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Clinical Cancer Research Vol. 10, 1047-1056, February 2004
© 2004 American Association for Cancer Research

Molecular Oncology, Markers, Clinical Correlates

Autoreactive, Cytotoxic T Lymphocytes Specific for Peptides Derived from Normal B-Cell Differentiation Antigens in Healthy Individuals and Patients with B-Cell Malignancies

Matthias Grube1, Katayoun Rezvani1, Adrian Wiestner2, Hiroshi Fujiwara1, Giuseppe Sconocchia1, Jan J. Melenhorst1, Nancy Hensel1, Gerald E. Marti3, Larry W. Kwak2, Wyndham Wilson2 and John A. Barrett1

1 National Heart Lung Blood Institute, 2 National Cancer Institute, NIH, and 3 Food and Drug Administration, Bethesda, Maryland


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Purpose: To investigate potential immunotherapeutic strategies in B lymphocytic malignancies we looked for CTLs recognizing CD19 and CD20 epitopes.

Experimental Design: Three CD19 and CD20 peptides binding to HLA-A*0201 were identified and used to detect peptide specific CTLs by a quantitative real-time PCR to measure IFN-{gamma} mRNA expression in 23 healthy individuals and 28 patients (18 chronic lymphocytic leukemia (CLL), 7 follicular lymphoma, 2 acute lymphocytic leukemia, and 1 large cell lymphoma). Peptide-specific CTLs were expanded in culture with CD40-activated B cells to test lytic activity in three patients.

Results: In healthy individuals, CD8+ T-cell responses were detected in one to CD1974–82, in three to CD20127–135, and three to CD20188–196. Seven of 27 patients (6 with CLL) had CD8+ T cells recognizing CD1974–82. Seven patients responded to CD20127–135 and three to CD20188–196. All were CLL patients. CD1974–82-specific CTLs from three patients were expanded over 4 weeks. These cells were HLA-A*0201 specific and lytic for peptide-loaded antigen-presenting cells but not to malignant or unpulsed B cells.

Conclusions: CTLs that recognize CD19 and CD20 epitopes exist in healthy individuals and may be increased in CLL patients. They are of low avidity and require high doses of peptide for activation. Strategies to increase T-cell avidity would be necessary for T-cell immunotherapeutic approaches using the peptides studied.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
CD8+ CTLs specifically recognize peptides derived from endogenous proteins presented by class I HLA molecules on the surface of malignant cells. T-cell-mediated immune reactions against autologous tumor cells have been described in a variety of malignant diseases (1, 2, 3, 4) . Several recent studies have identified tumor-reactive CD4+ and CD8+ T-lymphocytes in non-Hodgkin lymphoma and chronic lymphocytic leukemia (CLL; Refs. 5, 6, 7, 8 ). Furthermore, immunoglobulin idiotype vaccination in myeloma and non-Hodgkin lymphoma can induce immune responses to malignant B cells (9, 10, 11, 12) . However normal tissue-specific self-antigens, which are candidates for immunotherapy in solid tumors (13) , have not been widely explored in B-cell malignancies (14 , 15) . T cells reactive to self-antigens are mainly eliminated by negative thymic selection (16, 17, 18, 19) . However, the process is incomplete, leaving behind low-avidity T cells autoreactive to self-antigens (20 , 21) . The therapeutic potential of monoclonal antibodies targeting the B-cell differentiation epitopes CD19 and CD20 makes these molecules of particular interest to study as potential T-cell epitopes (22, 23, 24, 25, 26) . T cells, genetically modified to recognize CD19 or CD20 molecules through antibody ligands can efficiently eliminate their respective B-cell target (27, 28, 29, 30) . Peptides derived from CD20 have been used to generate CTLs to treat B-cell malignancies in mice (31) . CD19 and CD20 play an important role in the development, differentiation, and activation of B cells. CD19 is a signal-transduction molecule and CD20 is part of a multimeric receptor complex regulating cell cycle progression and B-cell differentiation. Both are expressed by B-progenitor cells, persist during all stages of B-cell maturation, and are lost on terminal differentiation to plasma cells (32, 33, 34) . CD19 and CD20 are largely restricted to normal and neoplastic B cells (although CD19 can be also found on dendritic cells). The majority of B-lineage malignancies express CD19 and CD20 (35 , 36) . These attributes make these molecules good candidates for T-cell immunotherapeutic research.

