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Cancer Research 68, 8076, October 1, 2008. doi: 10.1158/0008-5472.CAN-08-0387
© 2008 American Association for Cancer Research
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Immunology

CD8+ T-Cell Responses against Hemoglobin-β Prevent Solid Tumor Growth

Hideo Komita1, Xi Zhao1, Jennifer L. Taylor1, Louis J. Sparvero2,6, Andrew A. Amoscato2,3,6, Sean Alber4, Simon C. Watkins4, Angela D. Pardee5, Amy K. Wesa1 and Walter J. Storkus1,5,6

Departments of 1 Dermatology, 2 Surgery, 3 Pathology, 4 Cell Biology and Physiology, and 5 Immunology, University of Pittsburgh School of Medicine and 6 University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania

Requests for reprints: Walter J. Storkus, Departments of Dermatology and Immunology, University of Pittsburgh School of Medicine, W1041.2 Biomedical Sciences Tower, 200 Lothrop Street, Pittsburgh, PA 15213. Phone: 412-648-9981; Fax: 412-383-5857; E-mail: storkuswj{at}upmc.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 
Bone marrow–derived dendritic cells engineered using recombinant adenovirus to secrete high levels of IL-12p70 dramatically inhibited the growth of established CMS4 sarcomas in BALB/c mice after intratumoral administration. An analysis of splenic CD8+ T cells in regressor mice revealed a strong, complex reactivity pattern against high-performance liquid chromatography (HPLC)–resolved peptides isolated by acid elution from single-cell suspensions of surgically resected CMS4 lesions. Mass spectrometry analyses defined two major overlapping peptide species that derive from the murine hemoglobin-β (HBB) protein within the most stimulatory HPLC fractions. Although cultured CMS4 tumor cells failed to express HBB mRNA based on reverse transcription-PCR analyses, prophylactic vaccination of BALB/c mice with vaccines containing HBB peptides promoted specific CD8+ T-cell responses that protected mice against a subsequent challenge with CMS4 or unrelated syngeneic (HBBneg) tumors of divergent histology (sarcoma, carcinomas of the breast or colon). In situ imaging suggested that vaccines limit or destabilize tumor-associated vascular structures, potentially by promoting immunity against HBB+ vascular pericytes. Importantly, there were no untoward effects of vaccination with the HBB peptide on peripheral RBC numbers, RBC hemoglobin content, or vascular structures in the brain or eye. [Cancer Res 2008;68(19):8076–84]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 
In a previous study, we showed that intratumoral (i.t.) injection of syngeneic dendritic cells engineered to constitutively secrete interleukin-12p70 (IL-12p70) resulted in the sustained vitality of dendritic cells that were competent to promote the cross-priming of therapeutic CD8+ T cells in vivo (1). Of note, the protective CD8+ T-cell repertoire seemed to recognize a broad array of tumor MHC-presented peptide epitopes, although the identity of these peptides was not elucidated (1). In the present study, we show that at least one of these epitopes derives from stromal components (i.e., vascular pericytes) within the tumor microenvironment but not from the tumor itself.

In addition to tumor cells, the tumor cell microenvironment is sustained by numerous requisite stromal components, including blood vessels composed of vascular endothelial cells encased within a matrix of "mural" cells, also known as pericytes (2, 3). Pericytes serve to stabilize nascent tubules formed from vascular endothelial cells (VEC; refs. 2–4), with pericyte coverage of vascular structures greatest in the brain and eye, where edema could result in significant pathology (2, 5, 6). In contrast, pericyte coverage of blood vessels in tumors has been reported to be highly variable, resulting in a tortured, leaky vasculature (7). Notably, in experimental cases where pericyte coverage of vascular bodies falls below a critical threshold of 5% to 10%, endothelial cells may become susceptible to apoptosis, resulting in increased vascular permeability and hemorrhaging/aneurism (2). This is perhaps best exemplified in mice that fail to express the platelet-derived growth factor receptor-β (PDGFRβ), as these animals appear deficient in pericytes and exhibit aberrant vascularization (8).

Interestingly, Reisfeld and colleagues (9) have recently shown that immunization of wild-type mice with a recombinant DNA vaccine encoding PDGFRβ promotes the immune-mediated loss of NG2+ pericytes within PDGFRβneg solid tumors. Treated animals resist tumor progression and exhibit prolonged survival (9). Our current results suggest, surprisingly, that the self-antigen HBB may represent yet another pericyte-associated antigen within the tumor microenvironment, against which immunity can be effectively evoked, thereby limiting or ablating tumor growth in vivo.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosure of Potential...
 References
 
Mice. Female 6- to 8-wk-old BALB/c mice were purchased from The Jackson Laboratory and maintained in microisolator cages. Animals were handled under aseptic conditions per an Institutional Animal Care and Use Committee–approved protocol.

Cell lines and culture. CMS4 and MethA are chemically induced BALB/c (H-2d) sarcomas that have been described previously (1). The TS/A breast carcinoma and CT26 colon carcinoma, both H-2d, were purchased from the American Type Culture Collection (ATCC). These cell lines were free of Mycoplasma contamination and were maintained in complete medium [RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum, 100 units/mL penicillin, 100 µg/mL streptomycin, and 10 mmol/L L-glutamine (all reagents from Life Technologies, Inc.)] in a humidified incubator at 5% CO2 tension and 37°C.

