Originally published as JCO Early Release 10.1200/JCO.2006.05.9311 on August 14 2006

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T-Cell Immunity to the Folate Receptor Alpha Is Prevalent in Women With Breast or Ovarian Cancer

Keith L. Knutson, Christopher J. Krco, Courtney L. Erskine, Karin Goodman, Linda E. Kelemen, Peter J. Wettstein, Philip S. Low, Lynn C. Hartmann, Kimberly R. Kalli

From the Departments of Immunology, Oncology, Health Sciences Research and Surgery, Mayo Clinic College of Medicine, Rochester MN; and the Department of Chemistry, Purdue University, West Lafayette, IN.

Address reprint requests to Keith L. Knutson, PhD, Mayo Clinic, Department of Immunology, Guggenheim 3, 200 First St. SW, Rochester, MN 55905; e-mail: knutson.keith{at}mayo.edu

	ABSTRACT

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION Appendix Authors' Disclosures of... Author Contributions GLOSSARY REFERENCES

 
Purpose Studies have demonstrated that the generation of immunity to tumor antigens is associated with improved prognosis for many cancers. A candidate antigen is the folate receptor alpha (FR), which is overexpressed in breast and ovarian cancers. Our goal in this study was to attain a better understanding of the extent of endogenous FR immunity.

Methods Using a CD4⁺ T cell epitope prediction algorithm, we predicted promiscuous epitopes of FR, and tested for immunity in 30 breast (n = 17) or ovarian (n = 13) cancer patients and 18 healthy donors using enzyme-linked immunospot analysis.

Results Fourteen peptides were predicted, seven each from the carboxy- and amino-terminus halves of the protein. More than 70% of patients demonstrated immunity to at least one FR peptide. Patients responded to an average of 3 ± 0.5 peptides, whereas healthy donors responded to 1 ± 0.4 peptides (P = .004). Five peptides were recognized by more than 25% of patients. Responses to three peptides were higher (P < .05) in patients than in healthy donors, suggesting augmented immunity. Compared with healthy individuals, patients developed higher immunity to the amino-terminus half of the receptor (P = .03). There was no difference between each group in the responses to nonspecific (P = .2) and viral stimuli (P = .5). Lastly, patients demonstrated elevated levels of FR antibodies consistent with a coordinated immune response.

Conclusion These findings demonstrate that the FR is a target of the immune system in breast and ovarian cancer patients. Understanding which antigens are targeted by the immune system may be important for prognosis or immune-based therapies.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION Appendix Authors' Disclosures of... Author Contributions GLOSSARY REFERENCES

 
Immunity to tumor-associated antigens can be measured in breast and ovarian cancer patients, suggesting that the concept of pre-existent immunity is a reflection of an emerging antitumor response.^1-7 However, the ability to quantify immunity to tumor-associated antigens has been constrained by a paucity of tumor-specific epitopes and sensitive assays to measure reactivity. Thus, it has been assumed that only a small fraction of patients have pre-existent T-cell reactivity to tumor antigens. For example, Disis et al² have reported that only 11% of patients with HER-2/neu-overexpressing cancers have measurable natural T-cell immunity to HER-2/neu.

Recently, our capabilities of identifying immunogenic regions using predictive algorithms have improved and coupled with the emergence of sensitive T-cell assays, it is now suspected that immunity to tumor antigens is more prevalent in cancer patients than previously thought.⁸ For example, Rentzsch³ reported that more than 50% of breast cancer patients have pre-existent immunity to at least one of several tumor-associated antigens: HER-2/neu, MUC-1, CEA NY-ESO-1, and SSX-1. Data also suggest that the natural immunity contributes to tumor rejection. Goodell et al⁴ observed that ovarian cancer patients with p53-specific antibodies had significantly improved survival when compared with patients without detectable p53-specific antibody responses. These findings support the concept of identifying tumor antigens and designing antigen-specific immunotherapies to enhance immunity in cancer patients.