In this study we set out to determine whether cytotoxic CD8+ T-cell responses specific for peptides derived from CD19 and CD20 antigens occur in healthy individuals and patients with B-cell malignancies. We then used a sensitive quantitative real-time (RT) PCR (qPCR) technique to search for the anticipated low frequencies of circulating T cells recognizing peptides derived from these molecules. Our results show that low-avidity T cells recognizing both CD19 and CD20 circulate in normal individuals and in patients with B-cell malignancies with a notable increase in CD19-specific T cells in patients with CLL.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients and Donors.
After informed consent, cells from HLA-A2-positive patients with B-cell malignancies [18 CLL, 7 follicular lymphoma, 2 acute lymphocytic leukemia, 1 large cell lymphoma (non-Hodgkin lymphoma)], which were enrolled in clinical trials approved by the institutional review board and healthy individuals (n = 23) were obtained from leucopheresis products. Peripheral blood mononuclear cells were separated using Ficoll-Hypaque gradient-density (Organon Teknika Co., Durham, NC) and were subsequently frozen in RPMI 1640 complete medium (CM; 25 mM HEPES buffer, 2 mM L-glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin; Life Technologies, Inc., Gaithersburg, MD) containing 20% heat-inactivated FCS and 10% DMSO according to the standard protocols. Before use, cells were thawed, washed, and resuspended in CM supplemented with 10% human AB serum (HS) and rested overnight.

HLA-Typing.
High-resolution HLA-class I genotyping was performed by sequence-specific PCR using genomic DNA (HLA-Laboratory, Department of Transfusion Medicine, Warren G. Magnusson Center, NIH, Bethesda, MD).

Cell Lines.
T2 cells (American Type Culture Collection, Manassas, VA) are a HLA-A*0201-positive hybrid human cell line. They expresses very low levels of cell surface HLA-A 2.1 and are unable to present endogenous antigens because of the lack of most of the MHC class II region including the known TAP (transporter proteins for antigenic peptide) and proteasome genes (37) . C1R-A2 cells are a MHC-class I-defective LCL cell line, that expresses a transfected genomic clone of HLA-A2.1 (38 , 39) and were kindly provided by the laboratory of Dr. Jeffrey Schlom (National Cancer Institute, NIH, Bethesda, MD). The cells were maintained in CM supplemented with 10% FCS.

Peptide Predictions.
The protein sequence from CD19 was reviewed for 9-mer peptides that could potentially bind to MHC class-I molecules using the computer-based prediction analysis of H. G. Rammensee, (University of Tübingen, Tübingen, Germany; Ref. 40 ).4 In this analysis, peptides are ranked according to a score that takes the presence of primary and secondary MHC-binding anchor residues into account. The analysis was performed for MHC-class-I allele HLA*0201 because of the prevalence of this allele in the Caucasian population.

Peptide Synthesis.
All of the peptides used in this study were synthesized by Biosynthesis (Lewisville, TX) to a minimum of 95% purity as measured by high-performance liquid chromatography. The following peptides were used: CD1974–82 (HMRPLASWL), CD20127–135 (AISGMILSI), CD20188–196 (SLFLGILSV), Cytomegalovirus (CMV) peptide495–503 (NLVPMVATV) derived from the immunodominant pp65 protein and the synthetically modified gp100 peptide 209–217(2M; IMDQVPFSV) as a negative control. Peptides were dissolved in DMSO at a concentration of 5 mg/ml, further diluted in PBS, and stored at -20°C.

MHC Stabilization Assay.
Peptides were tested for their ability to bind to HLA-A2 molecules in a MHC-class I stabilization assay using the T2 cell line as described previously (41 , 42) . T2 cells were pulsed with 100 µM peptide and 5 µg/ml ß2 microglobulin (Sigma, St. Louis, MO) for 18 h at 26°C. CMV (pp65) and gp100 peptides were used as positive controls and the background expression of HLA-A2 was determined using DMSO as a negative control. The level of stabilized HLA-A2 on the surface of T2 was determined by using FITC-conjugated mouse antihuman monoclonal antibody (BB7.2; BD Biosciences PharMingen, San Diego, CA). The fluorescence index (FI) determined by fluorescence-activated cell sorting analysis for each peptide was calculated by the following formula: FI = (mean channel of fluorescence T2 + peptide)/(mean channel of fluorescence T2 without peptide). Peptides were considered to stabilize HLA-A2 molecules with high affinity, if the FI was >=1.5 and low affinity if the FI was between 1.1 and 1.49 at a peptide concentration of 100 µM. All of the assays were repeated a minimum of three times, and the results are given as means of replicate experiments.

CD8+ T-Cell Selection.
CD8+ T cells were isolated from the peripheral blood mononuclear cells of patients and healthy individuals by using a CD8-positive isolation kit (Dynal, Oslo, Norway). After detachment of the immunomagnetic beads by using DetachaBead (Dynal) a purity of at last 98% CD8+ T cells was determined by flow cytometry.

