Patients with chronic lymphocytic leukemia (CLL) who relapse after allogeneic transplant may achieve durable remission following donor lymphocyte infusion (DLI), showing the potency of donor-derived immunity in eradicating tumors. We sought to elucidate the antigenic basis of the effective graft-versus-leukemia (GvL) responses associated with DLI for the treatment of CLL by analyzing the specificity of plasma antibody responses developing in two DLI-treated patients who achieved long-term remission without graft-versus-host disease. By probing high-density protein microarrays with patient plasma, we discovered 35 predominantly intracellular antigens that elicited high-titer antibody reactivity greater in post-DLI than in pre-DLI plasma. Three antigens—C6orf130, MDS032, and ZFYVE19—were identified by both patients. Along with additional candidate antigens DAPK3, SERBP1, and OGFOD1, these proteins showed higher transcript and protein expression in B cells and CLL cells compared with normal peripheral blood mononuclear cells. DAPK3 and the shared antigens do not represent minor histocompatibility antigens, as their sequences are identical in both donor and tumor. Although ZFYVE19, DAPK3, and OGFOD1 elicited minimal antibody reactivity in 12 normal subjects and 12 chemotherapy-treated CLL patients, 5 of 12 CLL patients with clinical GvL responses were serologically reactive to these antigens. Moreover, antibody reactivity against these antigens was temporally correlated with clinical disease regression. These B-cell antigens represent promising biomarkers of effective anti-CLL immunity. Cancer Res; 70(4); 1344–55
- protein microarray
- antigen discovery
Although chronic lymphocytic leukemia (CLL) is most often controlled with combination chemotherapy (1), allogeneic hematopoietic stem cell transplantation (HSCT) remains the only potentially curative treatment for this disease (2, 3). It has been long appreciated that these curative responses rely on the ability of donor-derived immune effectors to recognize and eliminate leukemic cells. The existence of the graft-versus-CLL response has been supported by studies showing that (a) myeloablative allogeneic, but not autologous, HSCT consistently results in a plateau in survival after 1 year and the development of undetectable minimal residual disease (4); (b) disease remission is closely associated with immunologic competence [i.e., following taper of immunosuppression or development of graft-versus-host disease (GvHD); refs. 5, 6]; and finally (c) donor lymphocyte infusion (DLI) for posttransplant relapsed CLL effectively generates donor-derived tumor immunity against CLL (7). In this procedure, infusion of donor lymphocytes without further chemotherapy or radiation therapy results in remission in 30% to 50% of patients with relapsed CLL (8–10).
Because of its clear clinical efficacy in the absence of additional therapy, DLI is a promising system for elucidating the mechanism of donor-derived immunity to CLL. Identifying and characterizing the antigenic targets of DLI may provide insight into the basis of the antitumor effect of DLI. Although T cells clearly participate in graft-versus-leukemia (GvL) responses (11), several studies have implicated an important role for B-cell immunity in remission of hematologic malignancies following transplant and DLI (12–14). Some B-cell–defined antigens (15) have been shown to elicit T-cell responses, and effective tumor immunity likely comprises a coordinated humoral and cellular adaptive immune response.
To date, few CLL-specific target antigens have been identified (16–19). In the current study, we identify several novel candidate antigens through analysis of B-cell responses in CLL patients with clinically evident tumor immunity but without anti-host immune reactions following DLI. By using high-density human protein microarrays probed with plasma antibody from before and after DLI, we rapidly identified CLL antigens that elicit potent antibody responses in close temporal association with clinical response to DLI. Potential uses of these antigens include immune monitoring of CLL following therapy and even as immunogens for CLL-specific vaccines.
Materials and Methods
Preparation of cell and plasma samples
Serum or plasma samples were obtained from normal donors and patients enrolled on clinical research protocols that were approved by the Human Subjects Protection Committee at the Dana-Farber Cancer Institute (DFCI). Plasma was removed following brief centrifugation of whole blood and cryopreserved at −80°C until the time of analysis. Peripheral blood mononuclear cells (PBMC) from normal donors and patients were isolated by Ficoll/Hypaque density gradient centrifugation, cryopreserved with 10% DMSO, and stored in vapor-phase liquid nitrogen until the time of analysis.
