T-helper I immunity, specific for the breast cancer antigen insulin-like growth factor-I receptor (IGF-IR), is associated with increased adiposity
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- Cecil, D.L., Park, K.H., Gad, E. et al. Breast Cancer Res Treat (2013) 139: 657. doi:10.1007/s10549-013-2577-z
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Numerous lines of evidence demonstrate that breast cancer is immunogenic; yet, there are few biologically relevant immune targets under investigation restricting the exploration of vaccines to limited breast cancer subtypes. Insulin-like growth factor-I receptor (IGF-IR) is a promising vaccine candidate since it is overexpressed in most breast cancer subtypes, is part of a dominant cancer growth pathway, and has been validated as a therapeutic target. We questioned whether IGF-IR was immunogenic in cancer patients. IGF-IR-specific IgG antibodies were significantly elevated in early-stage breast cancer patients at the time of diagnosis as compared to volunteer donors (p = 0.04). Predicted T-helper epitopes, derived from the IGF-IR extracellular and transmembrane domains, elicited a significantly higher incidence of Th2 immunity in breast cancer patients as compared to controls (p = 0.01). Moreover, the magnitude of Th2 immunity was greater in breast cancer patients compared to controls (p = 0.02). In contrast, both breast cancer patients and volunteer donors demonstrated a similar incidence of Th1 immunity to IGF-IR domains with the predominant response directed against epitopes in the intracellular domain of the protein. As the incidence of IGF-IR type I immunity was not associated with a breast cancer diagnosis, we questioned whether other factors were contributing to the presence of IGF-IR-specific T-cells in both populations. While age was not associated with Th1 immunity, we observed a significantly greater magnitude of IGF-IR IFN-γ-secreting T-cells in obese subjects as compared to overweight (p < 0.001) or healthy-weight (p = 0.006) subjects, regardless of breast cancer diagnosis. No significant difference was observed for Th2 incidence or magnitude when stratified by age (p = 0.174, p = 0.966, respectively) or body mass index (p = 0.137, p = 0.174, respectively). Our data demonstrate that IGF-IR is a tumor antigen and IGF-IR-specific Th1 immunity may be associated with obesity rather than malignancy.
KeywordsIGF-IRBreast cancer antigenTh1Th2Obesity
Breast cancer has been shown to be immunogenic and clinical trials of cancer vaccines targeting the tumor antigen HER-2/neu (HER2) demonstrate encouraging clinical results in patients with both advanced and early-stage breast cancers [1, 2]. However, there is a paucity of biologically relevant antigens under investigation in breast cancer, and further immune targets need to be identified for additional breast cancer subtypes.
The development of a therapeutic immune response through active immunization requires an immune target that is expressed in a majority of breast cancers. Insulin-like growth factor-I receptor (IGF-IR) is an excellent candidate antigen as it is found upregulated in up to 55 % of breast cancers depending on subtype . Increased IGF-IR signaling is associated with a poor prognosis. In a study of over 400 breast cancer patients, the overall survival at 15 years was significantly increased in the patients whose tumors were negative for the active form of the receptor . Moreover, inactivation of IGF-IR signaling by small molecule inhibitors can result in a 50 % growth inhibition in murine models of human triple-negative breast tumor grafts .
We questioned whether IGF-IR was immunogenic in patients with breast cancer by evaluating for the presence of IGF-IR-specific IgG antibodies. As the presence of antibodies is predictive of a preexisting T-cell response , we further mapped the phenotype of IGF-IR-specific T-cell immunity across all domains of the growth factor receptor to identify class II epitopes that would be suitable immunogens for the development of a vaccine targeting IGF-IR. Our focus on defining IGF-IR CD4+ T-cell epitopes is based on investigations demonstrating that T-cell recognition of cancer occurs via cross-presentation . Vaccines that can elicit Th1 antigen-specific T-cells have the potential for activating antigen-presenting cells in the tumor microenvironment resulting in enhanced cross-priming and the development of epitope spreading to a multitude of tumor antigens .