The folate receptor (FR) is a glycosylphosphatidyl-inositol (GPI)-linked protein that is important in neurologic development and is overexpressed on nearly all ovarian cancers and a high proportion of breast cancers.^9-15 Overexpression is associated with increased tumor aggressiveness.^16-19 Immunity to FR is associated with neural defects in the developing embryo and cerebral folate deficiency syndrome in children, suggesting that the immune system is not completely tolerant to the protein.^20-25 Studies by Peoples et al^6,26 have revealed that tumor-associated lymphocytes in breast and ovarian cancer patients contain FR-specific cytolytic T cells.

In this study, using a predictive algorithm, a panel of FR-derived peptides representing potential T-cell epitopes was identified. Using lymphocytes from ovarian and breast cancer patients, the peptides were used for measuring pre-existing T-cell immunity in interferon gamma (IFN-) enzyme-linked immunospot (ELIspot) assays. Several peptides were identified to which immune responses were detected in a larger proportion of cancer patients compared with healthy donors.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION Appendix Authors' Disclosures of... Author Contributions GLOSSARY REFERENCES

 
Epitope Prediction
FR epitopes were predicted, as previously described²⁷ using RANKPEP (http://www.mifoundation.org; Table 1; Fig 1). The sequence for FR was from the Entrez Database (accession #P15328). Peptides were predicted that potentially bound to human leukocyte antigen (HLA) -DR1, -DR2, -DR3, -DR4, -DR5, -DR7, and -DR11 (Table 1). The algorithm predicted peptides of 15 residues. If a peptide was predicted to bind to at least three different HLA-DR alleles, it was selected. In some cases a peptide was extended beyond 15 residues, so that a predicted binding core was flanked by at least three amino acids. The peptides, 15 to 19 residues, were produced to more than 95% purity by high-performance liquid chromatography and mass-spectrometry at the Mayo Clinic (Rochester, MN). Routine testing of peptides demonstrate that they are negative for contamination by splenocyte blastogenesis assays.

View this table: [in this window] [in a new window]   Table 1. Predicted FR Peptides

View larger version (4K): [in this window] [in a new window] [PowerPoint Slide for Teaching]   Fig 1. Diagramatic view of the distribution of major histocompatibility complex class II epitopes within folate receptor alpha (FR). The bottom line represents the full length FR. The names associated with the small lines indicates the peptide name. Refer to Table 1 for the entire sequence.

Peripheral Blood Mononuclear Cell Preparation
Peripheral blood mononuclear cells (PBMCs) were isolated from patients' blood as previously described,²⁸ and cryopreserved in liquid nitrogen (20 x 10⁶/mL cells) in freezing media (RPMI-1640 with 12.5% human serum albumin, penicillin, streptomycin and 2 mmol/L glutamine).²⁹ The number of PBMCs purified from the blood of donors was 1.6 ± 0.07 x 10⁶ cells/mL whole blood for healthy donors and 1.0 ± 0.07 x 10⁶/mL for cancer patients (P < .0001).

ELISpot Analysis
A 10-day ELISpot for detecting low-frequency T cells was used to determine reactivity to the FR-derived peptides (Table 1) and was done in groups of two (two healthy donors, one donor/one cancer patient, or two cancer patients) essentially as previously described³⁰ (Appendix, online only).

Cytokine Flow Cytometry and Antibody Enzyme-Linked Immunosorbent Assay (ELISA)
Routine cytokine flow cytomety and ELISA methods were used (Appendix).

Statistical Analysis
The T-cell magnitude for each donor was summed across all 14 peptides, which, along with the response multiplicity, was compared with age using regression. The t test was used for means unless the data were not normally distributed, in which case the Mann-Whitney test was used. Fisher's exact test was used for comparing proportions. A proportion was considered elevated, relative to other peptides, if that proportion was statistically elevated relative to the mean proportion, 8.7%, which is the ratio of the total number of significant (P < .05, two-tailed t test) peptide-specific responses over the total number donors. Tests were performed using InStat (v.3.00), GraphPad Software (San Diego, CA). Changes were considered significant at P .05. Unless specified, one-tailed tests were used.