CD8+ T Cell in Vitro Stimulation.
To determine peptide-specific CD8+ T-cell reactivity, we measured the IFN-{gamma} mRNA gene expression by CD8+ T cells stimulated with candidate peptides. Multiple experiments to optimize assay conditions were performed with CD8+ T cells obtained from HLA-A*0201-positive, CMV-seropositive donors, stimulated with HLA-A*0201-associated CMV peptide (pp65)-pulsed C1R-A2 cells. After purification, 1 x 106 CD8+ T cells were plated in a 96-well flat-bottomed plate in 200 µl of CM supplemented with 10% HS and incubated overnight at 37°C, 5% CO2, to minimize background expression of IFN-{gamma} mRNA expression due to lymphocyte manipulation. CD8+ T cells were then stimulated in vitro with test peptides using an adapted protocol from previous studies (43) . Briefly C1R-A2 cells were washed three times in serum-free CM and incubated with test peptide at 50 µg/ml in CM at 37°C, 5% CO2, for 2 h. The peptide-loaded cells were then irradiated with 7500 cGy, washed once, suspended in CM containing HS, and added to the isolated CD8+ T cell at a 1:1 ratio. As controls, CD8+ T cells were either incubated with unloaded C1R-A2 cells (negative control), or with C1R-A2 cells and 5 µg/ml staphylococcus enterotoxin B (Sigma-Aldrich, St. Louis, MO; positive control). After 3 h of coincubation at 37°C, 5% CO2, cells were harvested for RNA-isolation and cDNA synthesis. Additional negative controls included HLA-A*0201-negative individuals, CMV seronegative HLA-A*0201-positive individuals and C1R-A2 cells pulsed with gp100 (209–2M) as irrelevant peptide.

RNA Extraction and cDNA Synthesis.
Total RNA was isolated from test samples using the RNeasy Mini kit (Quiagen, Valencia, CA) and stored at -80°C. For cDNA synthesis, 1 µg of total RNA was reverse transcribed into DNA with Advantage RT-for-PCR Kit (BD Biosciences, Clontech, Palo Alto, CA) and stored at -20°C.

qPCR.
Measurement of IFN-{gamma} mRNA gene expression was performed using an ABI Prism 7900 Sequence Detection system (Perkin-Elmer, Foster City, CA) as described previously (44 , 45) . The feasibility of this approach for the analysis of antigen-specific T-cell responses, both in peripheral blood lymphocytes and in tumor tissues, has been validated previously (46) . Primers for IFN-{gamma}, CD8, and Taqman Probes (Custom Oligonucleotide Factory, Foster City, CA) were designed to span exon-exon junctions to prevent transcription of genomic DNA. To create a standard curve, the cDNA was generated by reverse transcription using the same technique used for the preparation of test cDNA. IFN-{gamma} and CD8 cDNA was amplified by PCR using the same primers designed for the RT-PCR, purified and quantified by UV spectrophotometry. The number of cDNA copies was calculated using the molecular weight of each gene amplicon. Serial dilutions of the amplified gene at known concentrations were tested by RT-PCR. qPCRs of cDNA specimens, cDNA standards, and water as negative control were conducted in a total volume of 25 µl with Taqman Master mix (Perkin-Elmer), 400 nM primers, and 200 nM probe. Primer sequences were as follows: IFN-{gamma} (forward), 5'-AGCTCTGCATCGTTTTGGGTT; IFN-{gamma} (reverse), 5'-GTTCCATTATCCGCTACATCTGAA; IFN-{gamma} (probe), FAM-TCTTGGCTGTTACTGCCAGGACCCA-TAMRA; CD8 (forward), 5'-CCCTGAGCAACTCCATCATGT; CD8 (reverse), 5'-GTGGGCTTCGCTGGCA; and CD8 (probe), FAM-TCAGCCACTTCGTGCCGGTCTTC-TAMRA. The thermal cycler parameters were 2 min at 50°C, 10 min at 95°C, and 40 cycles each of 95°C for 15 s and 60°C for 1 min. Standard curve extrapolation of copy number was performed for both IFN-{gamma} and CD8. The calculated number of copies of IFN-{gamma} mRNA in each sample was normalized to the number of copies of CD8 mRNA by dividing the number of copies of IFN-{gamma} transcripts by the number of copies of CD8 transcripts. All of the PCR assays were performed in duplicates and reported as the mean. A 2-fold difference in gene expression was found to be within the discrimination ability of the assay.

