Development of Cellular Immune Responses against PAX 5 , a Novel Target for Cancer Immunotherapy

PAX5 is a member of the PAX family of developmental transcription factors with an important role in B-cell development. Its expression in normal adult tissue is limited to the hemopoietic system, but it is aberrantly expressed in a number of solid cancers and leukemias where it functions as an oncogene. We therefore hypothesized that anti-PAX5 immune responses could be used to target a number of malignancies without significant toxicity. We screened PAX5 peptides for the ability to bind HLA-A2 and identified a novel sequence, TLPGYPPHV (referred to as TLP). CTL lines against TLP were generated from peripheral blood of five normal HLA-A2–positive blood donors and showed specific HLA-A2– restricted killing against PAX5-expressing target cells. We generated high-avidity CTL clones from these lines capable of killing cells pulsed with <1 nmol/L of TLP and killing a range of PAX5-expressing malignant cell lines. I.v. injection of an anti-PAX5 CTL clone into immunodeficient mice bearing s.c. human tumors resulted in specific growth inhibition of PAX5-expressing tumors. This knowledge can be used for the therapeutic generation of CTL lines or the cloning of highavidity T-cell receptor genes for use in adoptive immunotherapy. [Cancer Res 2008;68(19):8058–65]


Introduction
The ultimate goal of cancer immunotherapy is the development of specific, sustained, and potent antitumor immune responses with minimal toxicity.Impressive clinical and immunologic responses have followed adoptive transfer of expanded lymphocytes derived from autologous melanoma tumors (1,2).Indeed, the study of melanoma tumor infiltrating lymphocytes has been instrumental in the identification of immunogenic tumor antigens and the subsequent development of cancer vaccine therapies.However, in many cancers, naturally occurring tumor infiltrating lymphocytes have not been characterized, and identification of tumor antigens is required, especially for poor-prognosis tumors for which novel treatment approaches are needed.Tumor antigens can also be discovered using ''reverse immunology.''Here a targetable cancer-specific protein is first predicted on the basis of its expression pattern and function, and then putative antigenic peptides are assessed for evidence of immunogenicity.Ideal putative tumor antigens have the following three characteristics: high expression in the tumor, low expression in normal tissues, and a critical role in oncogenesis.This ''candidate antigen'' approach has been used successfully for a number of potential immunotherapy targets lacking evidence of natural immunogenicity.For example, PSA (3), WT1 (4), CD45 (5), and CD19 (6) were not identified through analysis of naturally occurring tumor reactive lymphocytes but have all been used as targets in models of human immunotherapy.However, none of the above-mentioned antigens have all three of the hallmarks.
We and others have recently proposed the PAX family member PAX3 as a target for immunotherapy, and we have shown evidence of the potential clinical utility of generating anti-PAX3 immune responses (7).The PAX family comprises transcription factors with important developmental roles.The PAX5 protein meets the three criteria of an ideal putative tumor antigen.In hemopoiesis, PAX5 controls the identity of B lymphocytes throughout B-cell development from the pro-B to the mature B-cell stage (8).The developmental restriction of PAX5 to B cells has been deduced from the study of targeted PAX5 germ-line deletions.Pax5 À/À mice are born alive although growth retarded, and the most striking phenotypic deficit is the absence of functional B cells (9)(10)(11).All mutants fail to produce pre-B cells, B cells, and plasma cells due to a complete arrest in B-cell development at an early precursor B stage.Apart from altered morphogenesis of the midbrain, mice were phenotypically normal, suggesting a limited spectrum of functional developmental expression (9).Moreover, PAX5 expression is thought to be absent from most adult tissues (12).It is upregulated and highly expressed in B-cell malignancies (13)(14)(15) and a number of other cancers (12,16,17) including neuroblastoma (18).Recently, in a genome-wide screen, PAX5 was shown to be the most commonly mutated gene in pediatric acute lymphoblastic leukemia (19).PAX5 has been shown to be oncogenic in vitro (20).Moreover, overexpression of full-length PAX5 as a consequence of chromosomal translocation t(9;14)(p13;q32), which juxtaposes PAX5 with regulatory elements of the immunoglobulin heavy chain gene, is described in B-cell malignancies and is likely to function by inhibiting terminal B-cell differentiation (21)(22)(23).Developmentally, PAX5 can inhibit apoptosis (24).Recently, a number of alternate isoforms of PAX5 resulting from differential splicing have been described, although the relative oncogenic and developmental importance of these different forms has not been well characterized (25).Taken together, these findings suggest that PAX5 is likely to play a critical role in tumor maintenance.The implication is that PAX5-targeted immunotherapy is therefore less likely to result in immune escape variants with silencing of PAX5 expression.