	RESULTS

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Patients With Breast and Ovarian Cancer Generate Immunity to Multiple Peptide Epitopes in FR
The FR peptides predicted to be immunogenic were distributed throughout the receptor (Fig 1). Responses to phorbol myristate acetate (PMA)/ionomycin, the CEF peptide pool (CMV, EBV and Flu peptides), and an ovalbumin-derived control peptide were not different between the two populations (Fig 2A; Appendix). Figure 2 shows T-cell frequencies for each of the FR peptides for the patients (Fig 2B) and the healthy donors (Fig 2C). The mean frequencies for each of the peptides ranged from 0 to 124 T cells/million PBMC for the healthy donors and 1 to 162/million PBMC for the patients. The overall mean FR-specific T cell frequency (± SEM), considering all peptides, for patients was 74 ± 11 (n = 448) and for healthy donors was 46 ± 10 (n = 226; P = .05). Figure 2D-2E shows an ELIspot for patient 37 and healthy donor 15. Patient 37 demonstrated an FR56-specific response (mean, 183 spots/well; SEM, 17 spots/well; n = 3) which was higher (P = .0008) than no antigen (mean, 26; SEM, 2 spots/well) control. The responses to the CEF pool were not significantly elevated compared with control (P > .5). Donor 15 did not demonstrate elevated FR56-specific T cell (mean, 3; SEM, 1 spots/well; P > .05) compared to control (mean, 5; SEM, 1 spots/well), but did have an elevated CEF pool response (mean, 76; SEM, 5 spots/well, P = .0003).

View larger version (46K): [in this window] [in a new window] [PowerPoint Slide for Teaching]   Fig 2. Patients generate immunity to multiple folate receptor alpha (FR) epitopes. (A) Responses to controls. (B-C) T-cell frequencies (dot = one individual, bars = means). (D-E) Example ELIspot with graph. (F-I) Representative flow cytometry plots. (J) Number of epitopes responded to by each group. (K) Relational diagram comparing frequencies of each peptide between groups. (L) Frequencies/person for amino and carboxy pools. H, healthy control; P, patient, PMA/IONO, phorbol myristate acetate/ionomycin; CEF, CMV, EBV and Flu peptides; OVA, ovalbumin peptide; PBMC, peripheral blood mononuclear cell; FITC, fluorescein isothiocyanate; APC, allophycocyanin; PEP, phycoerythrin.

It was observed that ovarian and breast cancer patients demonstrated immunity to 3 ± 0.6 and 3 ± 0.7 FR-derived peptides, respectively (mean ± SEM; Fig 2J). These levels of reactivity were higher than those in healthy donors who responded to 1 ± 0.5 (mean ± SEM; n = 18) peptides. As shown in the relational diagram (Fig 2K), the calculated frequency of 11 of the peptides was increased in patients, whereas three were similar or decreased. The elevated T-cell frequencies observed in the patients were mostly confined to the amino terminus as shown in Figure 2L. The mean T-cell frequency per patient for the amino terminus peptides was 75 ± 17 (mean ± SEM), which was higher than the frequency observed in healthy donors (mean, 24; SEM, 11; P = .007). There was no difference in the mean T-cell precursor frequencies to the carboxy peptides (P = .1). Further, there were no associations between the T-cell frequencies and the number of HLA-DR alleles to which each peptide was predicted to bind. There were no effects of age (range, 25 to 73 years) on either the numbers of peptides that elicited an immunogenic response (r² = 0.006; two-tailed P = .6) or the magnitude of the T-cell responses (r² = 0.004; P = .7).

High Proportion of Breast and Ovarian Cancer Patients Have T-Cell Responses to FR
Figure 3A shows the percentage of patients who responded to each peptide, which ranged from 7% to 40%. Patients responded in higher proportions (P < .05) to FR30 (30%), FR56 (33%), FR113 (27%), FR138 (30%), and FR147 (40%; Table 2). Responses were more frequently observed in cancer patients than in healthy donors. Of the 14 peptides, four (FR30, FR56, FR113, FR238) generated responses in more patients than healthy donor counterparts (Table 2). Reactivity to three of these, FR30, FR56, and FR113, was observed in more than 25% of patients. Of the 14 FR peptides, FR56, was recognized more often (P = .05) by ovarian cancer patients than by breast cancer patients (Fig 3B). Overall, 69% and 76% of ovarian and breast cancer patients, respectively, demonstrate immunity to at least one eptiope of FR.