Generation of CD40-Activated B Cells.
CD40 activated B cells (CD40-B-cells) were generated from CD8-depleted peripheral blood mononuclear cells, as described previously (47) . In brief, human CD40L-transfected murine fibroblasts (LTK-CD40L) were lethally irradiated (7500 cGy), were subsequently plated on 6-well plates (BD Biosciences, Franklin Lakes, NJ) in CM supplemented with 10% FCS, and were cultured overnight at 37°C, 5% CO2. After washing the plates twice with PBS, we cocultured the peripheral blood mononuclear cells with fibroblasts at 2 x 106 cells/ml in the presence of interleukin (IL)-4 (4 ng/ml; Peprotech, Rocky Hill, NJ) and Cyclosporin A 0.7 µg/ml in Iscove‘s modified Dulbecco’s medium (Invitrogen), supplemented with 10% HS, 50 µg/ml transferrin (Boehringer Mannheim, Indianapolis, IN), 5 µg/ml insulin (Sigma Chemical Co., St. Louis, MO) and penicillin/streptomycin at 37°C, 5% CO2. Cultured cells were transferred to new plates with freshly prepared, irradiated fibroblasts every 3–5 days.

Before use, CD40-B cells were Ficoll-density centrifugated followed by washing twice with PBS to remove nonviable cells including remaining fibroblasts.

Expansion of Peptide-Specific CD8+ T Cells Using Peptide-Pulsed CD40-B-Cells.
Purified (>98%) CD8+ T cells (CD8+ T cell Isolation Kit; Miltenyi, Auburn, CA) from patients were stimulated with autologous, irradiated (7500 cGy), peptide-pulsed CD40-B-cells. Briefly, CD40-B-cells were washed three times in serum-free CM and were incubated for 2 h with test peptide at 50 µg/ml in the presence of 5 µg/ml human ß2 microglobulin (Sigma, St. Louis, MO) in CM at 37°C in 5% CO2. CD40-B-cells were then irradiated (7500 cGy), washed once, and added to the isolated CD8+ T cell at a ratio of T:CD40-B of 4:1 in CM containing 10% HS and recombinant human IL-7 (10 ng/ml; Peprotech). After 7 days in culture, a second stimulation was performed, and the following day, 20 IU/ml recombinant human IL-2 (Biosource International, Camarillo, CA) was added. After 14 days in culture, a third stimulation was performed, followed on day 15 by the addition of recombinant human IL-2. After a total of four stimulations, the peptide-stimulated T cells were obtained and tested for peptide-specific cytotoxicity.

Flow Cytometric Analysis.
CD8+ T cells, obtained after primary isolation, were stimulated with peptide-loaded antigen-presenting cells (APCs) and were stained for intracellular IFN-{gamma} production. Briefly, C1R-A2 cells were washed three times in serum-free CM and were incubated with test peptide at 50 µg/ml for 2 h in CM at 37°C and 5% CO2. C1R-A2-cells were then washed once, irradiated (7500 cGy), and suspended with 1 x 106 CD8+ T cells at a 1:1 ratio in CM containing 10% HS and 10 µg/ml Brefeldin A (Sigma). Cells were coincubated for 16 h at 37°C and 5% CO2. Unpulsed C1R-A2 cells and C1R-A2 cells pulsed with gp100 peptide (irrelevant peptide) were used as negative controls. Positive controls were performed by stimulating CD8+ T cells with 5 µg/ml staphylococcus enterotoxin B (Sigma). CD8+ T cells were then stained by incubation with phycoerythrin-conjugated monoclonal antibodies (Becton Dickinson, San Jose, CA). The intracellular staining for IFN-{gamma} was performed after adding FACS Lysing Solution and FACS Permeabilization Solution (Becton Dickinson) by using FITC-conjugated monoclonal antibodies (Becton Dickinson). Data acquisition was performed on FACSCalibur and was analyzed using CellQuest Software (Becton Dickinson).

Cytotoxicity Assay.
To determine peptide specific lysis, we used a semi-automated mini-cytotoxicity assay, as described previously (43 , 48) . Effector cells were diluted to different concentrations and plated in 40-µl, 60-well Terasaki trays (Robbins Scientific, Sunnyvale, CA) with six replicates per dilution. Target cells at a concentration of 2 x 106 cells/ml were stained with 10 µg/ml Calcein-AM (CAM; Molecular Probes Inc., Eugene, OR) for 60 min at 37°C. After washing three times in CM, cells were resuspended at 105 cells/ml in CM supplemented with 10% HS. Target cells (103) in 10 µl of medium were added to each well containing effector cells. Wells with target cells alone and medium alone were used for maximum and minimum fluorescence emission, respectively. After 4-h incubation at 37°C in 5% CO2, 5 µl of FluoroQuench (EB, Stain-Quench Reagent; One Lambda Inc., Canoga Park, CA) was added to each well and the trays were centrifuged for 1 min at 60 x g before measurement of fluorescence using an automated Lambda Fluoroscan (One Lambda Inc.). A decrease in the fluorescence emission is proportional to the degree of target cell lysis once the released dye is quenched by the hemoglobulin contained in the FluoroQuench reagent. The percentage of lysis was calculated as follows.