Processing and analysis of protein microarrays
Protein microarrays (Human ProtoArray v3, Invitrogen) from one printing lot were probed with patient plasma diluted 1:150 and processed according to the manufacturer's recommendations, as previously described (20). These microarrays contain ∼5,000 NH2-terminal glutathione S-transferase (GST) fusion human proteins generated in an insect cell line, with proteins spotted in adjacent duplicates on nitrocellulose-coated glass slides. Binding of plasma immunoglobulin to protein features on the array was detected using an Alexa Fluor 647–conjugated anti-human IgG (H and L chain; 1:2,000 dilution; Invitrogen). Protein microarrays were scanned and analyzed with correction for protein concentration, as previously described (20). Significant signal change was defined using the pretreatment and posttreatment Z scores, Zpre and Zpost, based both on a change in magnitude Zdelta = Zpost − max(0, Zpre) and in ratio Zmult = Zpost/max(1, Zpre) greater than a cutoff n. The cutoff n was set at 5 for significant interactions and 3 for borderline interactions. Both replicate spots were required to pass the test individually for an interaction to be considered significant. Candidate antigens were ranked based on the largest cutoff n that would identify them as significantly changed. Microarray data and calculated significance values are in the Gene Expression Omnibus repository under accession GSE11564.
Sequencing of candidate antigens from tumor and donor tissue
The genes encoding the candidate antigens were directly sequenced using the M13 forward and M13 reverse primers after gene-specific PCR amplification of genomic DNA and TA TOPO cloning (Invitrogen). Genomic DNA was isolated from patient tumor and donor-generated EBV cell lines using a Wizard kit (Promega), following the manufacturer's instructions. Flow cytometric analysis of patient tumor revealed all CD19+ cells to coexpress CD5, so tumor cells were immunomagnetically purified to >95% purity using CD19+ microbeads (Miltenyi). Sequences were aligned using Sequencher (Gene Codes Corp.).
Gene expression microarray data analysis
To compare gene expression of our collection of target antigens between normal B cells and CLL cells, raw data files (.CEL) generated on Affymetrix U95v2 microarrays by Klein and colleagues (21) were collected and normalized using Robust Multiarray Averaging. We used Resourcerer (22) to map gene identifiers of the panel of target antigens to Affymetrix probe set IDs. For those targets that mapped to multiple probe set IDs, the median of the expression values was used. Raw data files were also processed using Affymetrix MAS 5.0, and genes were defined as detectable if the detection call was “present” in ≥40% of samples for each cell type. The Fisher's exact test was used for comparison between groups.
Measurement of antigen-specific gene expression by quantitative PCR
To evaluate the gene expression of the candidate antigens, RNA was extracted (RNeasy kit, Qiagen) from normal PBMCs, immunomagnetically purified B cells from normal PBMCs (CD19+ microbeads; Miltenyi), and B-CLL tumor cells. CLL RNA was extracted from cryopreserved samples of peripheral blood or marrow that were known to contain >85% tumor cells. First-strand cDNA was generated from 2 μg of total RNA by using random hexamers (Pharmacia LKB Biotechnology) and reverse transcriptase (SuperScript, Life Technologies) according to the manufacturer's instructions. Antigen-specific gene expression was measured by quantitative real-time PCR (qRT-PCR; Taqman Gene Expression Assays, Applied Biosystems) using a 7500 Fast Real-Time PCR cycler (Applied Biosystems). Transcript expression was measured relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The significance of the difference in gene expression of target antigens between cell populations was evaluated using the exact Wilcoxon rank-sum test.
Generation of cell lysates and Western blotting
Whole-cell lysate was generated from patient samples by lysis with radioimmunoprecipitation assay buffer [1% NP40, 0.5% deoxycholate, 0.1% SDS, 125 mmol/L sodium chloride, 50 mmol/L HEPES (pH 7.4)] in the presence of protease and phosphatase inhibitors. Lysates (20 μg of total protein per lane) were subjected to gel electrophoresis using 4% to 15% gradient SDS-PAGE gels in Tris-glycine buffer and transferred onto nitrocellulose filters in Tris-glycine buffer containing 20% methanol. Antibodies specific to DAPK3 (1:500; Epitomics), MDS032 (1:1,000; Aviva Systems), SERBP1 (1:500; Abnova), ZFYVE19 (1:400; Abnova), and OGFOD1 (1:100; Abcam) were used to detect protein expression of the respective antigens by Western blot followed by incubation with horseradish peroxidase (HRP)–linked secondary antibodies. Antibody to β-actin (1:5,000; Sigma-Aldrich) was used as a control to ensure equal loading of lanes.