In vitro generation of bone marrow–derived dendritic cells. Dendritic cells were generated from bone marrow precursors isolated from the tibias/femurs of BALB/c mice, as previously described (1). Bone marrow cells were cultured in complete medium supplemented with 10% heat-inactivated fetal bovine serum, 1,000 units/mL recombinant murine granulocyte/macrophage colony-stimulating factor (rmGM-CSF) and 1,000 units/mL rmIL-4 (Peprotech) at 37°C in a humidified, 5% CO2 incubator for up to 7 d.

Viral vectors. The Ad.mIL-12p70 and control Ad.{psi}5 (empty) recombinant adenoviral vectors were produced and provided by the University of Pittsburgh Cancer Institute Vector Core Facility (a Shared Resource), as reported previously (1).

Adenoviral infection of dendritic cells. Five million (day 5 cultured) dendritic cells were infected at a multiplicity of infection of 50 with recombinant adenoviruses encoding mouse IL-12p70 (Ad.IL-12) or no cytokine (Ad.{Psi}5). After 48 h, infected dendritic cells (i.e., DC.IL12 or DC.{Psi}5, respectively) were harvested and analyzed for their phenotype and function, as previously reported (1). Culture supernatants were collected for measurement of mIL-12p70 production using a species-specific ELISA kit (BD Biosciences), with a lower level of detection of 62.5 pg/mL.

Synthetic peptides. Peptides HBB33-41 (LVVYPWTQR), HBB34-42 (VVYPWTQRY), HBB33-42 (LVVYPWTQRY), OVA257-264 (SIINFEKL), and β-galactosidase767-784 (β-gal; TPHPARIGL) were synthesized by 9-fluorenylmethoxycarbonyl (Fmoc) chemistry by the University of Pittsburgh Cancer Institute Peptide Synthesis Facility (a Shared Resource). Peptides were >96% pure based on high-performance liquid chromatography (HPLC) profile and mass spectrometric analysis performed by the University of Pittsburgh Cancer Institute Protein Sequencing Facility (a Shared Resource).

Animal experiments. For therapeutic experiments, BALB/c mice received s.c. injection with 5 x 105 CMS4 cells in the right flank on day 0. On day 7, mice were randomized into cohorts of 5 mice, each exhibiting average tumor sizes of ~20 to 30 mm2. On days 7 and 14, tumor-bearing mice were treated with i.t. injections of 1 x 106 adenovirus-infected dendritic cells (DC.{Psi}5 or DC.IL12) in a total volume of 100 µL PBS. Tumor size was then assessed every 3 to 4 d and recorded in mm2, determined as the product of orthogonal measurements taken using vernier calipers. Data are reported as mean tumor area ± SD.

For prophylactic experiments, BALB/c mice were immunized s.c. on the right flank with 100 µL PBS or 100 µL PBS containing 106 syngeneic DC.IL12 cells that had been untreated or prepulsed for 4 h at 37°C with the HBB33-42 peptide (LVVYPWTQRY) or 106 syngeneic DC.{psi}5 cells pulsed with the HBB peptide. Immunizations occurred on days –14 and –7, with mice subsequently receiving injections of CMS4 (5 x 105), MethA (2.5 x 106), TS/A (105), or CT26 (105) tumor cells in the left flank on day 0. As above for the therapeutic model, mean tumor sizes ± SD were evaluated every 3 to 4 d. In all cases, treatment groups contained 5 mice per cohort. For analysis of tumor cellular composition in repeat experiments, CMS4 tumors were surgically resected 14 d after tumor inoculation and prepared for fluorescence imaging, as described below. Also, in some experiments, vaccinated mice were depleted of CD4+ or CD8+ T cells by i.p. injection of 50 µg of monoclonal antibody (mAb) GK1.5 (ATCC) or 100 µg of mAb 53-6.7 (kindly provided by Dr. Zhaoyang Yu, University of Pittsburgh), respectively, in 100 µL PBS on days –3, –2, and –1. Confirmation of specific T-cell depletion was performed by analyzing splenocytes from treated mice by flow cytometry using FITC-labeled anti-CD4 and anti-CD8 mAbs (both from BD Biosciences) that were not sterically blocked by the corresponding mAbs used for in vivo depletion.

Natural peptide isolation. CMS4 tumors were harvested from untreated, control mice on day 28 posttumor inoculation, aseptically minced and digested with DNase, collagenase and hyaluronidase (all reagents Sigma-Aldrich), as previously described (10). After filtration through a 70-µm mesh (BD Biosciences), viable cells were washed five times with PBS by centrifugation. Peptides were acid eluted using citrate-phosphate buffer (pH 3.3) from this viable cell mixture, desalted, and consequently separated on reverse-phase HPLC (RP-HPLC), as described previously (11). Individual HPLC fractions (800 µL) were split into two duplicate aliquots (400 µL each) and then lyophilized to a volume of ~10 µL to effectively remove organic solvents (acetonitrile, trifluoroacetic acid). One series of aliquots were reconstituted in 100 µL of PBS and stored at –80°C until use for T-cell assays. The alternate series of aliquots was stored at –80°C and reserved for analysis by mass spectrometry (MS).

Mass spectrometry analysis of peptides. HPLC fractions recognized to the highest degree in vitro by CD8+ T-cell responses were analyzed by MS as previously described (11).