View larger version (16K): [in this window] [in a new window] [PowerPoint Slide for Teaching]   Fig 3. A high proportion of breast and ovarian cancer patients have T cell responses to folate receptor alpha (FR). (A) Proportions of each cohort responding to the individual epitopes. (B) Proportions of ovarian and breast cancer patients that responded to the individual peptides. (C) Distribution of responses among the amino or carboxy terminus halves of the FR.

Breast and Ovarian Cancer Patients Demonstrate FR-Specific Antibody Immunity. Patients demonstrated increased antibody immunity to FR (Fig 4A). Levels of FR-specific antibodies in patients were 68 ± 6 ng/mL (mean ± SEM; n = 18) which was significantly higher (P < .0001) than levels in the healthy donors (mean, 19; SEM, 8 ng/mL; n = 11). Antibody responses to tetanus toxin were equivalent (P = .3) between the two populations (patients, 30 ± 4 µg/mL; healthy donors, 27 ± 4 µg/mL; mean ± SEM, respectively; P = .3).

View larger version (15K): [in this window] [in a new window] [PowerPoint Slide for Teaching]   Fig 4. Patients with breast and ovarian cancer generate antibody responses to folate receptor alpha (FR). (A, B) Levels of antibody to the FR76 peptide and tetanus toxin (TT), respectively. Each bar shows the mean (± SEM) responses for both the patients (n = 19) and healthy (n = 11) volunteers. The P values were calculated using a two-sided t test.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION Appendix Authors' Disclosures of... Author Contributions GLOSSARY REFERENCES

A key finding in the current study is that immunity to FR is prevalent in breast and ovarian cancer patients. Immunity to tumor antigens is typically thought to be very low or undetectable for breast and ovarian cancer.² However, this understanding is limited by our capabilities to assess the tumor-specific immune response which, like infectious disease responses, consists of multiple effectors including CD8⁺ T cells, CD4⁺ T cells, and antibodies. Typically, immunity to tumor antigens has been assessed with MHC class I epitopes largely because of the availability of algorithms.^3,5 For example, in our prior studies in HER-2/neu–overexpressing cancer patients, we found that only approximately 13% of patients have pre-existent immunity to MHC class I-binding HER-2/neu peptide p369-377.³⁰ In another study, Rentzche³ found that 19% of breast cancer patients responded to a mixture of two HER-2/neu–derived MHC class I peptides. Similarly, immunity to an MHC class I MUC-1 peptide, which is overexpressed on up to 90% of breast tumors, is detected in only 32% of individuals.⁵ With the improved understanding of CD4⁺ T cells and MHC class II binding peptides, our capabilities of detecting tumor antigen-specific immunity have been greatly enhanced.²⁷

Another significant problem with detection of pre-existent immunity has been the inability of assays to detect low frequency immune responses.³² Recently developed assays such the IFN- ELIspot can have limits of detection as low as 1:100,000.^30,32 Thus, the development of newer epitope prediction capabilities coupled with improved assays will increase our understanding of the complex interactions of the immune system with tumors. Knowledge of the extent and prevalence of tumor antigen-specific immunity is important for identifying which antigens are naturally targeted by the immune system and for understanding why natural immunity fails to eradicate tumors. Several mechanisms are proposed to explain immune escape. Our laboratory has seen in the HER-2/neu mouse model of breast cancer that the generation of a T-cell response against tumors can result in cellular reprogramming accompanied by loss of antigens (including mouse FR) and acquisition of suppressive function (K.L. Knutson, unpublished observations).^33,34 Although this mechanism is plausible in breast cancer, which shows only 50% to 80% staining for FR, it seems unlikely in ovarian cancers, which retain FR expression in more than 90% of cases.¹⁷ Another strategy involves recruitment of immunoregulatory cells into the microenvironment to block activation and expansion of T cells.³⁵ Both ovarian cancer and breast cancers attract or induce regulatory T cells (Tregs) that suppress the function of the tumor antigen-specific T cells that migrate to the microenvironment.^36,37 For example, Curiel³⁶ found that ovarian cancers release chemokines which attract Tregs that block HER-2/neu-specific T cells and worsen patient survival. It appears that tumor antigen-specific T cells are rendered largely ineffective through a variety of mechanisms which permits the tumors to grow unimpeded despite their presence.

