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Immunology |
Transplantation Immunology Section, Blood and Marrow Transplant Department, University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030-4095 [J. J. M., C. W., R. E. C.], and Howard Hughes Medical Institute, Department of Microbiology and Immunology, Stanford University, Palo Alto, California, 94305 [P. P. L., M. M. D.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A recently developed technique to identify low-frequency peptide antigen-specific CTLs by flow cytometry uses fluorescence-labeled HLA-peptide tetramers as a staining reagent (6) . These tetramers are constructed by folding the HLA heavy chain, ß2-microglobulin, and peptide, followed by biotinylation at the COOH terminus of the HLA heavy chain and conjugation with fluorochrome-labeled avidin at a 4:1 molar ratio (6) . Using viral peptide tetramers, CTLs with LCMV-, EBV-, and HIV-reactive specificities have been identified in patients infected with these viruses (7, 8, 9) . Tetramer labeling has allowed sensitivity down to 0.02% CD8+ cells to identify antigen-specific CTLs, and correlative studies with conventional LDA show that LDA may underestimate the true number of peptide-specific CTLs (10) . This may occur because LDA relies on CTLs that can survive in culture for 2 weeks, whereas tetramer staining labels all peptide-specific CTLs regardless of their proliferative potential. This also calls into question which assay may best correlate with lytic function and possible clinical relevance.
In this study, we set out to determine the relationship between tetramer labeling of PR1-specific CTLs and the CTLp frequency determined by LDA and to adapt a cell-sorting technique using the PR1-tetramer to select CTLs that could be used in adoptive immunotherapy of myeloid leukemias. We first established short-term cultures of PR1-reactive CTLs from PBMCs of HLA-A2.1+ healthy donors. The number of PR1-reactive CTLp in the short-term cultures as measured by LDA underestimated by as much as 40-fold the number of PR1-specific CTLs measured using the PR1-HLA-A2.1 tetramer. Specific lysis of PR1-coated target cells by bulk culture CTL correlated best with the number of PR1-HLA-A2.1 tetramer positive cells contained in the culture. Moreover, the PR1-specific CTLs separated from a short-term culture by FAC sorting with the PR1-HLA-A2.1 tetramer showed 56% greater lysis of CML blasts compared to the nonsorted bulk culture CTLs, with no lysis of HLA-matched normal marrow progenitors at an effector to target (E:T) ratio of 10:1.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) as well as the paired HLA-identical healthy PBPCs from the donor for that patient (D1), were obtained before bone marrow transplant. Cells from three other unrelated HLA-A2.1+ healthy donors were also obtained from peripheral blood for the generation of the PR1-specific cell lines CTL1-3. The cells were separated using Ficoll-Hypaque gradient-density (Organon Teknika Co., Durham, NC) and subsequently frozen in RPMI 1640 complete (CM, Life Technologies, Inc., Gaithersburg, MD) supplemented with 20% heat-inactivated pooled human AB serum (Sigma Chemical, St. Louis, MO) and 10% DMSO according to standard protocols. Before use, cells were thawed, washed, and suspended in CM + 10% AB. HLA-A2 phenotype was confirmed using the BB7.2 mouse monoclonal antibody (ATCC, Rockville, MD).
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Cell Lines.
T2 cells (ATCC) are a hybrid human cell line that lacks most of the MHC class II region including the known transporter proteins for antigenic peptide and proteasome genes. They contain the gene HLA-A*0201 but express very low levels of cell surface HLA-A2.1 and are unable to present endogenous antigens (13
, 14) . These cells were maintained in culture in CM + 10% FBS (Atlanta Biologicals, Norcross, GA).
Generation of Peptide-specific T Cells from Healthy Human PBMCs.
PBMCs from three healthy HLA-A2.1+ donors (ND1-3, Table 1
) were stimulated in vitro with PR1 or pp65 using a protocol adapted from previous studies (2)
. Briefly, T2 cells were washed three times in serum-free CM and by incubating with peptide at 10 µg/ml for 2 h in CM. The peptide-loaded T2 cells were then irradiated with 7500 cGy, washed once, and suspended with freshly isolated PBMCs at a 1:1 ratio in CM supplemented with 10% AB. After 7 days in culture, a second stimulation was performed; and the following day, 10 IU/ml rhIL-2 (Biosource International, Camarillo, CA) were added. After 14 days in culture, a third stimulation was performed, followed on day 15 by the addition of rhIL-2. After a total of 20 days in culture, the peptide-stimulated T cells were harvested and tested for peptide-specific lysis of human bone marrow cells derived from a patient with CML (P1) or from PBPCs from an HLA-matched normal donor (D1). Peptide-specific CTLs were also tested for lysis of peptide-coated T2 cells.