Evaluation of CD8+ T-cell responses in the therapy model. Spleens were harvested from 2 mice per group 7 d after the second i.t. injection of dendritic cells (i.e., day 21 after tumor inoculation) and splenocytes were restimulated in vitro for 5 d with irradiated (50 Gy) CMS4 cells at a splenocyte to CMS4 ratio of 10:1. Responder CD8+ T cells were then isolated using magnetic bead cell sorting (Miltenyi Biotec). Finally, CD8+ T cells (105 cells) and syngeneic day 7 cultured dendritic cells (104 per well) and HPLC-fractionated peptides (10 µL/well) were added to individual wells of a 96-well tissue culture plate. Alternatively in some assays, CMS4 (104) or MethA (104) tumor cells (in the absence or presence of 10 µmol/L HBB peptides) were used as target cells. After 48-h incubation, culture supernatants were collected and analyzed for IFN-{gamma} release using a commercial ELISA (BD Biosciences) with a lower limit of detection of 31.5 pg/mL. Data are reported as the mean ± SD of duplicate determinations. In some assays, 10 µg/well of blocking anti-H-2Kd (31-3-4s; ATCC), anti-H-2Dd (34-4-21s; ATCC), and anti–H-2Ld (28-14-8; Santa Cruz Biotechnology) mAbs were added to replicate wells to discern the class I restriction element(s) used by responder CD8+ T cells.

Evaluation of CD8+ T-cell responses in the vaccine model. Lymph nodes were harvested from mice 5 wk after the second weekly s.c. vaccination with either PBS or DC + HBB33-42 peptide. Isolated lymph node cells were restimulated in vitro for 5 d with irradiated, naive H-2d splenocytes that had been prepulsed with the HBB peptide (1 µmol/L for 3 h at 37°C, then washed twice with PBS) at a responder to stimulator cell ratio of 10:1. Responder cells were consequently cultured with syngenic splenocytes pulsed with no peptide, 1 µmol/L irrelevant peptides (β-gal or OVA) or 1 µmol/L HBB33-42 peptide and analyzed for intracellular levels of IFN-{gamma} by flow cytometry using a BD FastImmune CD8 Intracellular IFN-{gamma} Detection Kit (BD Biosciences), per the manufacturer's protocol.

Peptide-binding assays. The HBB33-41, HBB34-42, and HBB33-42 peptides, as well as the positive control β-galactoside876-884 and negative control OVA257-264 peptides (12), were analyzed for their ability to stabilize H-2Ld class I complexes expressed by the T2.Ld cell line (kindly provided by Dr. Peter Cresswell, Yale University, New Haven, CT). Briefly, T2.Ld cells (106) were incubated in the absence, or presence, of synthetic peptides (0.01–1 µmol/L) at 37°C for 4 h, before being washed in PBS and stained on ice for 30 min using FITC-conjugated anti-H-2Ld mAb 28-14-8 (Santa Cruz Biotechnology). Stained cells were then fixed in 2% paraformaldehyde in PBS and analyzed using an Epics XL flow cytometer (Beckman Coulter, Inc.).

Reverse transcription-PCR. Reverse transcription-PCR (RT-PCR) was performed using the following primer pairs: mHBB (forward), 5'-TCAGAAACAGACATCATGGTGC-3'; mHBB (reverse), 5'-TAGACAATAGCAGAAAAGGGGC-3'; β-actin (forward), 5'-GGCATCGTGATGGACTCCG-3'; β-actin (reverse), 5'-GCTGGAAGGTGGACAGCGA-3'. Cycling times and temperatures were as follows: initial denaturation at 94°C for 2 min (1 cycle), denaturation at 94°C for 30 s, annealing at 60°C for 30 s and elongation at 72°C for 1 min (30 cycles), final extension at 72°C for 5 min (1 cycle). PCR products were identified by image analysis software for gel documentation (LabWorks Software; UVP) following electrophoresis on 1.2% agarose gels and staining with ethidium bromide.