In this study, we have identified an immunogenic HLA-A2restricted epitope sequence (TLPGYPPHV) in human PAX5.We show that this epitope is a potent stimulator of specific anti-PAX5 immune responses from peripheral blood of normal donors.Highavidity T cells reactive against the epitope can be generated from normal donors.This knowledge can be used for therapeutic generation of CTL lines or cloning of high-avidity T-cell receptor genes for use in adoptive immunotherapy.

Materials and Methods
Quantitative reverse transcription-PCR and transient transfection.
Total RNA from cell lines was generated using Trizol reagent (Invitrogen), and RNA from normal human tissues was purchased from Clontech.RNA (100 ng/AL) was reverse transcribed into cDNA using the SuperScript II Reverse Transcriptase Kit (Invitrogen) according to the manufacturer's protocol.Gene-specific primers and probes for the NH 2 terminus region of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and exon 8 of PAX5 were purchased from Applied Biosystems.PAX5 expression was normalized to GAPDH, and PAX5 quantification in cell lines and normal tissues was relative to its expression in PAX5-positive RH18 cells.Relative quantitation was determined by the 2 ÀDDCT method (Applied Biosystems Users Bulletin 2).Lipofectamine 2000 (Invitrogen) was used transiently to transfect HLA-A2-positive 293-T cells with a mammalian expression vector, pcDNA3.1/Hygro(+) (Invitrogen), containing the full-length PAX5 coding sequence (1,176 bp) together with 77 bp of the 5 ¶-untranslated region (UTR) and 2,026 bp of the 3 ¶-UTR sequence (kindly provided by Andreas Himmelmann, Internal Medicine, University of Zurich, Zurich, Switzerland).
Peptides.Two independent computer-based programs, BioInformatics and Molecular Analysis (BIMAS) 5 and SYFPEITHI, 6 were used to identify potential HLA-A*0201 binding peptides within human PAX5.Peptides were synthesized by Zinsser UK and purified to >95% by reverse-phase highperformance liquid chromatography.
Antibodies and pentamer staining.The phycoerythrin (PE)-labeled pentamer Pro5 (Proimmune) was titered and used at optimal concentrations (5-10 Ag/mL) to assay the PAX5-specific CD8 + T cells.For staining, 1 Â 10 6 PBMCs or 1 Â 10 5 T-cell clones were washed and resuspended in pentamer staining buffer consisting of Dulbecco's PBS, 0.1% NaN 3 , and 0.1% bovine serum albumin (BSA).The cells were stained with 10 AL of Pro5 for 20 min at room temperature, washed, and then stained in buffer with 5 AL of antihuman CD8-FITC (BD Biosciences) for 20 min at room temperature.The cells were washed in buffer and fixed in Dulbecco's PBS, 1% paraformaldehyde and run through a Cyan flow cytometer (DAKO).7-Amino-actinomycin D (BD PharMingen) was used to exclude dead cells.The pentamer-positive cells were analyzed on a two-color plot with CD8-FITC.Results are shown as percent pentamer-positive cells for each culture.Data were analyzed with Summit (DAKO).
Peptide-binding assay.T2 cells were used to determine binding of peptides to HLA-A2.T2 cells (2 Â 10 5 per well in 96-well plates) were incubated for 18 h with 0 to 100 Amol/L of each synthetic peptide, washed twice in PBS, and stained in the dark at 4jC for 30 min with 2 Ag/mL PE-conjugated anti-HLA-A2 (BB7.2) or PE-conjugated mouse IgG2 isotype control (both BD PharMingen).Fluorescence index was calculated as the mean fluorescence intensity.