Production of MHC/Peptide Tetramers.
The production of MHC/peptide tetramers was described in detail previously (6)
. Briefly, a 15-amino acid BSP for BirA-dependent biotinylation has been engineered onto the COOH terminus of HLA-A2. The A2-BSP fusion protein and human ß2-microglobulin were expressed in Escherichia coli and were folded in vitro with the specific peptide ligand. The properly folded MHC-peptide complexes were extensively purified using FPLC and anion exchange and were biotinylated on a single lysine within the BSP using the BirA enzyme (Avidity, Denver, CO). Tetramers were produced by mixing the biotinylated MHC-peptide complexes with PE-conjugated Neutravidin (Molecular Probes) at a molar ratio of 4:1. PR1 tetramers were validated by staining against a CTL line specific for PR1. CMV tetramers were validated by staining with PBMCs from a CMV-immune individual. Specificity was demonstrated by the lack of staining of irrelevant CTLs. By titrating positive CTLs into PBMCs from normal controls, we established our limit of detection to be as low as 0.01% of CD8+ cells. Each tetramer reagent was titered individually and used at the optimum concentration, generally 20–50 µg/ml.
Antibodies and Flow Cytometry.
Cytoplasmic proteinase-3 staining was determined by the following procedure: (a) cells were permeabilized and fixed with Ortho PermeaFix (Ortho Diagnostics, Raritan, NJ) according to the manufacturer’s directions; (b) the cells were stained with 5 µl of mouse antibody to human proteinase 3 (Accurate Chemical and Scientific Corporation, Westbury, NY) and incubated for 40 min at room temperature; (c) the cells were washed once with PBA (PBS with 0.1% NaN3 and 0.1% BSA); (d) the cells then were labeled with FITC-labeled goat antimouse IgG (PharMingen, San Diego, CA) for 30 min at room temperature and washed with PBA; and (e) the cells were then fixed with 1% paraformaldehyde and analyzed by flow cytometry.
Mouse monoclonal anti-HLA-A2.1 antibody BB7.2 was derived from culture supernatant of a hybridoma cell line (ATCC) and was not labeled.
For surface antigen staining, roughly 100,000 CTLs were incubated at 4°C for 1 h with the antibody. PR1-HLA-A2.1 tetramer was incubated at 50 µg/ml final concentration in PBS plus 2% FCS. After washing, cells were incubated with FITC-labeled CD8 (Caltag, Burlingame, CA) or with purified unlabeled CD8 (Caltag) plus APC-labeled goat antimouse (Caltag) before adding either FITC-labeled CD95 (Becton Dickinson, San Jose, CA) or FITC-labeled CD45RO (Becton Dickinson) for 30 min on ice. Cells were washed and fixed in 2% paraformaldehyde and analyzed on a FACScan (Becton Dickinson), and data were analyzed using CELLQuest (Becton Dickinson) software. Cell sorting was performed on the FACS Vantage (Becton Dickinson), and cells were selected for both PE-labeled PR1-HLA-A2.1 tetramer and FITC-labeled CD8 (Caltag) after gating on live cells.
CTL Precursor Frequency by Microtiter-LDA
The previously frozen PBMC samples were thawed and then washed three times in serum-free CM and counted. Nineteen and one-half million cells (7.5 million stimulator cells and 12 million responder cells) were prepared for the assay. One million additional PBMCs were used to generate PHA (Sigma Chemical)-stimulated blasts that were used as target cells on day 10 of the assay. One million unrelated HLA-disparate PBMCs (third party) were used as a positive control, with one-half of the total number used as stimulator cells and the other half as responder cells.
PHA blasts were generated by placing 106 cells in 1.0 ml of CM supplemented with 10% FBS (Sigma Chemical) in a 25-cm2 flask. Five µl/ml PHA were added, and the cells were cultured in 5% C02 at 37°C. On days 4, 6, and 8 of the culture, additional CM + 10% FBS with 5 µl/ml PHA (Sigma Chemical) and 500 IU/ml rhIL-2 (Biosource International) were added. Cells were counted, and cultures were maintained at 106 cells/ml. These cells were used as target cells on day 10 of the CTLp assay.