Our results also demonstrate that patients can generate immunity to multiple epitopes, suggesting that tolerance to FR is absent (ie, immunologic ignorance) or reversible (ie, anergy). This is in contrast to deletional tolerance that occurs for broadly expressed proteins in which antigen-specific T cells are deleted either in the thymus or the periphery.^38,39 However, when there is exclusive tissue distribution of an antigen, it appears tolerance is either reversible or nonexistent. For example, studies by Ohlen⁴⁰ in mice suggest that expression of a protein exclusively in the liver tolerizes potentially autoreactive T cells by induction of anergy rather than deletional tolerance. The results obtained in the current study showing multiepitope FR-specific immune responses suggest that the T-cell receptor repertoire targeting FR is largely intact and is maintained in normal healthy individuals by either ignorance or anergy. This is likely due to the fact that, in humans, expression of FR is limited to a few tissues, mainly kidney tubules.⁹ The observation that patients with breast and ovarian cancer apparently augmented immunity to the FR, particularly to epitopes in the amino terminal half of the molecule, shows that the immune system maintains a diverse T-cell repertoire that can be expanded in vivo.

Although the peptides used in the current study were predicted CD4⁺ T-cell epitopes, results suggest that some responses were caused by CD8⁺ T cells that were potentially activated by encompassed MHC class I peptides such as with peptide FR56. In addition, on comparison with studies by Peoples et al,²⁶ we found that another of the fourteen peptides we evaluated fully contained a previously identified HLA-A2-restricted epitope. That peptide, FR238, fully encompasses the HLA-A2 motif, FR 245 to 253. In the current study, we found that 23% of patients responded to FR238, whereas none of the healthy donors responded. Thus, although we did not examine for HLA-A2 expression in our subjects (likely 40%), the possibility exists that the patients were responding to the embedded HLA-A2–restricted epitope.³⁰ We have previously shown that patients can generate CD8⁺ T cells that recognize epitopes that are fully encompassed in HLA class II epitopes, presumably by internalizing the peptide, processing it in the endosomal compartment and presenting it as a shorter 9 amino acid peptide in the context of class I.³⁰ Our analyses suggest that the peptides we predicted may encompass epitopes that bind to other MHC class I molecules as well (eg, HLA-A3). Peptides that could generate both CD4⁺ and CD8⁺ T cells are useful for generating an effective antitumor immune response because several studies have shown that activating both T-cell subsets may be better than activating either alone.²⁷ Coupled with detection of FR-specific antibodies, the presence of both CD4⁺ and CD8⁺ T-cell immunity indicates that a coordinated immune response is being elicited in cancer patients, but that the response may be too limited to be effective.

The FR is a tumor-associated antigen that may have an important role in the biology of cancer, which may explain why its maintained in a high proportion of tumors.⁴¹ For example, decreasing FR expression in breast cancer cell lines reduces their proliferation rate.⁴² In addition, its high frequency of expression in ovarian cancer (> 90%) suggests that FR confers a growth advantage over tumor cells with reduced expression.¹⁷ Indeed, histochemical studies have shown that expression is positively associated with high histologic grade and advanced stage.¹⁷ Coupled with the observations that the T-cell repertoire is intact, these findings suggest that targeting the FR using immune-based approaches such as cancer vaccines may be advantageous because the immune system would target the most aggressive tumor cells. Targeting antigens that are important to the biology of the disease may reduce the risks of outgrowth of antigen-negative variants.⁴³

In conclusion, our results demonstrate that immunity to FR is prevalent in patients with breast and ovarian cancer. Understanding immunity to tumor-associated antigens should lead to a better understanding of how tumors interact and escape natural immunity. Furthermore, discovery of the epitopes of a tumor antigen such as the FR could lead to design and testing of strategies to augment tumor-specific immunity.