Human CTL line and clone generation.Unless indicated, all cytokines and granulocyte macrophage colony-stimulating factor (GM-CSF) were purchased from PeproTech.Fresh peripheral blood was centrifuged over Ficoll-Hypaque to obtain PBMC.Adherent cells (1.5 Â 10 6 per well in a sixwell plate) were cultured in 10% AB serum, interleukin (IL)-4 (30 ng/mL), and GM-CSF (100 ng /mL) for 7 d, with replenishment on days 3 and 5. On day 6, dendritic cells were matured with keyhole limpet hemocyanin (10 Ag/mL), CD40L (500 ng/mL; Biosource), and prostaglandin E2 (500 ng/mL; Cambridge Laboratories).CD8 T cells were stimulated twice at weekly intervals with autologous dendritic cells pulsed with peptide (10 Amol/L) and a further two stimulations with autologous CD40-activated B cells.Briefly, day 7 autologous matured dendritic cells were pulsed with 10 Amol/L peptide for 4 h, g-irradiated, and cocultured with autologous CD8 + T cells in the presence of IL-12 (20 IU/mL) and IL-7 (10 ng/mL), with a ratio of T cells/dendritic cells of 10:1.T cells had been positively selected from nonadherent PBMC using MACS beads (Miltenyi).T cells were restimulated under the same conditions for a second week.The next two further weekly stimulations used autologous peptide-loaded (10 Amol/L) CD40-activated B cells.Autologous CD40-activated B cells were generated as previously described (27).Briefly, CD40L stably transfected mouse fibroblast cells (t-CD40L À cells, from Dr. John Gordon, MRC Centre for Immune Regulation, University of Birmingham, Birmingham, United Kingdom) were g-irradiated at 100 Gy, plated at 3.5 Â 10 5 per well in six-well plates in basal Iscove's medium supplemented with 10% FCS, and incubated overnight.CD8 T-cell-depleted PBMC were added at 1 Â 10 6 /mL to 2 Â 10 6 /mL in the presence of 5.5 Â 10 À7 mol/L cyclosporin A (Sandoz Pharmaceutical) for 4 d.Fresh irradiated t-CD40L À cells were added every 4 d.The B cells generated were 75% CD19 positive.T cells were harvested after four rounds of stimulation and were further cloned by the limiting dilution method (at 0.4 and 1 cell per well) using g-irradiated allogeneic peripheral blood leukocytes at 1 Â 10 5 per well (25 Gy) and B-lymphoblastoid cell lines at 1 Â 10 3 per well (75 Gy) as feeder cells in RPMI medium, containing 250 IU IL-2 and 1 Ag/mL phytohemagglutinin.After 12 d, growing T-cell clones were tested by 51 Cr cytotoxic assay and subsequently expanded in RPMI medium.
T-cell expansion from leukemia patients.Peripheral blood samples were obtained from patients with chronic lymphocytic leukemia following local ethics committee approval.CTL from patient 1 and the HLA-A2negative patient were generated from CD19-depleted PBMC using dendritic cells from an allogeneic HLA-A2 + donor pulsed with PAX5 peptide (10 Amol/L) and stimulated for 1 wk with 10 ng/mL IL-7, following which 20 IU/mL IL-2 was added after 3 d of stimulation.CTL were then stimulated a further week with T2 cells pulsed with peptide and the same concentrations of IL-7 and IL2.The subsequent CTL from patients 2 to 4 were generated by stimulation of CD19-depleted T cells with PBMC pulsed with peptide (10 Amol/L), IL-7, and IL-2 as above for 1 wk.ELISPOT assay is conducted with 1 Â 10 4 cells per well and using T2 pulsed with PAX5 or nonrelated murine peptide as targets (10 Amol/L).