PBMC responder cells (from ND1-3) were plated into high-profile Terasaki Trays (Robbins Scientific, Sunnyvale, CA) at 6 dilutions (from 5 x 104 to 5 x 105 PBMCs/well) using 24 replicates at each dilution in CM + 10% human AB serum (Sigma Chemical). Third party responders were plated at a single dilution (5 x 104 PBMCs/well). Patient PBMC stimulator cells were either pulsed with peptide (PR1 or Flu as a positive control peptide) at 20 µg/ml or with no peptide for 90 min at 37°C, washed once with serum-free CM, and then irradiated with 2500 cGy. They were then plated into the wells containing the responder cells as well as 24 additional wells of stimulators alone. On days 3 and 7, 60 IU/ml IL-2 were added to each well. On day 10, the previously prepared PHA blasts from the same patient (as target cells) were either pulsed with PR1 or Flu at 20 mg/ml or no peptide for 90 min at 37°C, washed once with serum-free CM, and then labeled with Calcein AM (Molecular Probes, Eugene, OR) as described in the cytotoxicity experiments below. One thousand target cells per well were plated, lightly centrifuged at 800 rpm for 1 min, and then cultured in 5% C02 at 37°C for 4 h. Five µl of FluoroQuench EB Stain-Quench Reagent (One Lambda Inc., Canoga Park, CA) were added to each well, and the plates were analyzed for fluorescence emission using an automated CytoFluor II plate reader (PerSeptive Biosystems, Framingham, MA).
The mean plus three SDs of the 24 wells containing the stimulators alone was determined as the cutoff value for background fluorescence. Any experimental well less than the cutoff value was considered positive for lysis; and from the fraction of negative wells at the various responder cell dilutions, the frequency of CTLp was calculated using the maximum likelihood method based on Poisson probabilities.
CTL Cytotoxicity Assay.
A semi-automated mini-cytotoxicity assay was used to determine specific lysis as described previously (2)
. Effector cells (CTL1-3 cells) were prepared in doubling dilutions from 6 x 103 to 25 x 103 cells/well and were plated in 40-µl, 60-well Terasaki trays (Robbins Scientific) with six replicates per dilution. Target cells (T2 cells ± peptides, marrow-derived leukemic cells from P1 or PBPCs from D1) at a concentration of 2 x 106 cells/ml were stained with 10 µg/ml Calcein-AM (Molecular Probes Inc.) for 60 min at 37°C. After washing three times in CM + 10% AB, target cells were resuspended at 105 cells/ml (i.e., 103 target cells 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 (max) and minimum (min) fluorescence emission, respectively. After 4-h incubation at 37°C in 5% CO2, 5 µl of FluoroQuench (One Lambda, Inc.) were added to each well, and the trays were centrifuged for 1 min at 60 x g before the measurement of fluorescence (excitation at 485 nm, emission measured at 530 nm) using an automated CytoFluor II plate reader (PerSeptive Biosystems). A decrease in the fluorescence emission is proportional to the degree of lysis of target cells, once the released dye is quenched by the hemoglobin contained in the FluoroQuench reagent. The percentage of lysis was calculated as follows:
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) were stimulated with either PR1- or pp65-pulsed T2 cells as positive controls. The donor was seropositive for CMV by latex agglutination (data not shown). After 20 days in culture with weekly peptide restimulation, CTLs were tested for staining with the PR1-HLA-A2 tetramer and a pp65-HLA-A2 tetramer.
The specificity of the PR1-HLA-A2 tetramer is demonstrated by its ability to stain T lymphocytes from ND1 elicited with weekly PR1 stimulation but not lymphocytes from ND1 elicited with pp65. As shown in Fig. 1
, the PR1-HLA-A2 tetramer stained approximately 2% of PR1-stimulated lymphocytes and < 0.1% of pp65-stimulated lymphocytes, whereas the pp65-HLA-A2 tetramer stained 3% of pp65-stimulated lymphocytes and < 0.1% of the PR1-stimulated lymphocytes.
