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The Journal of Immunology, 2002, 169: 3407-3412.
Copyright © 2002 by The American Association of Immunologists

TCR Rearrangement in Lymphocytes Infiltrating Melanoma Metastases After Administration of Autologous Dinitrophenyl-Modified Vaccine1

Jayanthi Manne, Michael J. Mastrangelo, Takami Sato and David Berd2

Department of Medicine, Division of Medical Oncology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA 19107


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Administration of a vaccine consisting of autologous melanoma cells modified with a hapten, dinitrophenyl (DNP), induces T cell infiltration of metastatic sites. We have reported an analysis of these infiltrating T cells, indicating that certain TCR-V{beta} gene segments are greatly overexpressed. In this study, we investigate the rearrangement of the TCR-V{beta} as well as the junctional diversity in T cells infiltrating melanoma metastases following treatment with DNP vaccine. In 19 of 26 control specimens, V-D-J length analysis showed the expected polyclonal patterns. In contrast, postvaccine tumors from 9 of 10 patients showed dominant peaks of V-D-J junction size in one or more V{beta} families. Dominant peaks were seen most frequently in six V{beta} families (V{beta}7, 12, 13, 14, 16, and 23) and were never seen in seven others. Further analysis of the oligoclonal V{beta} products showed dominant peaks in the J region as well. Of particular interest was the finding that V{beta} and J{beta} peaks were similar in inflamed metastases obtained at different times or from different sites from the same patient. Although 6 of 10 patients expressed HLA-A1, there was no common pattern of TCR rearrangements among them. Finally, the amplified PCR products from seven of these specimens were cloned and sequenced and the amino acid sequence of the complementarity-determining region 3 was deduced. In six of seven specimens, the same complementarity-determining region 3 sequence was repeated in at least two clones and in five of seven in at least three clones. Our study indicates that DNP vaccine induces the expansion of particular T cell clones that may be agents of its antitumor effects.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Analysis of the TCR repertoire of lymphocytes extracted from blood or tissues can provide insights into the Ags that elicit them. Although a particular peptide structure may or may not elicit a highly restricted T cell response (1), a restricted response implies a limited array of Ags. Moreover, a change in the TCR repertoire following immunological treatment is an indication that the therapy is active, at least on the T cell level.

A number of reports have investigated modifications of the T cell repertoire in normal subjects and in various disease states, including autoimmune diseases, bone marrow and organ transplantation, and cancers (2, 3, 4, 5, 6). Büdinger et al. (7) showed preferential usage of TCR-V{beta} 17 by peripheral and cutaneous T cells in patients with nickel-induced contact dermatitis. Kuwana et al. (8) showed highly restricted TCR-V{beta} usage by autoreactive human T cell clones specific for DNA topoisomerase I in patients with systemic sclerosis. Nakajima et al. (9) reported accumulation of lesion-specific clonal TCR bands in the intestinal lesions of patients with Crohn’s disease by means of single-strand conformational polymorphism. Tanaka et al. (10) detected oligoclonal accumulation of T cells in inflamed skin areas of patients with atopic dermatitis, but not in normal skin. The T cells infiltrating tumor sites, either spontaneously or in response to treatment, have stimulated interest because they may reflect an in situ immune reaction directed to the malignant cells that is difficult to study by other techniques (2, 11).

Our group has been conducting clinical trials of a human cancer vaccine consisting of autologous tumor cells modified with the hapten, dinitrophenyl (DNP)3 (12, 13, 14). The rationale for this approach is the well-established observation that immunization with a hapten-modified protein can induce an immune response to the unmodified, natural protein even when that protein is a normal self-Ag. Thus, Weigle (15) observed that rabbits immunized with hapten-modified thyroglobulin developed Ab to natural thyroglobulin and subsequently autoimmune thyroiditis. Using a different approach to the same end, Neurath et al. (16) induced autoimmune colitis in mice by a single application of the hapten trinitrophenyl to the rectal mucosa.

We have reported that treatment of melanoma patients with the autologous DNP vaccine induces clinically evident inflammatory responses in metastatic sites (12). Biopsy of these inflamed tumors showed intense infiltration with T lymphocytes, predominantly CD8+. RT-PCR-based studies suggested that the infiltrating T cells produced IFN-{gamma} in situ (17). Finally, in some cases this response was followed by tumor regression (14).