Imaging of tissue sections. Tumor samples were prepared and sectioned as previously reported (13). All washing steps were performed using wash buffer [0.5% bovine serum albumin (BSA) in PBS; Sigma-Aldrich]. For staining, the sections were fixed in 2% paraformaldehyde (Sigma-Aldrich) at room temperature for 45 min, then incubated with 2% BSA for 45 min, washed, and blocked using goat anti-mouse Fab1 fragments (Jackson ImmunoResearch) in wash buffer overnight. For analysis of T-cell subsets, sections were incubated with FITC-conjugated anti-CD4 or anti-CD8β antibodies or matching isotype controls (all from BD Biosciences) for 1 h. For analysis of the CD31, SMA, and HBB markers, the tissue was first blocked in donkey serum (Sigma-Aldrich) for 40 min. One set of tissue was then incubated with monoclonal anti-mouse smooth muscle actin (SMA)-Cy3 (Sigma-Aldrich), rat anti-mouse CD31 (Millipore), and goat anti-mouse hemoglobin β (Jackson ImmunoResearch) for 1 h and then washed. The tissue was then treated with donkey anti-goat Ig-Alexa 488 (Invitrogen) along with donkey anti-rat immunoglobulin-Cy5 (Jackson ImmunoResearch) for 1 h and washed. Another second set of tissue was incubated with goat anti-mouse hemoglobin-β (HBB; Jackson ImmunoResearch) and with rabbit anti-mouse NG2 (Millipore) for 1 h, washed, and then treated with a combination of donkey anti-goat Ig-Alexa 488 (Invitrogen) and donkey anti-rabbit Ig-Cy3 (Jackson ImmunoResearch). After being coverslipped, slide images were acquired using an Olympus 500 scanning confocal microscope (Olympus America). Isotype control and specific antibody images were taken using the same level of exposure on the channel settings. Colocalization of probes was determined using a measure colocalization algorithm in Metamorph Imaging Software (Molecular Devices). For the analysis of brain and eye tissues, whole-body perfusion was performed using 10 mL of PBS followed by 10 mL of 2% paraformaldehyde (Sigma-Aldrich) through the left ventricle after cutting the posterior vena cava. Tissue was excised and immersed in 2% paraformaldehyde for 2 h. Fixed tissues were then immersed in 2.3 mol/L sucrose in PBS overnight at 4°C, before being frozen in liquid nitrogen–cooled isopentane and being stored at –80°C until sectioning. In addition to standard H&E staining, sequential tissue sections were analyzed by immunofluorescence microscopy. The CD31 and NG2 markers were coanalyzed using secondary goat anti-rat Ig-Alexa 488 (Invitrogen) and goat anti-rabbit Ig-Cy3 (Jackson ImmunoResearch) antibodies, respectively. For coanalysis of HBB and CD163 (primary antibody sc-33560 from Santa Cruz Biotechnology), we used secondary donkey anti-goat Ig-Alexa 488 (Invitrogen) and donkey anti-rabbit Ig-Cy3 (Jackson ImmunoResearch) antibodies, respectively. After being coverslipped, slide images were acquired using an Olympus BX51 microscope (Olympus America).

Analysis of RBC. RBCs were isolated by tail venipuncture from control mice or mice vaccinated twice with weekly s.c. injections of PBS or 106 DC.IL12 cells pulsed with the HBB33-42 peptide in their right flank. Blood was isolated 28 d after the first vaccination. RBCs were quantitated per milliliter of blood by hemacytometer count, with hemoglobin content estimated by optical absorbance measurements using the method of Kahn and colleagues (14).

Statistical analysis. Statistical differences between groups were evaluated using a two-tailed Student's t test or one-way ANOVA (StatMate III, ATMS Co.) as appropriate, with P values <0.05 considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosure of Potential...
 References
 
Intratumoral injection of syngeneic dendritic cells engineered to secrete mIL12p70 promotes tumor regression and amplification of systemic antitumor CD8+ T cells in vivo. To extend our previous work (1), syngeneic H-2d bone marrow–derived dendritic cells were engineered to secrete mIL-12p70 (i.e., DC.IL12) by infection with recombinant adenovirus (rAd). As shown in Fig. 1A , DC.IL12, but not control Ad{psi}5-infected dendritic cells or uninfected dendritic cells, secreted high levels (>1 ng/mL/106 dendritic cells/24 h) of IL-12p70. Only DC.IL12 (106 cells) when injected into s.c. established day 7 CMS4 sarcoma lesions (with an average size of 20–30 mm2) on days 7 and 14 proved competent to suppress tumor progression (Fig. 1B; P < 0.0001 on all days >14 versus DC.{psi}5 or uninfected dendritic cells), with complete tumor regression observed in 8 of 10 treated animals (versus 0 of 10 for either control dendritic cell treatment cohort).


Figure 1
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  		Figure 1. Intratumoral delivery of DC.IL12 is therapeutically effective against s.c. CMS4 tumors and promotes polyspecific CD8+ T-cell–mediated responses in vivo. A, day 5 dendritic cells (DC) were left untreated (control) or infected with rAd.{Psi}5 or rAd.IL-12 at MOI 50. After 48 h, culture supernatants were analyzed using an IL-12p70 ELISA. Results are representative of three independent experiments. B, control or gene-modified dendritic cells (106) were injected into established s.c. CMS4 tumors on days 7 and 14 post tumor inoculation and tumor growth was monitored through day 28. All cohorts contained 5 mice per group, with results representative of two independent experiments. C, on day 28, CMS4 tumors were resected from untreated mice and digested into single-cell suspensions; then, peptides were extracted by mild acid elution. Desalted peptides were then resolved into 50 fractions using RP-HPLC and a 10% to 100% acetonitrile gradient, with protein content monitored at 214 nm. D, after removing organic solvents by lyophilization and reconstitution with PBS, individual fractions were loaded onto syngeneic control dendritic cells and assessed for their ability to elicit IFN-{gamma} production (monitored by ELISA) from CD8+ splenic T cells isolated on day 28 from mice treated in B. Results depicted are representative of two independent experiments.

 
To determine the nature and diversity of specific CD8+ T-cell–mediated immunity in these animals, we resected untreated CMS4 lesions, generated single-cell suspensions, and then extracted peptides from MHC complexes expressed by these cells using mild acid extraction (10). These peptides were resolved into 50 fractions using reverse-phase high-performance HPLC (RP-HPLC; Fig. 1C). After lyophilizing these fractions to remove organic solvents, peptides were reconstituted in PBS and aliquots were loaded onto syngeneic dendritic cells to serve as antigen-presenting cells for splenic CD8+ T cells isolated from mice (as in Fig. 1B) 28 days posttumor challenge. An IFN-{gamma} ELISA performed on supernatants isolated from these cultures revealed that only DC.IL12-protected mice displayed pronounced CD8+ T-cell recognition of fractionated peptides, with three major bioactive peaks identified (Fig. 1D).