	Appendix

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION Appendix Authors' Disclosures of... Author Contributions GLOSSARY REFERENCES

 
Methods
Materials. Phorbol myristate acetate, human serum albumin, polyclonal human immunoglobulin G (IgG), tetanus toxin, and ionomycin were from Sigma (St. Louis, MO). Goat antihuman horseradish peroxidase (HRP) –conjugated antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). FITC-conjugated anti-CD4, PE-conjugated anti-IFN-, APC-conjugated anti-CD8, and all cytokine flow cytometry reagents were obtained from BD Biosciences (San Jose, CA). Hank's balanced salts solution, RPMI-1640-1640 and phosphate-buffered saline (PBS) were from Cellgro (Herndon, VA). Ficoll-Paque was from Amersham Biosciences (Uppsala, Sweden). The CEF viral peptide pool was from the National Institutes of Health (Bethesda, MD) AIDS Research and Reference Reagent Program.

Epitope prediction and synthesis. In select cases, the predicted epitopes were further analyzed using MHCPred (www.jenner.ac.uk) for encompassed HLA-A1 (0101), -A2 (0102), and -A3 (0301). These three HLA-A alleles were selected because they are those which are the highest among the white, African American, and Hispanic populations (Cao K, Hollenbach J, Shi X, et al. Hum Immunol 62:1009-1030, 2001). Peptides were considered to potentially contain embedded HLA-class I epitopes if the algorithm predicted regions with IC₅₀ values of 500 nmol/L or less (Knutson KL, Beebe M, Vielhauer G, et al. Proc Am Assoc Cancer Res 46, 2005 [abstr 5165]). Additionally, FR-derived T cell epitopes were also analyzed using B-cell epitope prediction algorithms ABCpred (www.imtech.res.in) and Antigenic (http://bioinfo.bgu.ac.il), to determine whether they would be useful for detecting antibodies.

Patients and donors. Healthy donors were recruited by means of local advertisement which gave the details of the blood draw. For the antibody enzyme-linked immunosorbent assay (ELISA) studies, sera were available from 11 healthy controls and 19 patients.

IFN- ELISpot analysis. On day 1, 2.5 x 10⁵ PBMCs/well were plated into 96-well plates in three-well replicates in 200 µL of RPMI-1640 containing L-glutamine, penicillin, streptomycin, and 10% fetal calf serum (T-cell medium) in the presence or absence of 10 µg/mL peptide antigen. The cells were incubated at 37°C, and interleukin 2 (IL-2; Zeptometric Inc, Buffalo, NY) was added to 10 U/mL on day 5. On day 8, 2.5 x 10⁵/well irradiated autologous PBMCs and 10 µg/mL antigens were added. On day 9, the cells were transferred to an anti–IFN--coated nitrocellulose (NC) plate (Millipore Corporation, Bedford, MA). The NC plate was incubated (37°C) for a further 20 to 24 hours followed by washing three times using PBS containing 0.05% Tween-20 (Sigma, St Louis, MO). The plate was then incubated for 2.5 hours at room temperature in PBS with 5 µg/mL biotinylated anti–IFN- antibody, washed in PBS, and further incubated with 100 µL/well avidin-HRP (Vector Laboratories, Burlingame, CA) for 2 hours at room temperature. After three washes in PBS, the plate was incubated with 100 µL/well HRP-colorimetric substrate (Vector Laboratories) for 20 to 30 minutes, rinsed with cool tap water, and allowed to dry completely. The nitrocellulose plates were read on an AID ELIspot reader (Cell Technology Inc, Columbia, MD; reader software v.3.1.1.). A positive response was defined as a frequency that was significantly (P < .05, two-tailed t test) greater than the mean of control no-antigen wells and detectable (ie, > 1:100,000). A 17-amino acid peptide (KISQAVHAAHAEINEAG) derived from chicken egg ovalbumin, produced in the same fashion as the FR peptides, was used as a control peptide. This peptide was predicted to bind to HLA-DR2 and -DR5 but not -DR1, -DR3, -DR4, -DR7, or -DR11. The anti–IFN- and biotinylated anti–IFN- antibody pair were obtained from Mabtech (Nacka Strand, Sweden).