Cytotoxicity assays.Target cells were labeled with 100 ACi Na 2 51 CrO 4 in cell culture medium containing 10% FCS for 60 min at 37jC.The cells were washed twice in culture medium.When required, the targets were pulsed with specific or nonspecific control peptides (0.0001-10 Amol/L) for 1 h, washed once in culture medium, and resuspended at 50,000 cells/mL.After 4 to 18 h of incubation, 25 AL of the assay supernatant were placed into a 96-well Lumaplate (Perkin-Elmer).Scintillant (100 AL) was added and radioactivity was counted using a Microbeta scintillation counter (Perkin-Elmer).The results are expressed as percent specific lysis, calculated as (experimental release À spontaneous release / total release À spontaneous release) Â 100.For blocking assays, 51 Cr-labeled tumor cells were treated with anti-HLA-A2 or isotype control monoclonal antibody (mAb) for 1 h at 37jC before addition to effector cells.For CD8 blocking, effector cells were incubated with anti-CD8 for 30 min at 37jC before addition to 51 Cr-labeled target cells.
Western blot analysis.Cell lines were lysed in 100 AL of radioimmunoprecipitation assay buffer (Pierce) containing protease inhibitor cocktail (Pierce).Cell lysates corresponding to 30 Ag of total protein were separated on 10% SDS polyacrylamide gels and transferred onto nitrocellulose membranes (Hybond enhanced chemiluminescence; Amersham Biosciences Europe).After blockage for 1 h in 5% milk powder in PBS, the membrane was washed and probed with rabbit anti-human PAX5specific mAb (1 Ag/mL; Invitrogen) diluted in 5% dry milk in PBS.Filters were developed with horseradish peroxidase-conjugated antirabbit IgG and enhanced by chemiluminescence (Roche).
Adoptive transfer.Groups of 6-to 8-wk-old ''triple knockout'' mice (RAG À/À common g chain À/À complement C5 À/À , completely deficient in T cells; ref. 28) were derived from the Institute of Child Health.In the first experiment, 5 Â 10 6 tumor cells were injected s.c.After 3 to 4 wk, with the tumors 2 to 3 mm in diameter, 10 Â 10 6 PAX5-specific T-cell clones were injected i.v.Mice were sacrificed 24 h and 7 d following adoptive transfer and organs were collected.In the subsequent tumor growth rate assays, 10 Â 10 6 SW480 tumor cells were injected s.c. in the back of mice.After 7 d, with the tumors reaching a size of f2 mm in diameter, 10 Â 10 6 T cells or PBS were injected i.v.Thereafter every 3 to 4 d, all mice received 25 Ag of IL-2 IP until the tumor volume size reached f500 mm 3 .For the analysis of tumor infiltrating cells, tissues were harvested 48 h after adoptive transfer, crushed and passed through a cell strainer, washed with PBS, resuspended in 0.1% BSA/PBS buffer, and incubated with antihuman CD8-FITC or isotype control antibodies before analysis by flow cytometry.Alternatively, tissues were fixed in formalin and analyzed by immunohistochemistry for the presence of infiltrating lymphocytes.Mice were treated in accordance with U.K. Home Office-approved protocols.

Results
High PAX5 expression is limited to hemopoietic cells and cancer.Previous workers have shown that PAX5 is expressed in early B-cell lineages during hemopoiesis (15).Expression in other adult tissues has not been well characterized.We measured PAX5 expression in a panel of adult tissues and cancer cell lines to confirm its limited tissue expression in normal tissues and its relative up-regulation in malignancy.We used quantitative reverse transcription-PCR (RT-PCR) using primers amplifying exon 8, which is thought to be present in most, but not all, splice variants (25).We also chose exon 8 because it encodes the epitope TLPGYPPHV, which was subsequently chosen for targeted immune response generation (see below).As expected, significant expression was seen in B cells and in spleen, presumably reflecting the presence of B cells here.In contrast, in 7 of 13 cancer cell lines tested, the expression at the RNA level was equal with or significantly greater than that seen in spleen, reflecting deregulation of PAX5 in these cells (Table 1).In the remaining nonhemopoietic normal human tissues, low-level expression was observed (range of 0.02-6.9% of the level seen in the relatively low expressing RH18 rhabdomyosarcoma cell line).We performed Western blot analysis of the cell lines to confirm that high-level expression, as shown by quantitative RT-PCR, corresponded with high protein expression (Supplementary Fig. S1).We concluded that there is a range of deregulated expression in human malignancy, including non-B-cell cancers, and that specific immune responses directed against PAX5 would not be predicted to cause autoimmune toxicity apart from B lineage hemopoietic cells.