Plasmid acquisition and in vitro translation of candidate antigens
To facilitate protein expression for target validation and characterization, DNA sequences encoding a subset of the candidate antigens were either acquired in or cloned into Gateway (Invitrogen) expression vectors. DNA sequences in Gateway vectors were obtained from PlasmID (Harvard Institute of Proteomics, Cambridge, MA; ref. 23), the Ultimate ORF collection (Invitrogen), or the Human Orfeome Collection (generous gift of Dr. Mark Vidal, Center for Cancer Systems Biology, DFCI, Boston, MA; ref. 24). Additional DNA sequences were purchased from the American Type Culture Collection, Open Biosystems, or the Resource Center of the German Human Genome Project or were directly PCR amplified from CLL tumor cell cDNA and cloned into the pDONR221 Gateway vector (Invitrogen). The majority of acquired cDNA clones originated from the I.M.A.G.E. Consortium (Lawrence Livermore National Laboratory; Supplementary Table S1; ref. 25). The inserts of all sequences in Gateway vectors were verified by sequencing and then shuttled into a Gateway-converted mammalian expression vector containing a T7 promoter and a COOH-terminal GST tag (gift of Wagner Montor, Harvard Institute of Proteomics) using LR Clonase (Invitrogen). Candidate antigens were expressed in vitro with rabbit reticulocyte lysate (TNT T7 Quick Coupled Transcription/Translation, Promega) using biotinylated lysine tRNA (Transcend tRNA, Promega). Reactions (50 μL) using 1 to 2 μg of template DNA and 2.5 μL of Transcend tRNA were incubated at 30°C for 90 min on a tabletop shaker at 950 rpm. A reaction using 1.5 μg of DNA encoding luciferase was performed with each synthesis batch as a positive control.
Immunoprecipitation of candidate antigens using patient plasma
Protein was immunoprecipitated using patient plasma, as previously described (20). In brief, 5 μL of reticulocyte lysate product were immunoprecipitated with 5 μL of serum or plasma at 4°C. To mirror the short incubation time period used for the protein array probing, and to differentiate strong antibody interactions from nonspecific cross-reactivity, we limited the immunoprecipitation reaction to 1 h. Immunoprecipitated products were isolated using 50% protein A–Sepharose CL-4B beads (GE Healthcare) and visualized by immunoblot. Protein was detected using immunoperoxidase-streptavidin (1:5,000 dilution; MP Biomedicals). The blots were developed with SuperSignal West Femto detection substrate (Pierce Biotechnology) and imaged with BioMax Light film (Kodak).
For some experiments, immunoprecipitation of a single candidate antigen against up to 48 serum or plasma samples was performed simultaneously to directly compare antigen-antibody affinity between plasma samples. Immunoprecipitation against GST was used as comparison for background serum reactivity. As a control and for comparing protein bands across immunoblots, a 1:440 dilution of one batch of luciferase reticulocyte lysate product was loaded onto one to two lanes on each immunoblot. Serum or plasma sample reactivity was compared against the range of reactivity evidenced by the 12 normal subjects, taking into account the difference in the strength of the control band on each Western blot.
DLI for CLL is associated with potent humoral immune responses
We identified two patients with relapsed CLL who remained in continuous remission more than 8 years after receiving CD8-depleted DLI (26). As shown in Table 1, both patients A and B failed multiple regimens of conventional chemotherapy before receiving myeloablative T-cell–depleted HSCT from HLA-matched sibling donors. Both relapsed with clinical disease within 5 years of transplantation and received DLI without any further chemotherapy or radiotherapy. Following DLI, both patients had a clear GvL response without acute or chronic GvHD, showing rapid conversion to full donor hematopoiesis and gradual disappearance of histologically evident marrow disease over 6 to 16 months.