Antibody ELISA. Antigen (10 µg/well) was prepared in 0.06 M carbonate buffer and added to ELISA microtiter plates for 24 hours. Plates were washed with PBS and blocked with 3% bovine serum albumin (BSA) PBS. One hundred microliters of diluted serums (1:125 for peptide and 1:40 for tetanus toxoid in 1%BSA-PBS) were added and the plates were further incubated for 2 hours at RT followed by washing with PBS/0.1% Tween-20. A 1:2000 dilution of anti–IgG-HRP was added to wells for 1 hour followed by washing and color development with tetramethylbenzidine (TMB). 3,3',5,5' TMB substrate was added (100 µL) to the wells. Color development was stopped with 50 µL of a 0.1 N HCl solution. For the standard curve, serial dilutions of human IgG were added to separate wells. As a control, a peptide derived from human collagen II, HII.71 (PPGLTGPAGEPGRQGSPGAD), was used.

Cytokine flow cytometry.PBMCs were cultured with peptide (10 µg/mL) for 7 days. IL-2 (20 U/mL) was added on day 3. After 7 days, the cells were distributed into a 96-well plate with fresh irradiated autologous PBMCs. Either medium alone or medium supplemented with phytohemaglutinin-L (20µg/well) or recall peptide (10 µg/well) was added to the appropriate wells for 29 hours. GolgiStop (BD Pharmingen, San Diego, CA) was added for last 5 hours followed by washing in PBS-0.5% BSA. The cells were resuspended in the same buffer containing anti–CD4-FITC and anti–CD8-APC for 30 minutes, followed by washes and fixing. The cells were permeabilized alone or with unconjugated antihuman IFN- (adsorption control) followed by washing and incubation with antihuman anti–IFN--APC for 30 minutes. The cells were washing, fixed, and analyzed previously described using a BD Biosciences FACscan flow cytometer and CellQuest Pro Software (version 4.0.2., BD Biosciences; Knutson KL, Disis ML. Hum Immunol 63:547-557, 2002). A response to antigen was considered positive if there was at least a 50% increase in IFN-⁺ cells and it was blocked by the adsorption control.

Statistics.The mean proportion method (Table 2) is used because the ELIspot does not provide a continuous read out due to (Cao K, Hollenbach J, Shi X, et al. Hum Immunol 62:1009-1030, 2001) the limits of detection and (Knutson KL, Beebe M, Vielhauer G, et al. Proc Am Assoc Cancer Res 46, 2005 [abstr 5165]) zero value assignment if not significantly different than control. The use of a mean proportion is a rigorous modification of a technique that is used in prior immunologic studies. For example, Goodell et al (Goodell V, Salazar LG, Urban N, et al. J Clin Oncol 24:762-768, 2006) recently used this strategy for detecting elevated levels of antibodies specific for oncoproteins in patients with ovarian cancer.

Results
Patients with breast and ovarian cancer generate immune responses to multiple peptide epitopes in FR. The mean number of spots per million PBMCs for the healthy donors in the PMA/ionomycin containing wells was 1,804 ± 426 (mean ± SEM; n = 18), which was not statistically different from the response in the patients (mean, 2,347; SEM, 440; n = 30; P = .2). The mean number of spots per million PBMC for the healthy donors in the CEF peptide–containing wells was 192 ± 76 (mean ± SEM), which was not statistically different from the response in the patients (mean, 196; SEM, 53; P = .3). The mean number of spots per million PBMCs for the healthy donors in the ova peptide–containing wells was 0 ± 0 (mean ± SEM), which also was not statistically different than the response in the patients (mean, 16; SEM, 8, P = .07). Lastly, the proportion of patients that responded to the CEF peptide pool (43%) was not different from the proportion of healthy donors who responded (33%; P = .4).

Breast and ovarian cancer patients demonstrate FR-specific antibody immunity. As an additional control, levels of antibodies to an irrelevant peptide, HII.71 were also assessed, showing no detectable antibodies in either population (data not shown).

	Authors' Disclosures of Potential Conflicts of Interest

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The authors indicated no potential conflicts of interest.