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) were stimulated with PR1-pulsed T2 cells. The T2 cells were incubated with 10 mg/ml PR1 at 37°C for 2–4 h, irradiated with 7500 cGy, and combined with PBMCs to induce PR1-specific CTLs as described previously (2). On the day the cultures were set up (day 0) and again after 20 days in culture with weekly restimulation, lymphocytes were tested for CTLp frequency against the PR1 peptide by LDA using a microtiter method. Lymphocytes on days 0 and 20 were also tested for the percentage of CTLs that stained with the PR1-HLA-A2 tetramer. Lymphocytes from day 20 were tested for specific lysis of Calcein AM-labeled T2 cells loaded or not loaded with 10 µg/ml PR1.
As shown in Table 2
, the CTLp frequency against PR1 on day 0 from each of the three donors ND1-3 ranged from as low as 1/345,000 to 1/15,781. At the end of 20 days of PR1 restimulation, the precursor frequency of PR1-specific CTLs had increased in each of the three cultures by as much as 2.5 logs, with the greatest increase in the culture established from ND1 (CTL 1). Although the percentage of PR1-HLA-A2 tetramer-staining CTLs was undetectable on day 0, by day 20, cultures from all of the three donors contained 2–8% PR1-specific CTLs.
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The number of PR1-specific CTLp on day 20 measured by LDA ranged from 9-fold (CTL 3) to 40-fold (CTL 2) less than the percentage of PR1-specific CTLs measured with the PR1-HLA-A2.1 tetramer. In Fig. 2
, the amount of specific lysis of PR1-coated T2 cells by all three of the CTL lines is plotted against the percentage of lymphocytes that stained with the PR1-HLA-A2 tetramer. Specific lysis correlated better with the percentage of PR1-specific CTLs contained in the lymphocyte bulk culture (R2 = 0.95) than with CTLp frequency determined by LDA (R2 = 0.76).
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shows the phenotype of the CTL2 cell line on day 20. All of the cells were labeled with unconjugated anti-CD8 plus the PE-labeled PR1-HLA-A2 tetramer and FITC-labeled anti-CD95 or CD45RO. A secondary antibody conjugated to APC and directed against the anti-CD8 antibody was used to gate only CD8+ cells. Of the total CD8+ lymphocyte population, 2.2% stained with antibody to CD95 (58% of all of the PR1-specific CTLs), which indicated an activated cell phenotype. Nearly all (91%) of the PR1-specific CTLs stained with antibody to CD45RO, which is consistent with a memory T-cell phenotype.
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). CTL2 cells were stained with the PR1-HLA-A2 tetramer and FITC-labeled antibody to CD8, and the dual-labeled lymphocytes were selected using a FACS Vantage cell sorter. Fig. 4
shows the phenotype of the sorted population. The yield of PR1-HLA-A2 tetramer+ cells was 78%, with 95% purity of PR1-specific lymphocytes as shown in Table 3
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) were readily lysed by bulk culture CTL2 cells (Fig. 5)
, whereas granulocyte-colony-stimulating-factor-mobilized PBPCs from an HLA-identical healthy donor (D1) showed only background lysis of less than 20%. By comparison, the PR1-HLA-A2 tetramer-sorted CTLs showed nearly 50% greater lysis of CML cells than the nonsorted CTLs at every E:T ratio tested, with no background lysis of normal PBPC, even at an E:T ratio of 20:1. The addition of antibody to HLA-A2.1 (BB7.2) during the 4-h cytotoxicity assay partially blocked lysis at every E:T ratio (data not shown), as demonstrated previously (2)
. The PR1-HLA-A2 tetramer-sorted cells were cultured in the presence of 20 IU/ml IL-2 for 48 h before testing for cytotoxicity.
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Proteinase 3 Is Overexpressed in CML Blasts.