MHC restriction of lytic activity was tested by blockade of HLA-A2 using monoclonal antibody BB7.2 and the corresponding isotype control (Becton Dickinson).

Statistical Analysis.
To determine specific response to stimulation, mRNA for IFN-{gamma} from CD8+ T cells stimulated with test-peptide versus unpulsed APC (background) was detected by qPCR. The IFN-{gamma} mRNA copy number was first corrected for CD8 mRNA. A cutoff value of 2.0 for the ratio of IFN-{gamma} mRNA obtained from CD8+ T cells stimulated with relevant test peptides to that obtained from CD8+ T cells stimulated with unpulsed APC was considered to be evidence of epitope specificity. The cutoff value was derived by analyzing IFN-{gamma} mRNA transcripts detectable in CD8+ T cells both from healthy donors and from patients stimulated with gp100 (209-2M; irrelevant peptide) to background. Analysis of these CD8+ T cells identified a mean ratio of 1.0 (range, 0.7–1.3) with 95 and 99% confidence intervals of 1.0 ± 0.89 and 1.0 ± 1.16, respectively, a SE of 0.06, and a SD of 0.20. The cutoff ratio (stimulation index) was estimated by adding the mean to three SDs, which equaled 1.6. To minimize the possibility of falsely considering CD8+ T cells immunoreactive, we accepted a 2-fold increase in stimulated:unstimulated IFN-{gamma} transcript ratio as evidence of epitope-specific reactivity.

Fisher’s exact test was calculated to determine, whether there was a statistically significant difference in T-cell response to test peptides between normal healthy individuals and patients. Statistical significance was achieved, when P < 0.05.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Selection and Binding Activity of Peptides to HLA-A*0201 Molecules.
Using the SYFPETHI computer-based prediction analysis we selected candidate 9-mer peptides that could potentially bind to MHC-class I molecules (40) . The peptides CD1974–82, CD20127–135, and CD20188–196 were synthesized and their binding to HLA-A*0201 molecules evaluated in the T2 cell assay. A FI between 1.11 and 1.49 was considered low-affinity binding and a FI greater that 1.5 high-affinity binding. As shown in Table 1Citation , T2 cells pulsed with positive control CMV (pp65) or gp100 peptide showed a FI of 2.3 or 2.2 respectively. Among the CD19- and CD20-derived peptides, pulsing with CD20127–135 led to the highest FI (1.6), whereas the other peptides showed a FI of 1.3 and 1.4 (CD1974–82 and CD20188–196) corresponding to a lower-affinity binding to the HLA-A*0201 molecules.


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  		Table 1 Peptides and predicted binding to HLA-A*0201

 
Identification of CD19- and CD20-Peptide-Specific CD8+ T-Cell Reactivity in Healthy Individuals and Patients with B-Cell Malignancies.
To determine whether CD8+ T cells recognizing CD19 and CD20 exist in healthy individuals and patients with B-cell malignancies, we looked for IFN-{gamma} mRNA production in peptide-stimulated CD8+ T cells using qPCR. We analyzed 23 healthy donors and 28 patients with B-cell malignancies (18 CLL, 7 follicular lymphoma, 2 acute lymphocytic leukemia and 1 large cell lymphoma). As controls, CD8+ T cells were stimulated either with CMV(pp65)-peptide or with staphylococcus enterotoxin B (positive controls) and gp100 (209–2M) peptide (negative control). A positive response was defined as >=100 IFN-{gamma} mRNA copies/104 CD8 copies and a stimulation index (SI) of >=2, where SI = IFN-{gamma} mRNA copies/104 CD8 copies in peptide-pulsed T2 cell cultures/unpulsed cultures.

CD8+ T-cell responses to CD1974–82 were detected in 1 of 21 healthy individuals with a SI of 2.6 (Table 2Citation ; Fig. 1ACitation ). Positive T-cell responses after stimulation with CD20-derived peptides were detected in 6 of 23 healthy individuals. Three responded to stimulation with CD20127–135 (donors 13, 15, and 16) with SI between 2.0 and 3.5 and three to stimulation with CD20188–196 (donors 14, 21, and 23) with SI between 2.2 and 2.3 (Table 2Citation ; Fig. 1, B and CCitation ). None of the healthy donors had a response to more than one peptide or to gp100 peptide.