Materials and methods
The use of human subjects was approved by the University of Washington Human Subjects Division. Volunteer control serum from 73 female donors was collected at the Puget Sound Blood Center, Seattle, WA (median age: 51, range 33–73 years). Volunteer controls met all criteria for blood donation. Serum samples from 94 breast cancer patients (100 % Stage I/II; median age 52, range 33–89 years) were obtained from individuals who consented to participate in the FHCRC/UW Breast Specimen Repository and Registry, Peggy Porter, MD, PI (IR file #5306). The serum was collected at the time of diagnosis and prior to definitive surgery. Peripheral blood mononuclear cells (PBMC) from 19 female volunteer controls (median age: 49, range 18–79 years) and 18 breast cancer patients (89 % Stage I/II, 11 % Stage III/IV; median age: 53, range 45–79 years) were collected and cryopreserved as previously described . All breast cancer patients had received definitive treatment for their disease at the time of collection. Data were available to calculate Body Mass Index (BMI) on 21 individuals.
Analysis of antibody immunity
IgG specific for IGF-IR was assessed by indirect ELISA as previously described with the modification that the Immulon 4HBX microtiter plates (Dynex) were coated overnight with 200 ng/ml human recombinant IGF-IR protein (R&D Systems) in carbonate buffer . Developed plates were read at 450 nm. The OD was calculated as the OD of the protein-coated wells minus the OD of the buffer-coated wells. The data are expressed as μg/ml IGF-IR-specific IgG. The mean and 2 standard deviations of the volunteer control population response, 0.09 μg/ml, defined a level above which a sample was considered positive. Positive and negative samples were assessed by Western blotting . 500 ng of recombinant IGF-IR protein (R&D Systems) was separated on SDS-PAGE gel and probed with anti-IGF-IR polyclonal antibodies (Santa Cruz Biotechnology, Inc.) or experimental sera. Specificity and sensitivity of ELISA were 100 and 75 %, respectively.
Analysis of peptide and protein-specific T-cell responses
Twenty IGF-IR peptides, (21 % of the protein), predicted to promiscuously bind human MHCII, were selected using web-based algorithms as previously described . The peptides were constructed and purified by high-performance liquid chromatography (>90 % pure; Genemed). PBMCs were evaluated by ELISPOT for antigen-specific IFN-γ and IL-10 production. For the IFN-γ ELISPOT, cells were plated at 2 × 105 per well (96-well plate) in medium with 10 μg/ml of the various IGF-IR peptides or HIVp17 (CPC Scientific) , PHA (1 μg/ml; Sigma-Alrich), CEF (2.5 μg/ml; AnaSpec), or medium alone for 7 days at 37 °C in 5 % CO2. On day 5, recombinant human IL-2 (10 U/ml; Hoffmann-La Roche) was added. A second in vitro stimulation (IVS) was performed on day 8 by adding 2 × 105 peptide-loaded (same concentrations as listed above) autologous irradiated (3000 rads) human PBMCs to the original culture and incubating for 24 h. 96-well nitrocellulose plates (Millipore) were coated with 10 μg/ml anti-human IFN-γ (clone 1-D1K; Mabtech). The washed nitrocellulose plates were blocked with 2 % bovine serum albumin in DPBS followed by 24h incubation with the PBMC culture. After extensive washing, 0.1 μg/ml biotinylated anti-human IFN-γ (clone 7-B6-1; MabTech) was added for 2 h. For the IL-10 ELISPOT , an anti-human IL-10-coated (2 μg/ml; BD Biosciences) nitrocellulose 96-well plate was blocked as described above. PBMC concentration and peptide stimulations were as described above, except that PHA was used at 20 μg/ml. After extensive washing, 4 μg/ml biotinylated anti-human IL-10 (BD Biosciences) was added for 2 h.