We have previously shown (2
, 3)
that both the amount of target cell lysis and the amount of CFU-GM inhibition correlate with the intracellular level of proteinase 3 in the leukemia cells. We, therefore, tested cells from P1 and D1 for the amount of cytoplasmic proteinase 3. Cells were first stained with PE-labeled anti-CD34 antibodies. Then, after permeabilizing the cell membrane, indirect staining was performed using a mouse antibody to proteinase 3 and a second FITC-labeled goat antimouse antibody, followed by flow cytometry. Small mononuclear PBPCs from the normal donor D1 were gated to analyze the CD34+ progenitors, and a separate gate for granulocytes was also examined. As shown in Fig. 6A
, proteinase 3 was expressed only in the granulocytes and not in the CD34+ progenitors of normal PBMCs from D1. However, bone marrow blasts from P1 expressed one log higher cytoplasmic proteinase 3 than normal granulocytes from D1, which normally express a maximal amount of proteinase 3.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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These studies suggested that PR1 might be a powerful therapeutic target for adoptive allogeneic cellular strategies to treat CML or other myeloid leukemias. We speculated that large numbers of such leukemia-selective CTLe might not be necessary because as few as 1 x 107 T cells/kg unselected allogeneic peripheral blood T cells are sufficient to induce remissions in patients with CML (with estimated leukemic burdens of 1010 to 1012 cells; Ref. 16 ). Leukemia-reactive CTLp within the unselected DLI could expand in vivo to sufficient numbers of CTLe to eliminate the leukemia. The observation that chronic phase CML responds more frequently to DLI than accelerated phase or blast crisis CML is consistent with the idea that leukemia growth outpaces the expansion of donor antileukemia CTLp (17 , 18) . Rapidly growing CML and acute myelogenous leukemia might be eliminated with lymphocyte infusions if a sufficient number of preselected leukemia-specific CTLe could be infused to overwhelm the kinetic growth of the leukemia while avoiding any graft-versus-host disease.
To better select only the leukemia-reactive CTLs, we studied whether a PR1-HLA-A2 tetramer could be used to identify PR1-specific CTLs in the PBMCs of healthy adult donors. Because we expected the number of CTLs with self-peptide specificity in healthy donors to be low and perhaps below the limit of detection by tetramer staining, we first examined the PR1-specific CTLp frequency in the PBMCs of healthy HLA-A2.1+ donors using a limiting dilution assay. Although the CTLp frequency was low in two donors, one donor had a surprisingly high frequency of 1 in 15,781. Still, the small number of PR-specific CTLs was below the detection threshold in peripheral blood when up to 100,000 cells were analyzed using the PR1-HLA-A2 tetramer. After only 20 days of in vitro expansion with weekly PR1 restimulation, however, the percentage of CTLs that stained with the PR1-HLA-A2 tetramer increased to between 2 and 8%.
The number of PR1-specific CTLp determined by LDA underestimated by as much as 40-fold the number of PR1-specific CTLs when examined by PR1-HLA-A2 tetramer staining in each of the three donors, although the two methods showed a strong correlation (R2 = 0.91). Although the amount of PR1-coated target-cell lysis by day-20 CTL lines did not correlate well with the CTLp frequency as determined by LDA (R2 = 0.76), it did show a strong correlation with the number of PR1-HLA-A2 tetramer-staining CTLs (R2 = 0.95). This suggests that tetramer staining identifies a higher percentage of CTLe than does LDA, perhaps because of the requirement of an adequate number of CTLp that must first survive and proliferate during the 10-day LDA assay.
When PR1-specific CTLs were selected by flow sorting using the PR1-HLA-A2 tetramer, a very homogeneous CTL population was collected. The tetramer-sorted CTLs showed significantly higher lysis of HLA-A2.1+ leukemia cells than nonsorted CTLs, with none of the background lysis of HLA-matched normal marrow cells observed using the nonsorted CTLs. Importantly, the tetramer-sorted CTLs could be easily expanded in vitro by another 10-fold over 3 days in the presence of 100 IU/ml of IL-2 while retaining peptide-specificity. Adding to our previous studies, proteinase 3 was overexpressed in both CD34+ and CD34- leukemia cell targets compared with both normal granulocytes and CD34+ cells.
It remains unclear why the PR1 self-peptide is retained within the T-cell receptor repertoire of healthy individuals who show no evidence of Wegener’s granulomatosis or other autoimmune vasculitis. This phenomenon has been observed by investigators studying other self-peptides, which strongly suggests that peripheral tolerance mechanisms must prevail to keep potentially self-reactive CTLs in check (19 , 20) . One postulated mechanism of peripheral T cell tolerance to self-antigens depends on the antigen activation threshold (21) . Therefore, the level of antigen expression by normal myeloid cells may be below the threshold necessary to trigger T-cell recognition and effector function. In the case that leukemia cells overexpress proteinase 3, the threshold of antigen expression may be exceeded resulting in T-cell activation. However, clonal deletion may result if there is a high avidity interaction between the T-cell receptor and the peptide-MHC combination, or anergy may result if the leukemia cells lack costimulatory molecules (21, 22, 23, 24) . Anergy may not be relevant when designing adoptive immunotherapy strategies using allogeneic CTLs because ex vivo T-cell activation would have already occurred during in vitro expansion, and a second stimulus from the leukemia cell would no longer be necessary. T-cell expansion strategies may also be designed to expand low avidity CTLe, thereby avoiding clonal deletion of reactive CTLs (21) .