In a study of the TCR {beta}-chain variable (TCR-V{beta}) region repertoire in tumor biopsy specimens of six melanoma patients who had been treated with DNP vaccine, we found that certain TCR-V{beta} families were overrepresented in inflamed tumors as compared with matched PBL (11). For example, V{beta}14 was overexpressed in three of six posttreatment metastases studied and accounted for 100% of the T cells in one of them. Molecular analysis of length and sequence composition of the complementarity-determining region 3 (CDR3) region and nucleotide sequencing of V{beta}14 transcripts in two of these samples demonstrated several T cell clones that were not detectable in pretreatment samples.

In this study, we present a more extensive analysis of the TCR-V{beta} rearrangements in melanoma metastases. The sample size has been expanded to more closely examine the differences between postvaccine metastases and control materials. Moreover, we have studied all of the samples by a more sensitive and specific technique, determination of the size distributions of the V-D-J junctional regions, which detects oligoclonality in a V{beta} whether or not that family is overrepresented.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Tumor samples and vaccine administration

The following specimens were studied: 1) melanoma metastases that developed inflammation following treatment of the patients with DNP vaccine (n = 10); 2) matched PBL samples obtained from these patients at approximately the same time as tumor excision (n = 10); 3) metastatic tumors excised from some of these patients before vaccine treatment (n = 5); and 4) melanoma lymph node metastases from randomly selected patients who had not been given vaccine (n = 11).

Tumor tissues were excised, maintained at 4oC, and delivered to the laboratory within 48 h of excision. The tumors were processed as previously described (18). In brief, cells were obtained by enzymatic dissociation with collagenase and DNase, aliquoted, frozen in a controlled rate freezer, and stored in liquid nitrogen in a medium containing 2.5% human serum albumin and 10% DMSO until needed. PBL were separated from blood by density centrifugation and cryopreserved in the same manner. Autologous, DNP-modified vaccines were prepared as previously described (14). In brief, cryopreserved tumor cell suspensions were thawed, irradiated (2500 cGy), and then modified with DNP by incubation with dinitrofluorobenzene (19). The haptenized cells were mixed with bacillus Calmette-Guérin and injected intradermally. All clinical protocols were approved by the Institutional Review Board of Thomas Jefferson University (Philadelphia, PA).

PCR and TCR-V{beta} analysis

Tumor cell suspensions or PBL were thawed and total cellular RNA was extracted using TRIzol according to the method of Chomczynski and Sacchi (20). Subsequently, 5–10 µg of total cellular RNA was reverse transcribed using oligo(dT), and cDNA synthesis was performed as recommended (Life Technologies, Gaithersburg, MD). Amplification was conducted in 50 µl of reaction mixture containing 200 ng of cDNA, 1.5 mM magnesium chloride, 200 µM of each dNTP, 50 pmol of one of the 24 V{beta} primers and a C{beta} primer, and 0.625 U of Taq polymerase (AmpliTaq Gold; PerkinElmer, Foster City, CA). The sequences of the V{beta} primers as well as the C{beta} primers used are listed in the study by Wei et al. (21). The PCR conditions consisted of an initial step of denaturation at 94oC for 10 min followed by 35 cycles of 94oC for 30 s, 60oC for 30 s, 72oC for 1 min, and a final extension of 10 min at 72oC in a GeneAmp PCR System 9700 thermal cycler (PerkinElmer). For each of the 24 V{beta}-C{beta} PCR, a negative control in the absence of cDNA was included. {beta}-Actin served as a standard to normalize for the quantity of mRNA subjected to PCR in the various samples. The PCR products of each of the 24 V{beta}-C{beta} reaction were separated by electrophoresis on 1.5% agarose gel and the sizes of the PCR products were determined; a 123-bp DNA ladder was used to check for fragments of the correct size.

Primer extension in runoff reactions

The 24 V{beta}-C{beta} reaction products of the 35 PCR cycles were subjected to one to three cycles of runoff reactions with C{beta} or J{beta} region primers labeled at the 5' end with the Joe or FAM fluorophores according to the manufacture’s protocols (PerkinElmer). For this purpose, we used 13 J{beta}-specific primers, the sequences of which are listed in Toyonaga et al. (22).