Both ELISPOT assays were developed as previously described . Positive responses were defined by a statistically significant difference (p < 0.05) between the mean number of spots from five replicates in the experimental wells and the mean number from no antigen control wells. Data are reported as the mean number of spots for each experimental antigen minus the mean number of spots detected in no antigen control wells ± SEM (corrected spots per well: CSPW) or as mean number of spots.
T-cell lines were generated from volunteer controls demonstrating significant responses to the selected epitopes. PBMCs were thawed, washed, resuspended at 3 × 106/ml, and stimulated with 10 μg/ml of the various IGF-IR peptides. The T-cells were subjected to a second IVS on day 8 and a third IVS on day 16 by adding equivalent numbers of peptide-loaded (10 μg/ml) autologous irradiated (3000 rads) PBMCs to the original culture. Recombinant human IL-12 (10 ng/ml; R&D Systems) and recombinant human IL-2 (10 U/ml) were added on day 5 and day 12, and IL-2 was added alone on days 20, 22, and 24. IFN-γ ELISPOT was performed stimulated with 10 μg/ml of the original IGF-IR peptides or cos-1 cell lysates transfected with human IGF-IR (IGF-IR) or vector alone (mock) (1 μg/ml), PHA (1 μg/ml), HIVp17 (10 μg/ml), or medium alone (No Ag). Cos-1 cells were transfected with pcDNA-3-hIGF-IR or pcDNA-3 alone (mock) using Polyfect reagent (Qiagen). IGF-IR expression was confirmed by a Western blot probing with anti-IGF-IR polyclonal antibodies (Santa Cruz Biotechnology, Inc). Cytokine levels were assessed according to the manufacturer’s instructions using the appropriate ELISA (eBioscience) on medium collected from the T-cell lines on day 10. Data are expressed as mean ng/ml ± SD of six separate donor expansions.
Receptor expression was documented in the expanded T-cells by adding APC-conjugated anti-human CD4 (clone OKT4, eBioscience) or PE-Cy7-conjugated anti-human CD3 (clone UCHT1, eBioscience). Intracellular expression of FOXP3 was documented after permeabilization and fixation with the FOXP3 Buffer Set (Biolegend) according to the manufacturer’s instructions and staining with PE-conjugated anti-human FOXP3 (clone 236A/E7, eBioscience) and anti-human CD4. Flow cytometry was performed on the FACSCanto and data analyzed using FlowJo software (BD Biosciences). Typically, 100,000 cells were collected per sample.
The unpaired, two-tailed Student’s t test (with Welch’s correction when variances were unequal) or Fisher’s exact test was used to evaluate differences. A p value of <0.05 was considered significant (GraphPad Software, Prism v.5.04).
IGF-IR antibodies are significantly elevated in breast cancer patients as compared to volunteer controls
IGF-IR epitopes derived from the extracellular and transmembrane domains of the protein are more likely to induce a higher magnitude Th2 response in breast cancer patients than volunteer controls
The breast cancer patients were also more likely to have a higher magnitude Th2 response to the ECD and TD of IGF-IR (mean, 13.1 CSPW; p = 0.03 and mean, 26.3 CSPW; p = 0.02, respectively) as compared to ICD [KD mean, 11.5 CSPW; CTD mean, 5.4 CSPW (Fig. 2b)]. Furthermore, the breast cancer patients demonstrated an overall greater magnitude of Th2 to TD epitopes than the volunteer controls (p = 0.02; mean, 26.3 vs.13.1 CSPW). This difference was evident even though mitogen-induced IL-10 secretion was decreased in the breast cancer patients as compared to the controls (p = 0.047) (Fig. 2b, Suppl. Fig. 1e). Similar to the incidence of response, the magnitude of the IGF-IR-specific Th2 responses did not differ between domains for the volunteer control population (p > 0.05 for all).