PAX5-reactive T-cell lines can be generated from the autologous repertoire.Using two computational algorithms (SYTHPETHI and BIMAS), we identified three sequences from human PAX5 predicted to bind HLA-A2 strongly (Table 2).However, on testing the affinity of these peptides in T2 binding assays, only the TLPGYPPHV peptide (hereafter referred to as TLP) showed significant stabilization of HLA-A2 on T2 cells (Fig. 1A).The TLP epitope is derived entirely from exon 8 of PAX5.Immune responses directed against TLP would therefore be predicted not to target normal body tissues (Table 1), with the exception of B cells, but to be cytotoxic against a range of PAX5 exon 8-expressing cancer types.
We next investigated whether the T-cell repertoire of normal blood donors contains CTL with avidity for the HLA-A2/TLP complex, by generating T-cell lines from buffy coat preparations from five normal blood donors.We generated CTL lines using CD8 positively selected cells, CD3 positively selected cells, or unselected PBMC as the starting cell population and used a standardized T-cell expansion protocol.This involved four stimulations, at weekly intervals, with peptide-pulsed antigen-presenting cells as described previously (7).In the first two stimulations TLP peptide is pulsed onto autologous dendritic cells, whereas in the third and fourth stimulations TLP-pulsed autologous B cells were used as antigen-presenting cells.The killing activity of all five CTL lines against TLP-pulsed T2 cells ranged from 10% to 80% at E/T ratio of 30:1 (Table 3).We assessed the frequency of TLP-reactive T cells in these CTL lines and in PBMC of HLA-A2-positive blood donors by staining with a PE-labeled pentamer comprising TLP and the HLA-A2 complex.The TLP-reactive T cells were undetectable in PBMC from normal donors, whereas 2% to 5% of cells in the best T-cell line (donor BC-24) were stained with the pentamer (Fig. 1B).To confirm that the donor BC24-derived CTL line recognizes endogenously processed PAX5, we transiently transfected 293-T cells (HLA-A2 positive, PAX5 negative) with a cDNA construct expressing full-length PAX5 (all exons included) and used the paired transfected and untransfected cell populations as targets in a standard chromium release assay.Specific killing was seen only against TLP-pulsed T2 cells or the PAX5-transfected cells (Supplementary Fig. S2), confirming that the TLP epitope can be intracellularly processed from the PAX5 polypeptide and presented on the cell surface in HLA-A2-positive human cells.
CTL clones directed against PAX5 can specifically lyse PAX5expressing cancer cells.To assess specific cytotoxicity against human PAX5-expressing cancer cells, we generated CTL clones from one of the TLP CTL lines ( from donor BC24) by limiting dilution.We initially screened for killing following addition of T2 cells pulsed with TLP peptide at concentrations of 0, 0.01, and 10 Amol/L by chromium release assay.Five high-avidity clones  from f300 screened were found to exhibit specific killing at 0.01 Amol/L and no killing at 0 Amol/L, and three were further characterized to determine the avidity of the interaction with the TLP:A2 complex.The clone with the highest avidity (clone 33) specifically lysed T2 cells pulsed with as little as 100 pmol/L peptide (Fig. 2A).We confirmed that these cells had the surface phenotype of CTL by staining with CD8 and an MHC pentamer specific for HLA-A2 and TLP (Fig. 2B) and by showing positive staining with pan a/h T-cell receptor antibody (data not shown).
Clone 33 also caused lysis of 293 cells transfected with PAX5, but not vector-transfected controls, confirming the specificity of its killing activity (Fig. 2C).Therefore, anti-PAX5 clone-33 was selected to determine the spectrum of the cytolytic activity of anti-PAX5 CTL.The highest specific killing in standard chromium release assays was seen against the HLA-A2-high and PAX5-positive cancer cell lines NALM6, RH18, and SW480 (Fig. 2D).Importantly, Raji (PAX5 positive, HLA-A2 negative), MEL5 (PAX5 negative, HLA-A2 low), and MDA-MB-231 or 293-T (PAX5 negative, HLA-A2 positive) were not killed by the clone, confirming its specificity (Fig. 2D; Table 4).LAN-1 and SH-SY5Y cells were only killed significantly after the addition of IFNg, which up-regulated class 1 MHC (Supplementary Fig. S3A).Cytotoxic specificity was further confirmed by showing that clone 33 killing of SW480 cells was inhibited by anti-CD8 and anti-MHC class 1 antibodies (Supplementary Fig. S3B).