To analyze the V-D-J junctional regions, we used the approach described by Even et al. (23). Accordingly, the runoff products from the V{beta}-C{beta} reactions were electrophoresed through 6% acrylamide sequencing gels on an ABI Prism 377 (Applied Biosystems) the presence of Genescan 500 marker labeled with TAMRA (401733) and ROX (401734) from PerkinElmer as a internal size standard. The raw data generated was further analyzed by Applied Biosystems Prism GeneScan and Genotyper analysis software from Applied Biosystems. The output was a histogram for each V{beta} family showing a frequency distribution of V-D-J junction lengths which infers the distribution of the lengths of the CDR3 region.

For each of the 24 V{beta} families, the histogram was examined and classified into one of three patterns: 1) the presence of a solitary peak or a dominant peak that was at least three times the height of any other peaks; 2) a Gaussian distribution of V-D-J lengths or the absence of any dominant peak as just defined; and 3) the absence of any PCR product.

Sequencing of the TCR

Amplified PCR products were cloned and sequenced to determine the sequence differences in the CDR3 region. The PCR products were cloned into the PCR-Script TM SK+ vector (Stratagene, La Jolla, CA); random clones were picked and the plasmid with insert DNA was sequenced by using the Taq Dye Deoxy Terminator Cycle Sequencing reaction on Applied Biosystems model 9600 DNA sequencing systems (PE Applied Biosystems). The forward and reverse primers T3 and T7, respectively, were used as sequencing primers. The CDR3 was defined according to Moss and Bell (24) and germline sequences were obtained from Toyonaga et al. (22).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
CDR3 size distribution patterns

Fig. 1Go is a summary of the analyses of V-D-J junction size distributions of amplified cDNA from 10 melanoma metastases that developed inflammation following treatment with DNP vaccine. Most of these were superficial metastases that showed clinical signs of inflammation (erythema, swelling, warmth), which was confirmed by histological examination showing infiltration of melanoma cells by T lymphocytes (25). One sample was a lung metastasis that was excised because it was solitary and stable in size; there was histological evidence of inflammation.



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  		FIGURE 1. V-D-J junction size distributions in metastatic melanomas that developed inflammation following DNP vaccine. Amplified DNA was copied in 24 runoff reactions with fluorescent C{beta}-specific primers. {blacksquare}, Single peak or dominant peak; , Gaussian distribution without dominant peak; {square}, no PCR product detected. SC, s.c. metastasis; LN, lymph node metastasis; LU, lung metastasis

 
In 9 of 10 of these inflamed tumor samples, dominant peaks in the V-D-J junction size distribution were demonstrated for one or more V{beta} families. Although no two samples were identical, the occurrence of dominant peaks was not completely random. For six V{beta} families (V{beta}7, 12, 13, 14, 16, and 23), dominant peaks of V-D-J junction size were seen in two or more samples. For seven other V{beta} families (V{beta}3, 5, 8, 18, 19, 21, and 24), dominant peaks were never detected. Serendipitously, 6 of the 10 patients expressed HLA-A1, and 5 of these were associated with HLA-B8. The V{beta}12 and V{beta}14 families showed dominant peaks in three of these specimens, respectively, but there were no other discernible similarities.

Dominant peaks among the V-D-J junction size distributions were found much less frequently in the control materials. Only 4 of 10 matched PBL, collected from the 10 tumor donors at the approximate time of tumor excision, showed dominant peaks, which, with two exceptions (V{beta}2 in patient 20297 and V{beta}20 in patient 20119), did not correspond to the clonally expanded V{beta} families in the inflamed tumors (Fig. 2Go). For five patients tumors obtained before vaccine treatment were available for analysis. As shown in Fig. 3Go, two of these metastases displayed a total of five dominant peaks; two of these (V{beta}6 in patient 20360 and V{beta}22 in patient 20319) appeared to have been lost in postvaccine tumors. Finally, we analyzed 11 randomly selected lymph node metastases from patients who never had been treated with vaccine (Fig. 4Go). With one exception (V{beta}15 in patient 110), the V-D-J junction size distributions were all polyclonal.