To identify the B-cell targets associated with CLL tumor regression in these two patients, we used plasma collected at 0 to 1 months before and 9 to 12 months after DLI as sources of antibody for probing high-density protein microarrays. The vast majority of array proteins were bound at comparable levels by pre- and post-DLI plasma antibodies based on raw fluorescence intensity. Using a concentration-dependent analysis, significant antigen-antibody interactions were identified, shown as black diamonds in Fig. 1 (20). These candidate antigens comprised <1% of spotted proteins. No proteins had significantly decreased antibody reactivity following immunotherapy.
Targets of the DLI-induced humoral immune response in patients with CLL
Analysis of the protein microarray data yielded 16 candidate DLI-associated antigens for patient A and 21 for patient B. These antigens were ranked based on the largest cutoff n that would identify them as significantly changed. Although most candidate antigens were identified in only one patient, the two proteins encoded by genes ZFYVE19 and C6orf130 were recognized by both patients. A third protein, MDS032, was identified as a candidate antigen for patient A and elicited borderline (n ≥ 3) antibody reactivity for patient B. The candidate antigens and the shared antigen MDS032, only borderline significant for patient B, are listed in Table 2. The candidate antigens as well as 26 interactions of borderline significance for patient A and 31 for patient B are further described in Supplementary Table S1.
We queried publicly available databases, including Entrez, UniGene, Harvester, and SymAtlas (27–29), for information on the function of the 35 identified candidate antigens, summarized in Table 2. Of the 25 known candidate antigens, 24 are intracellular and 18 are reported as tumor associated (27). Eight have DNA or RNA binding activity, four are part of the ubiquitination pathway, two are tumor suppressors, and one each is lymphocyte associated or functions in the cell cycle, spermatogenesis, or apoptosis.
Validation of serologic reactivity to candidate antigens
Significant microarray signals may result from true interactions, erroneous measurement, or interactions with misidentified proteins, such as due to improper synthesis or coprecipitation of additional proteins. To confirm that the serologic targets identified by microarray analysis represent true antibody-antigen interactions, we independently validated antibody reactivity against our screened candidate antigens. Figure 2A schematically summarizes our validation strategy. We expressed our candidate antigens as COOH-terminal GST fusion biotinylated proteins in a cell-free mammalian expression system, a different method of protein manufacture than used on the protein microarrays. We then immunoprecipitated the recombinant proteins with patient plasma and visualized immunoprecipitated proteins by immunoblot. In this manner, interactions between serum antibodies and properly folded and posttranslationally modified antigen were detected, and nonspecific interactions were excluded on the basis of size on immunoblot.
Using this assay, we successfully validated 22 of the 30 tested candidate antigen interactions, listed in Table 2. Eight interactions failed validation, and eight remained untested due to limited patient sample. Figure 2B shows representative results from these studies. Consistent with our bioinformatic analysis, post-DLI plasma from both patients A and B immunoprecipitated greater amounts of the shared antigens C6orf130, MDS032, and ZFYVE19 compared with pretreatment plasma. For SERBP1 and DAPK3, identified by patient A, and FAM122A and OGFOD1, identified by patient B, greater immunoprecipitated antigen was evidenced only by the post-DLI sample from the patient who identified the interaction. Equivalently high immunoprecipitation reactivity against the control EBNA1 construct was seen for all plasma samples tested. Interactions of higher significance were slightly more likely to be validated, with 88% of tested interactions with rank <10 validated, whereas only 57% of tested interactions with rank ≥10 were validated (Table 2).
A subset of candidate antigens is highly expressed in B lineage cells
To broadly survey the gene expression of our collection of antigens, we first compared the gene expression of our candidate antigens in primary CLL cells and normal B-cell subsets using existing gene expression microarray data generated on the Affymetrix U95v2 array (21). Probe sets for 18 of the 35 candidate antigens were present on this microarray. In these data, 5 of the 18 matched candidate antigens—LCP2, PDCD4, MKNK1, SRPK2, and CHD2—showed higher expression in CLL compared with normal B cells (P < 0.01) but none at the level of differential expression that was found for the most significantly upregulated 33 genes reported in that study.