Anti-PAX5 CTL activity against primary cells and primary tumors.To determine whether the PAX5-reactive CTL were also capable of specific recognition and lysis of primary human cancer cells, we made use of two primary pre-B leukemia samples (G7 and PL1), which were available to us and known to be HLA-A2 positive (data not shown) and PAX5 expressing (Table 1).PL1 cells had been engrafted in a nonobese diabetic-severe combined immunodeficient mouse model from a primary pre-B-cell leukemia sample (29), whereas G7 cells were a short-term in vitro culture from a patient with pre-B-cell acute lymphoblastic leukemia.Both of the primary patient leukemia samples expressed PAX5 exon 8 RNA (Table 1), and both were lysed in vitro by anti-PAX5 clone 33, with killing blocked by an anti-HLA-A2 mAb (Fig. 3A).To test the cytotoxicity against PAX5-expressing primary B cells, we positively selected CD19 cells from an HLA-A2-positive donor and tested them before and after 25 days of in vitro stimulation with CD40 ligand.Freshly isolated CD19-positive cells from the peripheral blood of HLA-A2-positive blood donors had higher PAX5 expression than CD40 ligand cultured B cells (Table 1).In accordance with this differential PAX5 expression, whereas killing of freshly isolated B cells was observed at a similar level to the primary leukemia samples, only a relatively low level killing of activated B cells was observed (Fig. 3B).
We next determined whether there was evidence of anti-PAX5 CTL activity in patients with PAX5-expressing leukemia.To this end, PBMC from HLA-A2-positive patients with chronic lymphocytic leukemia were stimulated with TLP peptide for 2 weeks and tested for specific IFNg secretion following coculture with TLPpulsed T2 cells.In four of four patients, there was an increase of IFNg secretion following addition of TLP peptide to T2 targets, and this was reversed following addition of an HLA-A2 blocking antibody.In an HLA-A2-negative chronic lymphocytic leukemia (CLL) patient, however, there was no evidence of increased IFNg secretion (Fig. 3C).Therefore, there is evidence of PAX5-specific T cells in the blood of HLA-A2-positive patients with PAX5expressing leukemia.
Anti-PAX5 CTL infiltrate PAX5-expressing tumor in vivo to inhibit tumor growth.To investigate whether the anti-PAX5 T cells were capable of infiltrating into PAX5-expressing tumors, we adoptively transferred 10 Â 10 6 clone 33 anti-PAX5 T cells into the tail vein of immunodeficient triple knockout (RAG À/À common g chain À/À complement C5 À/À ) mice bearing small palpable s.c.tumors of the PAX5-positive, HLA-A2-positive SW480 cell line.Mice were sacrificed at 48 hours or 7 days following T-cell infusion, and internal organs and tumor were analyzed for the presence of T cells by staining for CD8.At both time points, a detectable T-cell population was seen only in the tumor and, to a lesser extent, the Figure 3. Assessment of anti-PAX5 CTL in primary PAX5-expressing leukemia samples and freshly isolated CD19-positive B cells.A, killing of G7 and PL1 primary leukemia samples by clone 33 CTL is inhibited by prior incubation of targets with an HLA-A2 blocking mAb.B, killing of primary B cells by clone 33 anti-PAX5 CTL in a standard chromium release assay.As controls for CTL killing, T2 cells were pulsed with PAX5 peptide TLP or unrelated control peptide (P4).BC24 and BC27 refer to two buffy coats from two different HLA-A2-positive donors.C, short-term stimulation of T cells from peripheral blood of four HLA-A2 + CLL patients (left ) and one HLA-A2 À patient (right ) with antigen-presenting cells pulsed with PAX5 peptide (TLP) resulted in specific IFNg release following incubation with T2 cells pulsed with the same peptide, but no specific release following coculture with T2 cells pulsed with an irrelevant mouse peptide.IFNg release was inhibited by prior incubation of targets with an HLA-A2 blocking mAb.Columns, mean of triplicates; bars, SE. draining lymph nodes, but no significant numbers of T cells were seen in spleen, lung, liver, or kidneys (Fig. 4A).This is consistent with the T cells accumulating at the site of PAX5 overexpression.