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  		FIGURE 2. V-D-J junction size distributions in PBL. For each patient whose inflamed tumor was analyzed, PBL were obtained at about the same time. {blacksquare}, Single peak or dominant peak; , Gaussian distribution without dominant peak; {square}, no PCR product detected

 


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  		FIGURE 3. V-D-J junction size distributions in prevaccine metastases. For five patients, metastatic tissues were obtained before vaccine treatment. None showed clinical or histological signs of inflammation. {blacksquare}, Single peak or dominant peak; , Gaussian distribution without dominant peak; {square}, no PCR product detected. SC, s.c. metastasis; LN, lymph node metastasis

 


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  		FIGURE 4. V-D-J junction size distributions in metastases from control patients not treated with DNP vaccine. Lymph node metastases (n = 11) from patients who never had been treated with vaccine were randomly selected for analysis. All were lymph node metastases; {blacksquare}, single peak or dominant peak; , Gaussian distribution without dominant peak; {square}, no PCR product detected.

 
Comparison of two tumors from the same patients

For two patients, we were able to study two inflamed tumor specimens that had been excised from different anatomical sites. The results are summarized in Fig. 5Go. For patient 20063, the tumors were excised at two time points postvaccine, 4 mo apart. Both showed dominant V-D-J peaks in V{beta}7 and V{beta}10. For patient 20297, the tumors were excised at the same time postvaccine but from two different sites (abdomen and inguinal); both were clinically inflamed and had intense T cell infiltrates by histological examination. Dominant V-D-J peaks in the V{beta}4, V{beta}17, and V{beta}23 families were found in both specimens.



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  		FIGURE 5. V-D-J junction size distributions in two metastases excised from the same patient. For each patient, there were two inflamed tumor specimens that had been excised from different anatomical sites. For patient 20063, the tumors were excised at two time points postvaccine, 4 mo apart. For patient 20297, the tumors were excised at the same time postvaccine but from two different sites (abdomen and inguinal). {blacksquare}, Single peak or dominant peak; , Gaussian distribution without dominant peak; {square}, no PCR product detected. sc, s.c. metastasis

 
Specimens from these two patients were studied further by analysis of J{beta} subfamilies (Fig. 6Go), which confirmed the commonalities. For patient 20063, dominant peaks were observed in V{beta}7-J{beta}1.2 and V{beta}7-J{beta}1.3 in both postvaccine inflamed tumors, but not in matched PBL. For patient 20297, dominant peaks were observed in four V{beta}4 subfamilies, J{beta}1.1, 1.2, 2.1, and 2.4, in both tumor specimens, but were not seen in PBL. Analysis of V{beta}17 subfamilies also showed dominant J{beta} peaks but there were none common to both specimens. An example of the V-D-J size distribution histograms for patient 20297 is shown in Fig. 7Go. The dominant peaks seen in the tumor postvaccine tumors do not appear to be present in the matched PBL.



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  		FIGURE 6. V-D-J junction size distributions after amplification with J{beta} primers. For each patient, two inflamed tumors excised from different anatomical sites are compared with matched PBL. {blacksquare}, Single peak or dominant peak; , Gaussian distribution without dominant peak; {square}, no PCR product detected. SC/sc, s.c. metastasis

 


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  		FIGURE 7. Examples of histograms of V-D-J junction size distributions from inflamed metastases and PBL from patient 20297. Amplification with V{beta} primers revealed a dominant peak in V{beta}4 for both abdominal (Abd.) and inguinal (Ing.) metastases, both of which became inflamed after treatment of the patient with DNP vaccine, but a Gaussian distribution in matched PBL. Amplification with J{beta} primers showed dominant peaks in J{beta}2.1 and 2.4 for both tumors but not in PBL.

 
TCR transcripts of the selectively expanded clones

Seven postvaccine tumor samples that displayed dominant peaks in the V-D-J junction size distributions were further analyzed by cloning and sequencing of the V-D-J genes, from which were deduced the amino acid sequences of the CDR3 regions. From these 7 samples, 42 clones were obtained and all were sequenced. For each patient, we compared the CDR3 sequences found in the tumor with sequences found in the same V{beta} subfamily of matched PBL. The results are shown in Table IGo.