Because many of the candidate antigens were not represented on the gene expression microarray, we next directly quantified antigen-specific gene expression in normal and malignant B cells by using qRT-PCR. We performed gene expression studies for nine candidate antigens in three tissue sources: normal PBMC, normal B cells, and CD19+CD5+ CLL tumor cells. No significant difference in gene expression was observed among the three tissue groups for three of the antigens (PRKAA1, POLR3D, and SPZ1). We observed increased expression in normal and malignant B cells compared with PBMCs for the three shared antigens C6orf130, MDS032, and ZFVYE19 as well as for OGFOD1, whereas a third pattern, in which antigen-specific gene expression is significantly increased in CLL cells compared with normal B cells, was observed for DAPK3 and SERBP1 (Fig. 3A). Consistent with the notion that GvL-associated antigens may be B-cell lineage genes, the three shared antigens C6orf130, MDS032, and ZFYVE19 showed significantly higher expression levels in normal and malignant CD19+ B cells compared with PBMCs (P < 0.02). Western blotting of lysates from CLL B cells and normal B cells compared with PBMC confirmed these results (Fig. 3B). These results indicate that a subset of candidate antigens is both immunogenic and highly expressed in CLL.
Candidate antigens are not minor histocompatibility antigens
The immunogenicity of candidate antigens can arise from sequence differences between donor, host, and tumor or differential presentation of antigen epitopes by tumor cells. Sequence differences may exist between donor and host due to gene polymorphisms, resulting in minor histocompatibility antigens (mHA). Direct sequencing of 6 to 10 independent copies of both tumor and donor cell templates revealed the absence of sequence differences for ZFVYE19, MDS032, C6orf130, and DAPK3 between donor and CLL tumor cells. Thus, the selected subset of the identified antigens is not mHA but represents overexpressed CLL-associated or B-cell–associated antigens.
Clinical response to DLI is temporally associated with antibody responses to candidate antigens
After DLI therapy, both patients A and B showed normalization of peripheral blood lymphocytosis and disappearance of tumor cells from the marrow space (Fig. 4A). Leukemia cells occupied 25% of patient A's marrow intertrabecular space at 2 months after DLI but decreased to undetectable levels within 24 months. Similarly, CLL cells occupied 45% of the marrow intertrabecular space of patient B at 3 months after DLI but decreased to <5% tumor involvement by 9 months after DLI.
The development of humoral immunity was strongly correlated temporally with clinical response against CLL (Fig. 4A). To determine whether the kinetics of the humoral immune response against the candidate antigens correlates with the clinical response to DLI, we compared the disease burden with plasma reactivity against candidate antigens at various time points following DLI. We examined antigen-specific antibody reactivity over time by immunoblot following immunoprecipitation against DAPK3 and ZFYVE19 for patient A and against OGFOD1 and ZFYVE19 for patient B. Increasing antibody reactivity against the tested antigens correlated with decreasing tumor involvement in marrow, whereas comparable reactivity against the control EBNA1 antigen was observed for all plasma samples. For patient A, the antibody reactivity to ZFYVE19 was constant from before DLI to 4 months after DLI and then increased, peaking at 17 months and decreasing at 24 months. Antibody reactivity to DAPK3 for the same patient followed a similar pattern, increasing from its pre-DLI level from 4 months to 17 months before decreasing at 24 months. For patient B, the antibody reactivity to ZFYVE19 and OGFOD1 both increased at 6 and 9 months relative to the pre-DLI level, with samples for further follow-up not available.
The candidate antigens are broadly immunogenic in CLL patients with GvL
We examined patterns of serologic reactivity against GST fusion proteins of DAPK3, OGFOD1, and ZFYVE19 by testing 84 plasma samples collected from 72 patients with hematologic malignancies or normal volunteers (Fig. 4B). Five of 12 patients with long-term remission with minimal or no GvHD after HSCT developed new antibody reactivity after treatment against at least one of the three antigens. In addition to reactivity against DAPK3 and ZFYVE19 by patient A and OGFOD1 and ZFYVE19 by patient B, three additional patients (subjects 8–10) who underwent HSCT developed reactivity against one or more of the three antigens (Fig. 4B, first two panels, and C). Patients 8 and 9 underwent nonmyeloablative allogeneic HSCT, whereas patient 10 underwent myeloablative allogeneic HSCT. Posttreatment samples were selected at 1 year following transplant when immunosuppressive medications had been tapered and GvHD was not noted.