Mass spectrometric identification of murine HBB peptide as a putative "tumor-associated" CD8+ T-cell epitope. Given the strongest reactivity of protective CD8+ T cells against peptide(s) within HPLC fractions 19 and 20 (Fig. 1D), we prioritized analyses of these mixtures using a combination of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS and microcapillary-LC-ESI-tandem MS. Fraction 19 contained a prevalent peptide species of Mr 1,211.4 as determined by MALDI-TOF MS (Fig. 2A ). The Mr 1,211.4 peptide was consequently determined to represent the sequence VVYPWTQRY upon argon gas–mediated fragmentation into its daughter ion species monitored using microcapillary-LC-ESI tandem MS (Fig. 2B). Similarly, the dominant peptide species in fraction 20 exhibited a Mr 1,324.8 (Fig. 2C), yielding the MS/MS-deduced sequence XVVYPWTQRY (with X = isoleucine or leucine; Fig. 2D). Based on a consequent protein database search, these overlapping peptide sequences seem to derive from either the murine HBB1 or HBB2 chains (i.e., both chains contain the sequence LVVYPWTQRY).


Figure 2
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  		Figure 2. MS analyses of dominant peptide species in bioactive HPLC fractions. HPLC fractions 19 and 20 identified in Fig. 1D as exhibiting the greatest degree of recognition by protective antitumor CD8+ T cells were submitted for MS analyses (MALDI-TOF). Fraction 19 contained a predominant doubly charged peptide (m/z 606) that yielded a Mr of 1,211.4 as a singly charged species (A). MS/MS fragmentation of this doubly charged species was then performed by microcapillary LC-ESI tandem MS analysis. This revealed a deduced sequence of VVYPWTQRY for the Mr 1,211.4 peptide species (B). Similarly, fraction 20 contained a dominant doubly charged peptide (m/z 663) that yielded a Mr of 1,324.8 as a singly charged species (C). MS/MS analysis of the Mr 1,324.8 mass ion revealed a sequence of XVVYPWTQRY (X = leucine or soleucine; both with Mr 113; D). Based on protein database searches for identity, both sequences seem to derive from either murine HBB1 and/or HBB2 proteins, each of which contain the peptide sequence LVVYPWTQRY.

 
We subsequently noted that splenic CD8+ T cells isolated from tumor-bearing mice that had been effectively treated with DC.IL12 were (a) predominantly H-2Ld–restricted in their anti-CMS4 tumor reactivity and (b) unable to recognize the unrelated H-2d sarcoma MethA, unless this target cell line was prepulsed with the LVVYPWTQRY (HBB33-42) peptide (Fig. 3A ). In the latter case, CD8+ T-cell reactivity against the presented HBB33-42 peptide also seemed to be largely H-2Ld restricted based on results obtained in antibody blocking experiments (Fig. 3A). In additional experiments, we determined that each of the two 9-mer (i.e., HBB33-41 and HBB34-42), as well as the 10-mer (HBB33-42), peptides bound to H-2Ld with modest affinity when compared with a known H-2Ld–presented β-galactosidase876-884 peptide epitope (Supplementary Fig. S1).


Figure 3
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  		Figure 3. The HBB33-42 peptide is recognized by H-2Ld–restricted, splenic CD8+ T cells isolated from CMS4 tumor-bearing mice treated with DC.IL12 i.t. administration and such T cells are enriched in the spleens of mice vaccinated with DC.IL-12/HBB peptide. CD8+ splenocytes were harvested from CMS4 tumor-bearing mice treated with i.t. DC.IL12 therapy 28 d after tumor inoculation, then restimulated in vitro for 5 d with irradiated CMS4 tumor cells as outlined in Materials and Methods. A, responder T cells were then cocultured with CMS4 tumor cells or with the irrelevant H-2d MethA sarcoma cell line (in the absence or presence of HBB33-42 peptide) for 48 h. In replicate wells, blocking anti–H-2Kd, anti–H-2-Dd, or anti–H-2-Ld mAbs were added to determine the class I restriction elements used for specific CD8+ T-cell recognition of target cells. Specific T-cell responses were determined by quantitation of secreted IFN-{gamma} by ELISA. *, group differences with P < 0.05. B, intracellular staining for IFN-{gamma} was performed on control versus peptide-stimulated T cells isolated from the lymph nodes of control mice or mice vaccinated with DC pulsed with the HBB33-42 peptide. T cells were costained using anti-CD8 mAb and analyzed by flow cytometry, as outlined in Materials and Methods. Data are representative of two independent experiments.