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  		Table 2 CD8+ T-cell reactivities to peptides in healthy individuals

 


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  		Fig. 1. CD8+ T cell response to stimulation with the HLA-A*0201-restricted peptides. Selected T cells from healthy individuals (n = 23, {circ}), chronic lymphocytic leukemia (CLL) patients (n = 18, •) and patients with follicular lymphoma, large cell non-Hodgkin-lymphoma or acute lymphocytic leukemia (non-CLL, n = 10, {triangleup}) were incubated for 3 h with unpulsed antigen-presenting cells (APCs) or APCs pulsed with peptides. Peptides used were CD1974–82 (A), CD20127–135 (B) and CD20188–196 (C). Values represent the stimulation index (SI) of single experiments where SI is determined by the ratio of IFN-{gamma} mRNA copy number obtained from CD8+ T cells stimulated with relevant test peptides to that obtained from CD8+ T cells stimulated with unpulsed APCs. A cutoff value of 2.0 was considered to be evidence of epitope specificity. p, statistical difference of T-cell reactivity between CLL-patients and, respectively, non-CLL-patients and normal individuals (p = n.s., not significant).

 
Clinical data for the patients with B-cell malignancies are shown in Table 3Citation . A CD8+ T-cell response to CD1974–82 was observed in 7 of 27 patients with B-cell malignancies (SI range, 2.1–7.1; Table 4Citation ; Fig. 1ACitation ). Positive T-cell responses after stimulation with CD20-derived peptides were detected in eight patients. Seven of 24 patients had a positive response to stimulation with CD20127–135 (SI range, 2.3–3.0). Three of 26 patients showed a response to CD20188–196 (SI range, 2.3–3.7; Table 4Citation ; Fig. 1, B and CCitation ). In two patients (patients 3 and 16), there was a positive response to both CD1974–82 and CD20127–135 peptides. Two patients showed a positive response to all three peptides (patients 5 and 8). No patient responded to stimulation with gp100 peptide.


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  		Table 3 Patients: clinical properties

 

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  		Table 4 CD8+ T-cell reactivities to peptides in patients with B-cell malignancies

 
It was notable that six of seven positive CD8+ T-cell responses against CD1974–82 occurred in CLL-patients. A CD1974–82 CD8+ T-cell response was significantly more frequent in the CLL group compared with normal donors (Fig. 1ACitation ; Table 5Citation ).


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  		Table 5 Summary of CD8+ T-cell reactivities in healthy individuals, chronic lymphocytic leukemia (CLL) patients, and patients with other B-cell malignancies

 
Similarly, positive CD20127–135 and CD20188–196 responses occurred only in the CLL patient group. However, the frequency of positive responses did not differ significantly from that found in normal individuals. Overall the CD19 peptide induced the greatest CD8+ T-cell reactivity [SI, 3.8 compared with 3.2 (CD20188–196) and 2.6 (CD20127–135)].

Peptide specificity determined by qPCR was confirmed previously using intracellular cytokine assay and tetramer analysis (49) . In six healthy donors, IFN-{gamma} mRNA expression (qPCR) after CMV pp65 495–503 stimulation was compared with intracellular IFN-{gamma} production and tetramer assay. There was strong correlation between qPCR, intracellular cytokine assay, and tetramer assay (R2 = 0.92 and 0.78, respectively). The qPCR assay was the most sensitive, detecting 1/105 CMV pp65-specific CD8+ T cells (data not shown). Good correlation between the analysis of CD8+ T-cell reactivity by flow cytometry (detection of intracellular IFN-{gamma} expression) and by qPCR is shown in Figs. 2Citation and 3Citation after stimulation with CD1974–82 in patient 1 (representative experiment).



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  		Fig. 2. Flow cytometric analysis of CD8+ T-cell response to stimulation with CD1974–82 in patient 1. Peptide-reactive T cells were identified by intracellular IFN-{gamma} expression (IC IFN-G) after stimulation with peptide-pulsed antigen-presenting cells. A and B, the CD8/IFN-{gamma} profile of CD8+ T cells after coincubation with unpulsed (A) and peptide-pulsed (B) antigen-presenting cells (C1R-A2). Frequencies of IFN-{gamma}-positive T cells are shown as percentages of the total number of CD8+ T cells. PE, phycoerythrin.

 


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  		Fig. 3. Analysis of CD8+ T-cell response against CD1974–82 by quantitative real-time PCR (qPCR) in patient 1. Data are shown as absolute numbers of IFN-{gamma} mRNA copies after 3-h stimulation of CD8+ selected T cells with unpulsed (left) or peptide-pulsed (right) antigen-presenting cells (C1R-A2). Error bars, the SD.