In contrast, we observed only occasional instances of reactivity for these three antigens among normal donors or other control groups. Only 1 of 12 normal volunteers was reactive to OGFOD1, and none of 12 to the other two antigens. Only 1 of 9 post-HSCT CLL nonresponder patients and 1 of 10 untreated CLL patients were reactive to both DAPK3 and ZFYVE19. Similarly, only 2 of 12 chemotherapy-treated CLL patients displayed reactivity against DAPK3, and 1 of 12 against ZFYVE19. No evidence of reactivity was observed among nine chronic myelogenous leukemia patients successfully treated with DLI or among eight allotransplanted CLL patients who developed GvHD, suggesting that the antigens are broadly immunogenic in patients with CLL who have developed GvL responses.
Identification of target antigens remains a priority in cancer immunology. Few CLL-associated antigens have been previously described, and of these, few have been shown to consistently generate cytotoxicity against primary CLL cells (16, 17, 19, 21, 30). To identify CLL antigens associated with the GvL effect, we have focused on naturally immunogenic antigens associated with long-lasting immunity to CLL induced by DLI. Antibody-based approaches for target antigen discovery are attractive because of their ease of use and the mounting evidence that antibody responses correlate with meaningful clinical responses to treatment. For example, Miklos and colleagues (12) reported a highly significant correlation between relapse-free survival and the presence of antibody responses against Y-encoded minor histocompatibility antigens. Other investigators have reported antigen-specific B-cell responses as reliable markers of clinical response following vaccination (31). To this end, we dissected the immune responses of two patients who achieved long-term disease remission without clinically apparent GvHD and identified 35 unique antigens that elicited significant antibody reactivity following, but not before, DLI.
Although our candidate CLL-associated antigens are predominantly intracellular proteins, they are potentially reliable markers for T-cell targeting. Our recent studies show that treatment-associated tumor antigens that elicit high-titer B-cell responses are also targets of CD8+ T-cell immunity (32). Intracellular antigen may complex with antibody for enhanced delivery of antigen through FcγR-mediated pathways to antigen-presenting cells, thus augmenting T-cell responses (33). For example, vaccination with the intracellular tumor antigen NY-ESO-1 consistently results in antigen-specific antibody production that participates in cross-priming of this antigen to T cells (34). Our findings thus provide a rich source of candidate CLL antigens, and our future studies will explore the extent to which antigen-specific antibody responses contribute to the development of coordinated T-cell responses against our most promising targets.
Our candidate antigens were identified using high-density protein microrarrays, a powerful emerging technology. This immunoproteomic screening platform enables high-throughput screening of thousands of proteins for interactions and measures the molecular biomarker end points of disease more directly than DNA microarray technology (35). High-density protein microarrays offer several distinct advantages over conventional screening platforms. All proteins spotted on protein microarrays are distinct open reading frames (ORF) expressed in a eukaryotic system that preserves potentially antigenic posttranslational modifications. Protein microarrays also offer a degree of standardization across experiments because the same proteins of comparable concentration are printed across multiple microarrays. On the other hand, they are unlikely to contain antigens that are specific to a tumor or to an individual, such as personal immunogenic tumor-restricted mutations. Protein microarrays are also not yet well established; we therefore devoted extensive efforts to developing a novel bioinformatic analysis (20) as well as a reliable method for independent validation of identified antigens. Because errors in the manufacture of protein microarrays have been described, which could lead to misidentified antigens on high-throughput screening (36), we developed an immunoprecipitation-based validation assay to sensitively detect the interaction of antibodies with properly folded protein antigens with size determination via immunoblot.