	Author Contributions

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION Appendix Authors' Disclosures of... Author Contributions GLOSSARY REFERENCES

 

Conception and design: Keith L. Knutson, Linda E. Kelemen, Philip S. Low, Lynn C. Hartmann, Kimberly R. Kalli Financial support: Keith L. Knutson, Lynn C. Hartmann, Kimberly R. Kalli Administrative support: Keith L. Knutson, Lynn C. Hartmann, Kimberly R. Kalli Provision of study materials or patients: Keith L. Knutson, Karin Goodman, Lynn C. Hartmann, Kimberly R. Kalli Collection and assembly of data: Keith L. Knutson, Christopher J. Krco, Courtney L. Erskine, Karin Goodman, Peter J. Wettstein, Lynn C. Hartmann, Kimberly R. Kalli Data analysis and interpretation: Keith L. Knutson, Courtney L. Erskine, Linda E. Kelemen, Peter J. Wettstein, Lynn C. Hartmann, Kimberly R. Kalli Manuscript writing: Keith L. Knutson, Christopher J. Krco, Linda E. Kelemen, Philip S. Low, Lynn C. Hartmann, Kimberly R. Kalli Final approval of manuscript: Keith L. Knutson, Christopher J. Krco, Linda E. Kelemen, Philip S. Low, Lynn C. Hartmann, Kimberly R. Kalli

 

	GLOSSARY

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MHC class I (major histocompatibility complex I):

MHC class I molecules are found on almost every nucleated cell of the body. In humans, these are often referred to as HLA (human leukocyte antigen) molecules. These molecules act as "signposts" by displaying fragments of protein antigens on the cell surface. The presented fragments (epitopes) can be derived from self or nonself (viral) antigens. MHC class I molecules presenting epitopes are recognized by cytotoxic T cells. When a nucleated cell is aberrant (malignant or virally infected), intracellularly derived epitopes that are foreign to circulating T cells will be presented, leading to immune activation. In humans, there are three major loci that encode MHC class I molecules—HLA-A, HLA-B, and HLA-C.

MHC class II (major histocompatibility complex II):

MHC class II molecules are found only on a few types of cells in the body, most notably, antigen-presenting cells such as macrophages and dendritic cells. Like MHC class I, these molecules present fragments of protein antigens. Unlike MHC class I, MHC class II peptides are usually derived from extracellular antigens that may be released by bacteria or tumors. MHC class II molecules presenting epitopes are recognized by helper T cells. In humans, there are three major loci that encode MHC class II molecules—HLA-DR, HLA-DP, and HLA-DQ.

Epitope:

Region within an antigen that has the potential to give rise to an antibody response. With respect to protein antigens, epitopes may be defined on the basis of primary, secondary, or tertiary structure of the molecule and, consequently, maybe exposed or hidden within the molecule.

ELISpot:

Enzyme-linked immunospot that is exquisitely sensitive to assay minute amounts of mediators that are produced by cells. Typically, cells are deposited on a membrane coated with an antibody specific for a given protein. The protein of interest is captured directly around the secreting cell and is detected with an antibody specific for a different epitope. Coupled with colorimetry, the cells are visualized by specialized plate readers. Thus, the molecule is assayed before it is diluted in the supernatant, captured by receptors of adjacent cells, or degraded.

ELISA (enzyme-linked immunosorbent assay):

ELISA is an immunoassay that uses an enzyme linked to an antibody or antigen as a marker to detect a specific protein (ie, an antigen or antibody) that has been absorbed onto a solid-phase surface (eg, plastic).

	ACKNOWLEDGMENTS

 
We thank Nora Disis, MD, of the University of Washington (Seattle, WA) for providing some specimens, Shay Park for assistance, and the Mayo Immune Monitoring Core for performing the ELIspots.

	NOTES

 
published online ahead of print at www.jco.org on August 14, 2006.

Supported by Commonwealth Foundation for Cancer Research and National Institutes of Health/National Cancer Institute award, K01CA100764.

Terms in blue are defined in the glossary, found at the end of this article and online at www.jco.org.

Authors' disclosures of potential conflicts of interest and author contributions are found at the end of this article.

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Submitted January 27, 2006; accepted May 26, 2006.

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