 
In Vitro Expansion of Peptide-Specific CD8+ T Cells with Recombinant Peptide.
To determine whether CD1974–82-specific T cells could be expanded in vitro, B cells stimulated with CD40L and loaded with CD1974–82 were used to weekly restimulate autologous CD8+ T cells in media containing 10 units/ml IL-2 and 10 ng/ml IL-7. Three patients (patient 1, 8, and 26), in whom we were able to detect antigen-specific T cells, were studied. The cytotoxicity of the expanded T cells was then tested in a Calcein-AM release assay. Cytotoxicity against CD1974–82-pulsed T2 cells ranged between 24 and 48% at E:T-ratios between 30:1 and 70:1 in patients 1, 8, and 26 (Fig. 4, A–C)Citation . Negative controls (T2 cells pulsed with gp100 peptide or K562 cells) showed no lysis. Lysis of the T2-loaded target was partial after treatment with a monoclonal antibody against HLA-A2 confirming that the CTLs were HLA-A2 restricted (Fig. 5)Citation . However, there was no significant lysis of CD40-activated autologous B cells (Fig. 4)Citation nor lysis of unstimulated B cells or allogeneic HLA-A2-positive EBV-LCL (data not shown). These observations suggest that, despite peptide specificity of the CD8+ cells, the expression of peptide on the unpulsed B cells was too low to activate lytic activity.



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  		Fig. 4. Cytotoxicity assay to show lytic activity of CD1974–82-specific CD8+ T cells after 4 weeks expansion. CD8+ T cells from patient 1 (A), patient 8 (B), and patient 26 (C) were weekly stimulated using CD40-activated, peptide-loaded autologous B cells. Cytotoxic responses to T2 cells pulsed with CD1974–82 ({diamondsuit}), gp100-peptide ({blacksquare}), and unpulsed T2 cells ({diamond}) are shown. K562 cells ({blacktriangleup}) were used as control to rule out natural killer cell activity. To analyze lytic activity against B cells, autologous CD40-activated B cells ({square}) were used as target cells.

 


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  		Fig. 5. Inhibition of CD1974–82-specific target cell lysis by anti-HLA-A2 monoclonal antibody (mAb). The cytotoxic activity of CD1974–82-specific CD8+ T cells (patient 1) against peptide-pulsed antigen-presenting cells (T2 cells {diamondsuit}) occurs in a MHC-restricted manner and is inhibited by the addition of anti-HLA-A2 mAb ({blacktriangleup}). ({triangleup}) indicates lytic activity after addition of a control (isotype)-antibody (Ab).

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Recent studies in non-Hodgkin lymphoma of the B-cell type indicate the presence of autologous CD4+ and CD8+ T-lymphocytes reactive against malignant B cells (5, 6, 7, 8 , 50) . Because malignant B cells can act as APCs (51) , they could induce T-cell responses directly or indirectly via professional APCs. Recently it has been shown, that dendritic cells induce proliferation and antitumor-responses against B-cell lymphomas and CLL (52, 53, 54, 55) . Here we set out to determine, whether the proteins CD19 and CD20, abundantly expressed by many malignant B cells, could induce such cytotoxic CD8+ T-cell responses to B-cell leukemias and lymphomas, in patients with B-cell malignancies and in normal individuals.

We studied the B-cell differentiation antigens CD19 and CD20, because these molecules play a critical role in the development, differentiation, and activation of B cells. They are tissue-restricted to B-lineage cells and are expressed in the majority of B-cell malignancies (35 , 36) . We selected 9-mer peptide sequences (CD1974–82, CD20127–135, and CD20188–196) on the basis of HLA-A2 binding as candidates for the detection and the expansion of antigen-specific CTLs. Interestingly, despite relatively weak binding of these peptides to MHC molecules, all three were immunogenic. Using a qPCR assay that has 10-fold greater sensitivity than tetramer analysis (49) , CD8+ T cells recognizing CD1974–82, CD20127–135, and CD20188–196 were detected in 5, 21, and 13%, respectively, of normal individuals. The presence of self-reactive T cells, that have escaped thymic deletion in the periphery, does not necessarily imply functional autoimmunity: In the thymus, T cells with a high binding affinity to self-MHC/self-peptide complexes are eliminated from the T-cell repertoire by clonal deletion (21 , 56 , 57) . However, T cells with low-avidity for self-antigens escape clonal deletion and enter the post-thymic compartment (58, 59, 60) . We assume, that the CD19- and CD20-specific T cells described here were of low avidity, because T-cell responses to peptide were detected only after pulsing the APCs with high concentrations of peptide (50µg/ml). (In preliminary experiments, peptide doses of 0.1 or 1µg/ml never elicited T-cell responses).