 
Mice vaccinated against the HBB33-42 peptide are protected against consequent tumor challenge. To directly assess the tumor relevance of HBB-derived peptides, naïve BALB/c mice were vaccinated with syngeneic DC.IL12, DC.IL12 pulsed with the HBB33-42 peptide (DC.IL12/HBB), DC.{psi}5 pulsed with the HBB peptide (DC{Psi}5/HBB), or control DC pulsed with the HBB peptide (DC/HBB) on days –14 and –7, before challenging these animals on day 0 with either CMS4 sarcomas or unrelated H-2d tumors, including MethA (sarcoma), TS/A (breast carcinoma), or CT26 (colon carcinoma). We were not only able to show specific induction of CD8+ Tc1 responses in DC/HBB vaccinated mice versus control vaccinated mice via intracellular staining for IFN-{gamma} (Fig. 3B) but also showed that only mice immunized with DC.IL12/HBB were protected against tumor establishment/progression (Fig. 4A ). Notably, protection was observed not only against CMS4 tumors but also against s.c. challenge with the unrelated H-2d sarcoma (MethA), breast carcinoma (TS/A), or colon carcinoma (CT26) cell lines (Fig. 4A). Animals vaccinated with control dendritic cell–based vaccines (DC.IL12 alone or DC.{psi}5/HBB) exhibited progressive lesions in all cases. Interestingly, even the DC.IL12/HBB vaccinated group of CMS4-bearing mice developed small palpable lesions on day 7, which subsequently resolved over the following 7 to 10 days. Extended experiments using specific depleting antibodies in vivo confirmed that immune protection was dependent on CD8+ T cells but not CD4+ T cells (Fig. 4B).


Figure 4
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  		Figure 4. Mice vaccinated with the HBB33-42 peptide reject a subsequent challenge with HBBneg tumor cell lines. A, BALB/c mice (5 mice per group) were either not vaccinated or vaccinated with 106 DC.IL12 alone ({blacktriangledown}), 106 DC.IL12 cells pulsed with the HBB33-42 peptide (bullet), or 106 DC.{Psi}5 cells pulsed with the HBB peptide ({circ}) on days –14 and –7. On day 0, mice were challenged with the CMS4 (5 x 105), MethA (2.5 x 106), TS/A (105), or CT26 (105) tumor cell lines and monitored for up to 4 wk. Data are representative of two independent experiments performed in each case. Tumor growth in naïve, nonvaccinated animals was indistinguishable from that observed for mice vaccinated with DC.IL12 alone (data not shown). B, the experimental protocol in A above was repeated with the exception that depleting anti-CD4 or anti-CD8 mAbs were injected i.p. into mice on days –3, –2, and –1 relative to CMS4 tumor challenge. Tumor size for each cohort was then compared versus untreated control mice on day 14. *, significant group differences, P < 0.05. C, to determine the HBB (versus control β-actin) mRNA expression status of the CMS4, MethA, TS/A, and CT26 tumor cell lines, RT-PCR was performed and compared with peripheral blood mononuclear cells (PBMC) obtained from normal mice via tail venipuncture. -cDNA, control RT-PCR performed in the absence of template mRNA.

 
In vivo targets of anti-HBB CD8+ T cells are not tumor cells themselves but rather tumor-associated stromal cells. Although these peptides clearly derived from murine origin and could not have been the result of bovine protein contamination (i.e., fetal bovine serum used to culture CMS4 tumors; bovine HBB chains contain the sequence LVVYPWTQRF; Genbank accession no. NP_776342), we also noted in RT-PCR analyses that cultured CMS4, MethA, TS/A, and CT26 tumor cells fail to express message transcripts for HBB (Fig. 4C). Furthermore, MS inspection confirmed that HBB-derived sequences were not present in detectable quantities in (acid-eluted) peptides harvested from CMS4 tumor cells grown in vitro (data not shown).

These results suggested that either (a) tumor cells turn on HBB transcription as a consequence of growth in vivo or (b) an alternate, nontumor cell type(s) present within the tumor microenvironment may present HBB-derived peptides and constitute a clinically relevant target for protective antitumor immunity. We attempted to address this using immunohistochemistry in two ways. First, we theorized that the composition of early CMS4 lesions in HBB peptide immunized mice would differ from that of control vaccinated mice, in that the HBB+ target cell population might be selectively depleted. Second, we directly analyzed control CMS4 (untreated) lesions for expression of HBB protein in an attempt to colocalize it with stromal cell subsets.

In Fig. 5A , we noted that day 14 CMS4 lesions in mice preimmunized with DC.IL12/HBB (as in Fig. 4A) contained higher frequencies of CD8+ T cells (Fig. 5A, b) and comparable levels of CD4+ T cells (Fig. 5A, d) when compared with control cohorts (Fig. 5Aa, c). We also observed that CMS4 tumors in the DC.IL12/HBB vaccinated cohort of mice contained fewer CD31+ vascular structures versus control tumors (Fig. 5A, f versus A, e; Fig. 5B; P < 0.0001) and that the CD31+ vessels in the DC.IL12/HBB immunized group exhibited a reduced level of coverage by SMA+ pericytes versus controls (Fig. 5A, f versus A, e; Fig. 5C; P < 0.001). Because CD31+ vessels can be compromised in the absence of sufficient pericyte coverage (2), this could suggest pericytes as a logical primary immune target for HBB-specific CD8+ T cells in these animals. When performing costaining analyses, we noted that HBB protein (a) did not seem to be expressed by CMS4 sarcoma cells in situ (Fig. 5A, g, h), (b) seemed to be expressed by SMA+ pericytes and/or CD31+ VEC (Fig. 5A, g, h), and (c) was comparatively depleted in the tumors of DC.IL12/HBB vaccinated versus control vaccinated mice (Fig. 5A, h versus A, g). Further immunohistochemical analyses using progressive day 14 CMS4 tumors harvested from control, untreated mice suggested that HBB expression was primarily associated with SMA+ and/or NG2+ pericytes, and less so with CD31+ vascular endothelial cells (Fig. 6A and B ). Fluorescence confocal microscopy also revealed that many perivascular HBB+ cells within CMS4 tumors coexpressed CD163 (Fig. 6C), a scavenger receptor for the haptoglobin-hemoglobin complex (14, 15).