A potential pitfall concerning adoptive immunotherapy with only the single peptide PR1-specific CTLs is that this may allow development of resistance by leukemia cells that mutate or down-regulate expression of proteinase 3. However, tetramers used to study immune reactions against EBV have demonstrated that CTLs with single-epitope reactivity may comprise 44% of the entire CD8+ T-cell population in peripheral blood at the height of an effective immune response to the virus (8) . If similarly high numbers of PR1-specific T cells could be infused into patients with leukemia, this might have the effect of overwhelming the leukemia before selection pressure could result in resistance.
This study highlights the utility of PR1-HLA-A2 tetramers to identify leukemia-reactive CTLs. Because LDA assays are time-consuming and difficult to perform, this technique offers the advantages of more rapid identification of CTL and better correlation with effector function against leukemia. Tetramers also allow for the selection of a homogeneous population of CTLs that may be useful as adoptive immunotherapy in the relapse setting after BMT or as prophylaxis in high-risk patients for early relapse. We are currently using PR1-HLA-A2 tetramers to investigate the clinical relevance of PR1-specific T-cell expansions in patients after allografting and after DLI. In the future, tetramers could also be used to add back or deplete selective T-cell immunity to many antigens after marrow allografting or as a tool to tolerize T cells to minor antigens that contribute to graft-versus-host disease.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported in part by a Translational Research Award (LSA 6148-99) from the Leukemia Society of America (to J. J. M.). 
2 To whom requests for reprints should be addressed, at Section of Transplantation Immunology, Department of Blood and Marrow Transplantation, University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Box 65, Houston, TX 77030-4095. Phone: (713) 792-8750; Fax: (713) 794-4902; E-mail: jmolldre{at}notes.mdacc.tmc.edu
3 The abbreviations used are: HLA, human leukocyte antigen; aa, amino acid; CFU-GM, granulocyte-macrophage colony-forming unit; CML, chronic myelogenous leukemia; CTLp, precursor CTL(s); CTLe, effector CTL(s); LDA, limiting dilution analysis/analyses; PBMC, peripheral blood mononuclear cell; PBPC, peripheral blood progenitor cell; CM, 25 mM HEPES buffer, 2 mM L-glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin; CMV, cytomegalovirus; ATCC, American Type Culture Collection; FBS, fetal bovine serum; IL, interleukin; rhIL-2, recombinant human IL-2; BSP, substrate peptide; PHA, phytohemagglutinin; DLI, donor lymphocyte infusion.
Received 12/22/98. Accepted 3/29/99.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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E. J. Novak, A. W. Liu, J. A. Gebe, B. A. Falk, G. T. Nepom, D. M. Koelle, and W. W. Kwok Tetramer-Guided Epitope Mapping: Rapid Identification and Characterization of Immunodominant CD4+ T Cell Epitopes from Complex Antigens J. Immunol., June 1, 2001; 166(11): 6665 - 6670. [Abstract] [Full Text] [PDF]
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 Y. M. van der Geld, P. C. Limburg, and C. G. M. Kallenberg Proteinase 3, Wegener's autoantigen: from gene to antigen J. Leukoc. Biol., February 1, 2001; 69(2): 177 - 190. [Abstract] [Full Text]
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R. F. Storb, R. Champlin, S. R. Riddell, M. Murata, S. Bryant, and E. H. Warren Non-Myeloablative Transplants for Malignant Disease Hematology, January 1, 2001; 2001(1): 375 - 391. [Abstract] [Full Text] [PDF]
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, tetramer-sorted CTL2 versus P1;
, nonsorted CTL2 versus P1;
, tetramer-sorted CTL2 versus D1;
, nonsorted CTL2 versus D1.