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  		Table I. Sequence analysis of TCR {beta}-chain transcripts in melanoma metastases following DNP vaccine

 
In six of seven tumors (including two tumors from patient 20297), the same CDR3 amino acid sequence was repeated in at least two clones. Moreover, identical sequences were found in three of three clones (V{beta}12-J2.1) from specimen 20113, five of seven clones (V{beta}23-J2.1) from specimen 20254, and three of six clones (V{beta}7-J2.6) from specimen 20249. In the abdominal metastasis from patient 20297, all six clones were V{beta}4-J1.1, comprising one of two CDR3 sequences. This sequence was not detected in the inguinal metastasis from this patient; instead, three of five clones were V{beta}4-J2.1, which was still consistent with the J{beta} overexpression pattern observed in these two specimens (Fig. 6Go). It is noteworthy that none of the repeated sequences were found in matched PBL (data not shown).

Clinical correlations

Four of these 10 patients had measurable metastases that developed inflammation after DNP vaccine treatment. In three of these four cases, the tumors regressed at least partially; these antitumor responses have been described and documented (14). The other six patients were clinically melanoma free when DNP vaccine administration was initiated, but developed recurrent metastases that were inflamed by clinical and histological criteria. Following resection of these tumors, three patients have died and three have remained alive and melanoma free at 7.1, 7.7, and 8.4 years, respectively. Thus, it appears that the infiltration of metastases by clonally expanded T lymphocytes was associated with a favorable clinical response and prognosis.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Conventional studies of in vitro propagated T cells derived from lymphocytes infiltrating human cancers can provide important information about immune responses to particular tumor Ags. However, often the clinical relevance of the results is uncertain, because the effector cells may not be representative of the original T cell population (26). PCR-based analysis of the TCR repertoire of tumors has the advantage of providing an in situ profile of tumor-infiltrating lymphocytes without the need for their in vitro growth and expansion.

It has been demonstrated in several animal models that T cells responding to tumor-associated Ags utilize a restricted TCR repertoire (27, 28, 29). These observations have been extended to human tumors by Ferradini et al. (2), who showed that T cells infiltrating a metastatic melanoma undergoing immunological regression were clonally expanded. They studied a patient who was exhibiting spontaneous regression of a large soft tissue metastases that was accompanied by clinical signs of inflammation and massive T cell infiltration. These T cells greatly overexpressed V{beta}16, and 38 of 45 cDNA clones had the same V-D-J rearrangements (V{beta}16 J2.1).

Some investigators have reported clonal expansion of T cells infiltrating untreated and nonregressing human cancers. However, this phenomenon has been reported mainly in primary tumors (3, 4, 5, 30, 31). Whether clonally expanded T cells spontaneously infiltrate metastases is less clear. Studies of melanoma metastases are limited by the fact that, nodal metastases aside, they are characterized by absence of an inflammatory response (32, 33). In particular, s.c. metastases rarely have lymphocytic infiltration, and the percentage of lymphocytes in cell suspensions made from these tumors is usually <10%. In a previous study of melanoma metastases excised before and after vaccine administration (11), we reported that six of six prevaccine tumors displayed differences in the expression in a total of 22 V{beta} gene families when compared with matched prevaccine PBL. Only 4 of 22 of these V{beta} gene families were overexpressed in postvaccine lesions as well, and in all four cases the expression was increased by vaccination.

In this article, we again demonstrate oligoclonality in T cells from melanoma metastases before treatment, but at a much lower frequency. Only 3 of 16 tumors excised before vaccine or from untreated patients displayed dominant CDR3 peaks in a total of 6 V{beta} families. The different results are attributable to the difference in technique: Whereas a dominant peak in the size distributions of the V-D-J junctional regions implies clonal expansion of T cells, the mere overexpression of a particular V{beta} family, particularly at a low level, could occur as a result of polyclonal expansion as well. The presence of dominant clones in pretreatment metastases could signify a baseline T cell response to one or more melanoma Ags, but in most cases this appears to be qualitatively different from that induced by the DNP vaccine.

There is evidence that cytokine therapy can induce clonal expansion of T cells at the tumor site. Willhauk et al. (34) compared melanoma metastases that appeared to be regressing after treatment with IL-2 plus IFN-{alpha} with tumors that were not responding. Although seven of seven responding tumors contained T cells showing overexpression of some V{beta} families, zero of five nonresponding tumors did so. It is noteworthy that the TCR pattern of the overexpressed V{beta} gene families was diverse even when patients with matching HLA-A phenotypes were compared. Similarly, Kumar et al. (35) reported dominant CDR3 peaks in three liver metastases of colon carcinoma following IL-2 treatment and in only one of four controls. Although Puisieux et al. (36) could not find expansion of any particular V{beta} families following treatment with a chemoimmunotherapy regimen containing the same cytokines, the concomitant administration of cytotoxic drugs in their regimen may have compromised the T cell response.