T cells reactive against CD1974–82 were also detected in patients with B-cell malignancies but predominantly in CLL (35 versus 5%). T-cell responses to CD1974–82 were significantly more frequent and greater in amplitude than in normal individuals. CD20127–135 but not CD20188–196 recognizing T cells were also found twice as frequently in the CLL group compared with the normal individuals (47 versus 21%), suggesting that peptide CD20188–196 was less immunogenic than CD1974–82 and CD20127–135. Although T-cell responses to CD19 and CD20 were commonest in CLL, insufficient numbers of patients with other B-cell malignancies were tested to determine whether there was a statistically greater probability of CLL patients developing responses to B-cell antigens. The increased reactivity to self-peptides in CLL compared with normals could be due to either direct or indirect T-cell stimulation from CLL or dendritic cells presenting quantitatively larger amounts of CD19 and CD20 because of the characteristic increase in B cell production in CLL. However, because CLL cells only weakly express critical adhesion and costimulatory molecules (61, 62, 63) , CD19 and CD20 peptide epitopes are more likely to be presented by professional APCs. Although the expression of CD19 and CD20 on CLL cells is somewhat decreased, the massive leucocytosis in CLL could counteract any decreased antigenic potential from these molecules (64 , 65) . It is possible that CLL patients had the best CD19 and CD20 responses because of a higher tumor load than patients with lymphomas. Unlike high-avidity T cells, which can be eliminated from the repertoire by high concentrations of antigen, stimulation of low-avidity T cells by these self-antigens could elicit increased frequencies of persisting non-autoreactive T cells. However, T cells analyzed in this study were derived from peripheral blood. Additional studies to look for T cells recognizing CD19 and CD20 epitopes in lymph node biopsies would be worthwhile.

Because of the significant increase in CD19 responses in CLL, we studied T-cell responses to this peptide further in three patients (2 CLL and 1 large cell lymphoma). After 4 weeks of stimulation with 50µg/ml recombinant CD1974–82, peptide-specific, MHC-restricted cytotoxicity of peptide-pulsed APCs (T2 cells) occurred in all three cases. However these T-cell lines were not cytotoxic to unpulsed autologous or allogeneic B cells whether or not they were activated by CD40L. This suggested that the peptides studied were not naturally presented, or that antigen density is below the threshold required to cause a cytotoxic response in the CD8+ T cells. Activation of B cells by CD40L has recently been shown to effectively up-regulate expression of costimulatory molecules in normal and malignant B cells including CLL cells, making it unlikely that the CLL targets failed to deliver costimulatory signals (61, 62, 63) . Furthermore, CD40-activated CLL B cells were capable of expanding T cells in culture.

In conclusion, these studies indicate that T-cell responses to peptides derived from CD19 and CD20 molecules are common in both normal individuals and patients with CLL but involve only low-avidity T cells, requiring high doses of peptide to stimulate them. Our findings suggest that the T-cell repertoire is thus shaped in CLL by the elimination of any high-avidity CLL-specific T cells and the expansion of low-avidity T cells, recognizing common and highly expressed molecules such as CD19 and CD20 during the course of CLL. This suggests a form of T-cell repertoire shaping by CLL similar to that described for proteinase 3 antigen PR1 in patients with chronic myelogenous leukemia (50) . Development of T-cell immunotherapy approaches to CLL using CD19 or CD20 peptide antigens would, therefore, require strategies to increase T-cell avidity to generate effective B-cell-specific cytotoxic T cells. Because nonmalignant B cells would also be recognized by CD19- and CD20-specific T cells, it would be necessary to limit the in vivo persistence of specific T cells to avoid continuous B-cell depletion using, for example, IL-2-dependent T cells, or T cells coexpressing a suicide gene such as the herpes simplex virus thymidine kinase (HSV-TK).


   ACKNOWLEDGMENTS
 
We thank Dr. Ernst Holler for his support and encouragement and Dr. Mathias Oelke for his support and helpful discussions.


   FOOTNOTES
 
Grant support: The Dr. Mildred-Scheel-Stiftung Deutsche Krebshilfe Foundation (to M. G.).

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.

Requests for reprints: John Barrett, NIH, 9000 Rockville Pike, Building 10, Room 7C 103, Hematology Branch, Bethesda, Maryland 20892. Phone: (301) 402-3296; Fax: (301) 435-8655; E-mail: barrettj{at}nih.gov

4 Internet address: http://www.uni-tuebingen.de/uni/kxi/. Back

Received 8/21/03; revised 10/20/03; accepted 10/22/03.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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