Figure 5
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  		Figure 5. Regressing CMS4 tumors in HBB peptide–vaccinated mice exhibit elevated CD8+ T-cell infiltration and reduced expression of CD31 VEC, SMA+ pericytes, and HBB protein in situ. The experiment outlined in Fig. 4A was repeated and CMS4 tumor lesions were surgically resected from mice vaccinated with DC.IL12/no peptide versus DC.IL12/HBB peptide on day 14 posttumor challenge. A, after sectioning, tissues were evaluated for CD8+ T cells (a, b), CD4+ T cells (c, d), CD31+ VEC (e-h), SMA+ pericytes (e-f), and HBB (g, h) using multicolor confocal immunofluorescence. Data obtained from unvaccinated mice were comparable with that derived from control DC.IL12/no peptide vaccinated mice (data not shown). The number of CD31+ vessels per high-power field (HPF; B) and the percentage of CD31+ vessels with associated SMA+ pericytes (C) was quantitated for e versus f; *, P < 0.05.

 

Figure 6
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  		Figure 6. Pericytes are the major cell population within the progressor tumor microenvironment expressing HBB. A, CMS4 tumors growing progressively in untreated BALB/c mice were harvested on day 28, sectioned, and costained for the pericyte markers NG2 or SMA versus HBB, then analyzed by two-color confocal immunofluorescence. B, the percentage of CD31+ (from Fig. 5A, g) or NG2+ or SMA+ cells (from Fig. 5A, g and panel A of this figure) coexpressing HBB was quantitated. *, significant group differences (P < 0.05). C, the CMS4 tumor-associated vasculature was analyzed for coordinate expression of HBB versus CD163 (a hemoglobin scavenger receptor) using immunofluorescence microscopy, as outlined in Materials and Methods. Data are representative of three independent experiments.

 
DC.IL12/HBB peptide-vaccinated mice do not exhibit RBC-associated abnormalities or tissue pathology in the brain or eye. Because the HBB33-42 peptide used to vaccinate mice represents a normal, "self" protein sequence that is also expressed by RBC, it was entirely possible that HBB-specific CD8+ T cells could mediate autoimmune anemia. However, we did not detect any significant difference in the numbers of RBC/mL of blood or the level of hemoglobin/dL of peripheral blood in DC.IL12/HBB vaccinated animals versus control (PBS) vaccinated mice (Supplementary Fig. S2).

Although there were no obvious behavioral alterations in DC.IL12/HBB vaccinated mice that would suggest impairment of brain/visual functions, given the importance of pericytes to the integrity of blood vessels in the brain and eye (2, 5, 6), it was also important to rule out pathologic consequences of HBB-based vaccines targeting these organs. To further corroborate a lack of vascular alterations in the brain and eye, tissues were harvested from control untreated mice or mice vaccinated with DC.IL12 only versus DC.IL12/HBB. We noted no discernable inflammatory infiltrates or alterations in tissue-associated vasculature (Supplementary Fig. S3) and we failed to observe expression of CD163 on microglial cells (Supplementary Fig. S4), which has been reported to represent a marker of encephalitis (14). Interestingly, unlike the tumor stroma, we were not able to detect expression of perivascular HBB in either brain or eye tissues (Supplementary Fig. S4).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosure of Potential...
 References
 
In extending our previous evaluation of the effectiveness of i.t. administration of DC.IL12 in promoting the cross-priming of therapeutic immunity (1), we identified HBB as a tumor microenvironment–associated antigen recognized by T cells. In particular, the HBB33-42 peptide and its two derivative 9-mer peptides seem capable of binding to H-2Ld (that seems to serve as the dominant restricting allele for protective T cells in the CMS4 model) and could each represent (at least cross-reactive) epitopes for CD8+ T effector cells. Although our attempts to generate stable H-2Ld/HBB peptide tetramers have failed to date, using an intracellular staining assay for IFN-{gamma} production as a single-cell readout of "clonal" T-cell reactivity, we have shown that anti-HBB33-42 CD8+ T cells are enriched (representing up to 5% of all CD8+ T cells; Fig. 3B) in the spleens of DC.IL12/HBB peptide immunized mice versus control vaccinated mice.

Although this is the first report for CD8+ T-cell epitope(s) derived from HBB, a recent publication describes the biochemical identification of CD8+ T-cell epitopes derived from {gamma}-globin (16). Like HBB in our model, {gamma}-globin is a normal, "self" protein target to which tolerance would be presumed to be in effect. However, in contrast to our findings in which HBB seems to be expressed by stromal components and not the tumor itself, {gamma}-globin seems to represent an endogenous tumor cell product, with juvenile myelomonocytic leukemia cells being directly recognized by specific CD8+ T cells (16).