There are few published analyses of the effect of human tumor vaccines on the T cell repertoire infiltrating metastatic sites (37). Sensi et al. (11) reported our initial study of the TCR-V{beta} repertoire in melanoma metastases that developed inflammation following treatment of patients with autologous DNP-modified vaccine. Posttreatment tumors from six of six patients overexpressed from one to three V{beta} families. CDR3 length analysis of two tumors showed oligoclonal peaks, one of which was confirmed to be clonal by cDNA sequencing. Moreover, T cells from one of these samples were expanded in vitro and found to be cytotoxic for the autologous melanoma cells.

In this article, we have expanded the studies to 10 melanoma patients, 3 of whom were also analyzed by Sensi et al. (11). Moreover, we studied all of the specimens with a more sensitive and specific technique—analysis of the length of the CDR3, which allows for the detection of expanded TCR clones within each V{beta} family, regardless of whether a particular family is overexpressed. In 9 of 10 metastases that developed inflammation following treatment of patients with DNP vaccine, we detected clonal expansion in one or more V{beta} families. These findings were validated by direct sequencing of the TCR. Of particular interest were the results of analysis of tumors obtained from the same patient following vaccine treatment but from two different sites. CDR3 peaks were detected in V{beta}7 and 10 in both specimens from patient 20063 and in V{beta}4, 17, and 23 in both specimens from patient 20297. These results imply that the T cell response to the paired tumors of each patient may have been elicited by similar antigenic determinants.

Our results indicate that the administration of DNP vaccine induced the expansion of T cell clones that were undetectable before treatment. Since these clones infiltrated the tumor sites and were not found in PBL, they were presumably elicited by melanoma-associated Ags. Moreover, the TCR rearrangement data and CDR3 sequencing reported here appear to correlate with clinical findings. Of the four patients with measurable metastases, three had tumor regression following administration of DNP vaccine (14). Of the six patients who were clinically tumor free when DNP vaccine administration was initiated but who subsequently developed recurrent metastases that were inflamed, three are long-term survivors postvaccine. Since the tumors were selected for study only because they developed inflammation, the data support the argument that the DNP vaccine-induced inflammatory response, mediated by clonally expanded T cells, has biological significance.

There are technical limitations that could affect these interpretations. Our criterion for designating oligoclonal expansion in a given V{beta} family was strict: There had to be a clearly defined dominant peak in the histogram of CDR3 size distributions. Consequently, we may have overlooked TCR clones that were present at a low frequency in a polyclonal background. Thor Straten et al. (38) suggested that the picture may be less clear when a more sensitive technique for defining TCR clonality is used. Analyzing PCR products by denaturing gradient gel electrophoresis, they detect as many as 60 different clonotypes in each of 6 s.c. melanoma metastases that were apparently growing progressively. These clonotypes were not found in matched PBL. To determine the significance of these findings would require application of this technique to metastases that were undergoing immunological regression.

Although a variety of human cancer vaccines are in clinical trials, none has yet been validated in a pivotal study. We would argue that the results presented here provide support for our approach of using autologous tumor cells that have been modified by a hapten and supplement the immunological and clinical results previously reported (12, 13, 14). The search for cross-reacting tumor Ags should not obscure the possibility that each human cancer may have unique Ags and that these may be more important in eliciting an antitumor T cell response.


   Acknowledgments
 
We acknowledge the technical assistance of Carmella Clark and Jason Kuchar and the assistance of research nurse Ellen Bloome.


   Footnotes
 
1 This work was funded by Public Health Service Grant CA-39248 and by a research grant from AVAX Technologies, Inc. Back

2 Address correspondence and reprint requests to Dr. David Berd, Thomas Jefferson University, 1015 Walnut Street, Suite 1024, Philadelphia, PA 19107. E-mail address: d_berd{at}mail.jci.tju.edu Back

3 Abbreviations used in this paper: DNP, dinitrophenyl; CDR3, complementarity-determining region 3. Back

Received for publication March 8, 2002. Accepted for publication July 10, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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