To investigate whether T-cell infiltration could inhibit tumor growth, further adoptive transfer experiments were performed in which SW480 tumor-bearing mice were treated with clone 33 T cells and given IL-2 i.p. every 3rd day to promote T-cell survival.Control mice were given nonspecific activated T cells and IL-2, or IL-2 alone.Remarkably, whereas IL-2 alone and nonspecific T cells had no significant effect on tumor growth, clone 33-treated mice developed only small tumors up to 25 days after tumor challenge (Fig. 4B).At 48 hours following adoptive transfer, an infiltrate of lymphocytes surrounded the outside of the tumor in both clone 33-and control CD8 cell-treated mice, but this was not apparent in the untreated tumors; fluorescenceactivated cell sorting staining indicated a larger number of infiltrating T cells in clone 33-versus control CD8-treated mice (Fig. 4C).Therefore, T cells were attracted toward SW480 tumors, but only PAX5 targeted T cells were capable of preventing tumor outgrowth.After 25 days, inhibition of tumor growth was not associated with any histologic evidence of organ damage in clone 33-treated mice as shown by normal morphologic appearances of liver, spleen, and kidneys and normal peripheral blood appearance compared with untreated controls (data not shown).Importantly, in parallel adoptive transfer experiments, clone 33 had no effect on the in vivo growth of the PAX5-negative HLA-A2-positive tumor cell line MDA-MB-231 compared with IL-2 alone-treated mice (data not shown).

Discussion
PAX5 is herein identified as a potential target for cancer immunotherapy by virtue of its limited normal developmental expression but high-level expression in a range of adult and pediatric cancers.Central to the argument for identification of a clinically useful new tumor antigen is the demonstration of a marked differential expression between neoplastic and normal tissues.The low expression levels in normal tissues we have shown are generally supportive of potential clinical use.No tissues have the high degree of expression seen in B-cell malignancies apart from hemopoietic tissues (normal B cells, spleen, and bone marrow) and testis (an immune privileged site).We hypothesize, but have not formally proved, that the high expression in bone marrow and spleen reflects B hemopoiesis.
It would therefore be important to investigate potential hematologic toxicity before clinical use.Previous workers have shown that PAX5 is critical for B-cell specification and for maintenance of B-cell phenotype, such that loss of PAX5 is tumorigenic for B-cell leukemia (15,30).We have not been able to evaluate the toxicity of PAX5 immunotherapy in a murine model

Cancer Research
Cancer Res 2008; 68: (19).October 1, 2008 with targeting of the human TLP epitope because its antigenicity is restricted to human HLA-A2.The clinical consequence of PAX5targeted therapy resulting in destruction of normal B cells might be the requirement for long-term immunoglobulin replacement.Another concern about PAX5 immunotherapy would be the emergence of PAX5 antigen loss variants among B cells predisposing to B-cell malignancy.These issues will require formal testing in appropriate animal models (e.g., humanized HLA-A2 transgenic mice).Given the expression observed in normal bone marrow, it is important to confirm that PAX5 is not seen in normal hematopoietic stem cells.However, Pax5 À/À mice have normal hemopoiesis apart from B cells, strongly suggesting that PAX5 has little significant expression outside of B-cell hemopoiesis.
Our in vivo data show that anti-PAX5 CTL accumulate in the tumor site of PAX5/HLA-A2-positive tumor cells.Moreover, accumulation is associated with a significant slowing of tumor growth despite the lack of T-cell help.It is interesting to speculate that in the immunocompetent setting, it would be possible to boost anti-PAX5 immune responses using peptide vaccinations to achieve long-term disease control.Further work is also required to show the therapeutic potential in a larger number of cancer types.
