic response to bacterial infection (59). Peptide 106 has a Arg residue homologous to Lys, with the Asp residues identical to peptides that induce cross-reactive cellular responses in bacterial studies (59). The molecular details of how this occurs are not as yet known. Further analysis of binding affinities of peptide and carbohydrate with I-Ad will be illuminating. However, immunization with sLex did not induce T lymphocytes that reacted with peptide 106 nor itself, an effect of sLex being a T cell-independent antigen.

Immunization with peptide 106 induces cellular responses that are not achievable by immunization with carbohydrate alone. Nevertheless, the induction of optimal systemic anti-tumor immunity involves the priming of both CD4+ and CD8+ T cells specific for tumor-associated antigens. Although cellular responses generated by the peptide mimeotope may enhance CTL induction, vaccination with peptide alone appears not to be completely sufficient in the effector phase when challenged with a very high tumor burden (19). Our CTL data using effectors from peptide-only-immunized mice on Meth A cells as targets (Fig. 6BGo) confirm this fact with marginal CTL activity observed. Consequently, we did not observe complete tumor protection in our previous study (19).

Our results are very narrow with regard to the breadth of carbohydrate directed cellular responses. However, constituents of sLex and sLea are proposed to be influential to the metastatic properties of a variety of human tumor cells. Consequently, further efforts to optimize and isolate the carbohydrate moieties associated with presented glycopeptides may facilitate vaccine applications for eradication of metastatic lesions by both antibody-dependent lysis and cellular responses. This possibility has yet to be proved with the appropriate models but suggests that for certain carbohydrate antigens, peptide mimetics might augment cellular responses other than delayed-type hypersensitivity like responses (60) in future vaccine applications.


   Acknowledgments
 
This work was supported by NIH grant AI45133 and by US Army Material Command grant DAMD 17-01-0366. We thank Charlotte Read Kensil of Aquila Biopharmaceuticals, Inc. (Framingham, MA) for supplying the QS-21.


   Abbreviations
 
APC antigen-presenting cell
CTL cytotoxic T lymphocyte
LeY Lewis Y
MAP multiple antigen peptide
Meth A cells methylcholanthrene-induced fibrosarcoma
MMC mitomycin C
OVA ovalbumin
sLex sialylated Lewis x
sLea sialylated Lewis a

   Notes
 
Transmitting editor: M. Miyasaka

Received 29 January 2001, accepted 20 July 2001.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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B. Monzavi-Karbassi, P. Luo, F. Jousheghany, M. Torres-Quinones, G. Cunto-Amesty, C. Artaud, and T. Kieber-Emmons
A Mimic of Tumor Rejection Antigen-Associated Carbohydrates Mediates an Antitumor Cellular Response
Cancer Res., March 15, 2004; 64(6): 2162 - 2166.
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