IGF-IR epitopes derived from the C-terminal domain induce Th1 immunity at an equivalent incidence, but with a greater magnitude of response in volunteer controls as compared to breast cancer patients
Th1, especially those cells secreting IFN-γ, has been shown to be associated with an anti-tumor response . The presence of IGF-IR-specific antibodies and a predominant Th2 response directed against IGF-IR Th epitopes caused us to question whether IGF-IR-specific Th1 could even be detected in the peripheral blood of breast cancer patients. Th2 inhibits the proliferation of Th1 . All but one IGF-IR epitopes stimulated significant IFN-γ secretion in some breast cancer patients and volunteer control donor PBMCs (p < 0.05 compared to HIVp17) (Suppl. Fig. 2). In contrast to IGF-IR-specific Th2 responses, both the breast cancer patients and volunteer control donors had a similar incidence of Th1 immunity to each of the protein domains (p > 0.05). Moreover, individuals in both breast cancer (44 %, p = 0.03) and volunteer controls (59 %, p < 0.001) responded at a greater incidence to epitopes in the CTD than epitopes in the ECD (breast cancer, 29 %; volunteer control, 28 %).
IGF-IR epitope-specific Th responds to naturally processed and presented IGF-IR protein
IGF-IR-specific Th1 is found at a greater magnitude in the peripheral blood of obese as compared to healthy-weight and overweight individuals regardless of a breast cancer diagnosis
IGF-IR Th2 immunity appeared to be more prevalent and of higher magnitude in patients with breast cancer inferring the immune response was potentially associated with the diagnosis of malignancy. IGF-IR-specific Th1 immunity had no such association; breast cancer patients and volunteer controls demonstrated an equivalent incidence of IGF-IR-specific Th1 with 49 % of responses (42 % volunteer control and 56 % breast cancer) at the level of a CEF response (Suppl. Fig. 2). For this reason, we explored additional factors, known to potentially impact immunity, as a potential etiology for the presence of detectable IGF-IR-specific Th1 in both populations.
Data presented here demonstrate that IGF-IR is immunogenic. Patients with breast cancer have detectable antibodies directed against IGF-IR. PBMCs from both volunteer controls and breast cancer patients demonstrated evidence of IGF-IR-specific T-cell immunity. In breast cancer patients, Type II immunity was more prevalent and of a higher magnitude directed against the ECD than the intracellular portions of the protein. In contrast, both volunteer controls and breast cancer patients had measurable IGF-IR-specific Th1 directed against the ICD. Of note, the magnitude of IGF-IR-specific Th1 immunity was not associated with a cancer diagnosis, but rather an individual’s BMI.
IGF-IR-specific antibodies were found in breast cancer patients. It is known that protein overexpression in breast cancer is directly associated with increasing levels of endogenous antigen-specific antibody immunity. As an example, 82 % (18/22) of breast cancer patients with high HER2 expression levels exhibited HER2-specific antibodies, whereas antigen-specific antibodies were not detected in any patient (0/22) with low protein expression . Upregulation of IGF-IR expression is detected in nearly all subtypes of breast cancer, thus providing a source for increased IGF-IR-specific B-cell activation in patients as compared to volunteer controls . The detection of an IGF-IR-specific humoral response indicates an antigen-specific cellular response as cognate T-cell help is required for immunoglobulin class switching from IgM to IgG . T-cells, particularly Th2, secrete cytokines, such as IL-10 which drive B-cell clonal expansion and antibody production. IL-10 can double B-cell proliferation and increase IgG secretion tenfold in previously activated B-cells [13, 14]. The development of antigen-specific Th2 in breast cancer patients is not unexpected since accumulating evidence demonstrates that the breast tumor microenvironment is dominated by immune suppressive factors, resulting from a chronic inflammatory state . Elevated levels of tumor-infiltrating Th2, producing high levels of IL-4 and IL-13, are found in breast cancer compared to benign tissue [24–26]. Chronic Th2 and B-cell activation can potentiate an immune suppressive tumor microenvironment through cytokine and immunoglobulin production, ultimately resulting in inhibition of the Th1- and CTL-mediated anti-tumor immune response [27, 28].