The ability to generate effective anti-PAX5 CTL for all of the normal blood donors tested was slightly surprising and maybe suggests a lack of tolerance to PAX5, confirmed by the presence of anti-PAX5 T precursors in four of four patients with CLL.However, the precursor frequency of anti-TLP T cells in the peripheral blood of normal HLA-A2-positive donors was below the level of detection by pentamer staining.Future studies to prospectively to collect samples and test for precursor frequency at diagnosis will be of interest.
Anti-PAX5-directed T-cell therapy has potential clinical application in a range of adult and pediatric malignancies.Especially attractive is the prospect of generation of vectors for gene therapy encoding high-affinity T-cell receptors directed against PAX5, with a view to transducing the patient's autologous repertoire and adoptively transferring the redirected cells back into the patient.The clone with the highest affinity described in this study (specific killing observed at as little as 100 pmol/L pulsing of T2 cells) might be of sufficiently high affinity for clinical benefit in this setting, but further in vivo evaluation is required.

Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.

Figure 1 .
Figure 1.Generation of anti-PAX5 CTL from the autologous repertoire of normal HLA-A2-positive blood donors.A, HLA-A2 binding of three PAX5 peptides was measured using TAP-deficient T2 cells and conformation-dependent HLA-A2-specific antibodies.Binding efficiency of PAX5 peptides was compared with that of the known HLA-A2-binding peptide flu matrix.A PAX3 mouse peptide was a negative control.Columns, mean of triplicates; bars, SE.B, representative PAX5 peptide pentamer staining of donor BC24 PBMC, or CTL line (after four stimulations).The dot plots show staining with PE-labeled HLA-A*0201/PAX5-2 (TLPGYPPHV) pentamer.The percentages of pentamer-reactive T cells are given in the top right quadrants.

Figure 2 .
Figure 2. Killing activity of anti-PAX5 CTL clones.A, isolation of high-avidity CTL clones with specific killing of PAX5-expressing target cells.A, killing of T2 cells pulsed with TLP peptide by T-cell clones isolated from donor BC24 bulk CTL line.B, TLP/A2-specific MHC pentamer staining of CTL clone 33.C, standard 51 Cr assay to evaluate CTL killing by clone 33 of 293-T cells transfected with either vector or full-length PAX5 cDNA.D, PAX5 CTL clone 33 killing activity of different tumor cell lines by a standard 51 Cr assay.SW480, RH18, and NALM6 are PAX5 + , HLA-A2 + ; 293-T and MEL5 are PAX5 À , HLA-A2 + cell lines; and Raji is PAX5 + and HLA-A2 À .Bars, SE of triplicates.

Figure 4 .
Figure 4. Anti-PAX5 CTL infiltrate PAX5-expressing tumors and inhibit tumor growth in vivo .Immunodeficient mice with small palpable tumors received an i.v.injection of 10 Â 10 6 PAX5-specific CTL clone 33 or sorted activated CD8 + T cells from an HLA-A2 + donor, or PBS only.Each mouse then received 20-Ag IL-2 until sacrifice.Each group had four to five mice.A, flow cytometric analysis of CD8 + T cells in different organs and tumors following anti-PAX5 CTL clone 33 injection.B, tumor growth rate as measured by callipers.C, middle and left, representative H&E stains showing representative lymphocytic infiltration (arrows ) in the periphery of tumors treated with clone 33 or sorted CD8 cells; magnification, Â200.Right, flow cytometry of total disaggregating tumor following staining with antihuman CD8 antibody.Columns, mean of triplicates; bars, SE.

Table 2 .
Predicted affinities of selected PAX5 peptide epitopes for HLA-A2 as determined by SYFPEITHI and BIMAS algorithms

Table 3 .
CTL NOTE:51Cr release assay assessed killing against T2 pulsed with PAX5 or nonrelated peptide, and percentage specific killing is derived by subtracting killing of T2 pulsed with unrelated A2-binding peptide.Abbreviation: BC, buffy coat.

Table 4 .
Killing in cytotoxicity assays of cancer cell lines by clone 33 anti-PAX5 CTL and correlation with PAX5 and HLA-A2 expression