Arguably, the most intriguing aspects of the current study are that (a) HBB, an antigen most strongly affiliated with erythropoiesis (17), may be expressed by cell types within the tumor microenvironment to which specific CD8+ T cells may be targeted in a class I–restricted manner, (b) CD8+ T cells of a "therapeutically relevant" functional avidity may be evoked against this self-antigen in the absence of overt autoimmune pathology affecting RBC or normal vasculature in the brain and eye, and (c) DC.IL12/HBB-immunization results in specific immunity that protects against consequent challenge with unrelated HBBneg tumor cell lines (CMS4, MethA, TS/A, and CT26) of disparate histologies. Hence, tumor cells need not express HBB themselves for T-cell–mediated protection to occur. This does not mean that protection occurs in a completely tumor-independent manner, because clearly tumor cells may condition the microenvironment in a way that (uniquely) promotes HBB peptide presentation by stromal cells, such as NG2+ and/or SMA+ pericytes. This hypothesis is further supported by the observed lack of expression of HBB protein by perivascular cells in normal brain/eyes (Supplementary Fig. S4).

Although we believe that pericytes are indeed the HBB+ cells targeted by protective CD8+ T-cell–mediated immunity in our model, based on the preferential depletion of this cell type in the tumor lesions of mice vaccinated with DC.IL12/HBB. However, we have not formally shown that pericytes actually transcribe and translate HBB mRNA in situ. Still, this would not be surprising given reports of unexpected HBB+ cell types, such as lipopolysaccharide-activated macrophages (18) or alveolar type-2 epithelial cells (19). Furthermore, hemoglobin transcription can be turned on under hypoxic conditions (20, 21), such as those encountered in the tumor microenvironment, which could affect tumor-associated pericytes (or VEC). A preliminary RT-PCR analysis for HBB mRNA expression in flow-sorted NG2+ versus CD31+ cells isolated from progressive CMS4 tumors yielded positive signals for both types of cells (Supplementary Fig. S5). However, these sorted cells were only 76% to 86% pure and this conclusion must remain cautiously interpreted until confirmatory experiments can be performed on even cleaner cell populations.

An alternate or additional possibility is that pericytes may cross-present HBB-derived peptides in H-2Ld complexes but fail to express HBB mRNA. Pericytes and VEC are active in caveolin-1–dependent transcytosis of exogenous proteins (22, 23). Hence, it is feasible that serum HBB protein may be captured from the lumen of vascular vessels or as a result of vascular permeability (24), resulting in VEC/pericyte presentation of HBB peptides in our model. HBB uptake could be specifically mediated by CD163 (a hemoglobin scavenger receptor; ref. 25), which we have shown to be expressed by tumor-associated perivascular cells. It is also important to note that because the HBB-specific antibody probe that we used in this study does not recognize the immunogenic HBB33-42 peptide itself (data not shown), it is also formally possible that tumor-associated antigen-presenting cells may serve as HBB cross-presenters to tumor-infiltrating effector CD8+ T cells (25, 26).

Thus, why should HBB be considered a logical and relevant "tumor-associated" target antigen? Although conventionally linked with oxygen transport in RBC, hemoglobin mediates a more primordial function in protecting cells against oxidative and nitrosative stress (27, 28) and has been shown to be intimately involved in nitric oxide metabolism (28, 29). By being produced within, or taken up by, cells within the tumor microenvironment (such as pericytes or VEC), the associated angiogenic/vasculogenic processes might be expected to be stabilized, leading to progressive tumor growth. Interestingly, a COOH-terminal fragment of HBB has been reported to be specifically increased within head-and-neck squamous cell carcinomas in a disease stage-dependent manner, which may support the enhanced presence and proteolytic processing of HBB during disease progression (30). This could also suggest that the "unstable" NH2 terminus of the protein is selectively degraded/processed and more likely to yield MHC class I–presented epitopes (such as the HBB33-42 peptide defined in this study).

Overall, these results and recent results from others (9, 31–34) support the likely clinical safety and efficacy of combinational vaccine approaches targeting the tumor-associated vasculature. Although similar concepts in the targeting of VEC using vascular endothelial growth factor receptor inhibitors (among others) and pericytes using the PDGFR kinase inhibitor ST1571 have resulted in VEC apoptosis, inhibited tumor growth and prolonged survival in orthotopic murine models of ovarian carcinoma (35), specific CD8+ T cells prompted by vaccines would be presumed to provide a more stable, durable mode of protection to patients with active disease or those at high-risk of recurrence. Importantly, although we did not observe any acute toxicities in the blood, brain, or eyes of DC.IL12/HBB–vaccinated mice, off-target effects could result from such approaches and prospective studies should investigate whether such vaccines inhibit wound healing or promote any deleterious destabilization of normal vascular structures over extended periods of time.

Finally, it is important to acknowledge that (i.t. delivered and vaccine) DC.IL12 but not control DC.{Psi}5 served as competent "adjuvants" in eliciting anti-HBB CD8+ T cells in our model system. This is consistent with prior reports documenting the critical role of IL-12p70 in breaking operational tolerance to self-antigens (36, 37) and supports the clinical use of DC.IL12 in cancer vaccine formulations targeting the induction of type-1 T-cell responses against nonmutated antigens/epitopes presented by tumor cells and/or stromal cells within the tumor microenvironment.


   Disclosure of Potential Conflicts of Interest
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
References
 
W.J. Storkus: IVT Advisory Board. The other authors disclosed no potential conflicts of interest.


   Acknowledgments
 
Grant support: NIH grants R01 CA 57840 and P01 CA 100327 (to W.J. Storkus).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Mark Ross for excellent technical support.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

Received 1/31/08. Revised 7/ 9/08. Accepted 8/ 7/08.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 

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