Overall, 22 of 30 (73%) tested antigen interactions were experimentally confirmed to elicit greater antibody reactivity in the post-DLI compared with pre-DLI plasma. Characterization of several of the validated target antigens led us to the insight that many of the identified targets of GvL are encoded by nonmutated B-cell lineage genes. Increasing numbers of mHAs have been described over the years, including some with selective B-cell expression (37). Screens for GvL-associated antigens following HSCT have been biased to identify mHAs because their primary criterion for selection is differential cytolytic T-cell reactivity against host tissue compared with donor cells. These studies show that alloimmunity is a fundamentally important mechanism underlying GvL. However, they neglect the potential contribution of immunity against other classes of tumor-associated antigens that have been defined in other nontransplant tumor systems, such as nonmutated differentiation, aberrantly expressed antigens (38), or even self-antigens that are immunogenic when presented by tumor but are normally sequestered from immune detection (39). Our current studies therefore reveal that classes of antigen other than mHAs (i.e., nonmutated tumor-associated antigens) comprise a component of effective GvL responses. These results are consistent with recent studies by Nishida and colleagues (40) showing that, in addition to T-cell responses against alloantigens, T-cell reactivity against CLL-specific antigens can be detected following nonmyeloablative HSCT in association with clinical response.
The discovery that the targets of effective anti-CLL immunity include nonmutated antigens that are highly expressed in CLL suggests that these antigens may have broader anti-CLL immunologic utility beyond the donor-host pairs from which they were identified. Although patients A and B developed reactivity against largely different panels of antigens, a subset of antigens elicited humoral immunity in both patients. Moreover, close to half of our cohort of 12 CLL patients with GvL responses reacted against at least one of the three candidate antigens tested. These results suggest that, as a collection, these antigens would be useful for monitoring immune responses against CLL after immune-based therapies. For example, several ongoing studies are examining the effects of vaccination with whole autologous tumor in which the specific immunogens are unknown (41, 42).
We selected for further characterization three antigens identified by both patients' sera and three antigens found to be highly expressed in CLL. Of the shared antigens, C6orf130 is a predicted ORF in chromosome 6. MDS032 (USE1) is an uncharacterized hematopoietic stem/progenitor cell protein that localizes to the endoplasmic reticulum and functions in the formation of a SNARE complex (43). ZFYVE19 is a zinc finger FYVE domain-containing protein, which has been reported as an in-frame fusion partner to the mixed-lineage leukemia gene (44). The FYVE domain mediates recruitment of proteins to membranes containing phosphatidylinositol 3-phosphate, particularly endosomes. Among the three nonshared antigens, DAPK3 (death-associated protein kinase 3) plays a role in apoptosis (45) and spermatogenesis (46). SERBP1 (SERPINE1 mRNA binding protein 1) is significantly overexpressed in ovarian epithelial cell tumors, with increased expression in advanced disease, suggesting a role in tumor invasion and metastasis (47). OGFOD1 (2-oxoglutarate and iron-dependent oxygenase domain containing 1) has been reported in tumor cell pseudopodial protrusions (48).
In closing, our identification of nonmutated and commonly immunogenic CLL antigens reveals that immune responses following effective immunotherapy are directed, at least in part, to antigens present on CLL tumor cells. As the identified candidate antigens represent immunogenic tumor-associated antigens and not alloantigens, these may be attractive targets for immunotherapy directed at CLL outside of the context of allogeneic transplantation.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
We thank Roslyn Gerwin and Beth Witten for expert technical assistance, Drs. David Hill and Mark Vidal for providing plasmids, and the DFCI transplant team and Pasquarello Tissue Bank for providing clinical samples.
Grant Support: Howard Hughes Medical Institute Medical Research Training Fellowship (O. Marina) and Department of Defense grant W81XWH-07-1-0080, Miles and Eleanor Shore Award, National Cancer Institute grant 5R21CA115043-2, and Early Career Physician-Scientist Award of the Howard Hughes Medical Institute (C.J. Wu). C.J. Wu is a Damon-Runyon Clinical Investigator supported (in part) by the Damon-Runyon Cancer Research Foundation grant CI-38-07.
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.
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).
Presented in part as a simultaneous oral session at the 49th Annual Meeting of the American Society of Hematology, December 9, 2007, Atlanta, Georgia.
- Received August 25, 2009.
- Revision received November 11, 2009.
- Accepted December 10, 2009.
- ©2010 American Association for Cancer Research.