Th2 was predominantly directed against epitopes in the ECD/TD, suggesting that these sequences may be more tolerogenic. ECD proteins can be shed from the cell surface during homeostatic apoptosis that occurs regularly during development and aging [29, 30]. This exposure may result in more frequent immune presentation of extracellular epitopes leading to tolerance. Studies in animal models of autoimmunity have shown that when intact apoptotic cells are injected, antigen-specific tolerance occurs . In contrast, inoculation with heat-denatured cells elicits an inflammatory immune response and activation of Th1 . The heat-denatured cells release their intracellular components due to rupture of the plasma membrane . The intracellular proteins may be less frequently presented, reducing the opportunity to stimulate tolerance . This notion is consistent with our results demonstrating that IGF-IR-specific Th1 was predominantly directed against epitopes in the ICD.
We detected a similar level of IGF-IR-specific Th1 in volunteer control donors and breast cancer patients, which has been reported for other tumor antigens such as p53 . Studies of p53-specific T-cells demonstrated that these cells were of a memory phenotype, raising the question as to the etiology of antigen exposure in subjects unaffected by cancer. Investigators cited the ubiquitous role of p53 as an oncogene in many cancers and suggested that Type I p53 precursors could represent a previous exposure to cancer and successful immune surveillance. In our study, we first considered age as an explanation for the elevated IGF-IR-specific Th1. Increased levels of inflammatory cytokines have been shown to positively correlate with aging. In a study examining 73 healthy individuals, those over 60 years exhibit increased levels of IL-1β, TNFα, and IL-6 compared to younger individuals . Additional investigations have shown that precursor CD4+ T-cells in aging individuals are predominantly committed to a Th1 phenotype . However, as reported here, the incidence and magnitude of IGF-IR-specific Th1 were not associated with the age of the individual. We explored adiposity as an etiology of the observed immune responses as recent evidence suggests that obesity is also associated with Type I inflammation. Th1 and activated CD8+ T-cells are the predominant adaptive immune cell infiltrates in adipose tissue . Further, a recent study described a greater number of circulating Th1 in obese individuals compared to overweight and healthy-weight individuals . We found a higher magnitude IGF-IR-specific Th1 immune response in obese subjects compared to overweight and healthy-weight subjects. Why would IGF-IR be a target for the adaptive immune response in obesity? Overexpression of self-antigens during oncogenesis is associated with enhanced immunogenicity, as discussed above . IGF-IR-specific cellular immunity may have been primed by an overexpression of the receptor as a result of increasing adiposity. Studies in humans and animal models of obesity indicate that adipocytes first increase cell size in response to excess energy intake, then increase their number and begin to secrete growth factors which stimulate the proliferation and eventual maturation of pre-adipocytes [38, 39]. Pre-adipocytes isolated from subcutaneous adipose tissue express thirty-fold more IGF-IR protein than mature adipocytes . Collectively, increased antigen exposure and the upregulation of pro-inflammatory cytokines, including IL-12p40 and IL-18, observed in obese adipose tissue may result in the priming of Th1 to adipose-related self-antigens, such as IGF-IR .
Evidence of a preexistent Th1 response in breast cancer patients may allow more facile vaccine boosting of that response in patients whose tumors express IGF-IR. Indeed, in a clinical trial targeting p53 in 17 colorectal cancer patients, 33 % of patients with preexisting immunity demonstrated increased levels of antigen-specific T-cells after vaccination, whereas no augmentation of immunity was observed in any patient without preexisting p53-specific T-cells . Our data suggest IGF-IR-specific immunity may also be a marker of inflammatory obesity.
This work was supported by a grant from the Ovarian Cancer Research Fund, 17624550-36370-A, and a Department of Defense Postdoctoral Fellowship, W81XWH-10-1-0700. MLD is supported by the Athena Distinguished Professorship of Breast Cancer Research.
Conflict of Interest
MLD discloses funding received from Seattle Genetics, stock ownership in VentiRx and Epigenomics AG, and a consultant/advisory role at VentiRx and EMD Serono. All other authors disclose no conflict of interest.
All authors declare that the experiments comply with current U.S. laws.