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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Environ Mol Mutagen. Author manuscript; available in PMC 2011 February 11.
Published in final edited form as:
PMCID: PMC3037545
NIHMSID: NIHMS225920

Clonal Expansions of 6-Thioguanine Resistant T Lymphocytes in the Blood and Tumor of Melanoma Patients1

Abstract

The identification of specific lymphocyte populations that mediate tumor immune responses is required for elucidating the mechanisms underlying these responses and facilitating therapeutic interventions in humans with cancer. To this end, mutant hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficient (HPRT-) T-cells were employed as probes to detect T-cell clonal amplifications and trafficking in vivo in patients with advanced melanoma. Mutant T-cells from peripheral blood were obtained as clonal isolates or in mass cultures in the presence of 6-thioguanine (TG) selection, and from tumor-bearing lymph nodes or metastatic melanoma tissues by TG-selected mass cultures. Non-mutant (wild-type) cells were obtained from all sites by analogous means, but without TG selection. cDNA sequences of the T-cell receptor (TCR) beta chains (TCR-β), determined directly (clonal isolates) or following insertion into plasmids (mass cultures), were used as unambiguous biomarkers of in vivo clonality of mature T-cell clones. Clonal amplifications, identified as repetitive TCR-β V-region, complementarity determining region 3 (CDR3), and J-region gene sequences, were demonstrated at all sites studied, i.e., peripheral blood, lymph nodes, and metastatic tumors. Amplifications were significantly enriched among the mutant compared with the wild-type T-cell fractions. Importantly, T-cell trafficking was manifest by identical TCR-β cDNA sequences, including the hyper-variable CDR3 motifs, being found in both blood and tissues in individual patients. The findings described herein indicate that the mutant T-cell fractions from melanoma patients are enriched for proliferating T-cells that infiltrate the tumor, making them candidates for investigations of potentially protective immunological responses.

Keywords: human melanoma, T cells, T cell receptors, tumor immunity, molecular biology, hypoxanthine-guanine phosphoribosyltransferase

INTRODUCTION

Studies of hypoxanthine-guanine phosphoribosyltransferase (HPRT) mutations in human peripheral blood T-cells have often revealed evidence of clonal amplifications within the mutant (MT) but not wild-type (WT) fractions [O’Neill et al., 1994]. Several explanations have been offered for this, e.g., T-cell replication in response to exogenous antigen, restricted genomic instability, or, in some individuals, selection by purine analogue therapy [reviewed in Albertini RJ, 2001]. Indeed, using T-cell mutations as mechanistic probes, each has been shown to produce clonality of T-cell mutants [Albertini, et al., 1998; Allegretta et al., 1990; Gmelig-Meyling et al.,1992; Dawisha et al., 1994; Holyst et al., 1994; Van den Berg et al., 1995; Ansari et al., 1995; Falta et al., 1999; Finette et al., 2000]. Although clonality does not seriously bias quantitative results of mutant frequency determinations when group mean values for normal individuals are considered [O’Neill et al, 1994], it can have a large effect when evaluating individual values. As quantitative mutation studies in humans was the reason for developing the HPRT cloning assay in the first place, factors that tend to accentuate clonality must be understood. A corollary to this, however, is that interpreting clonality allows this phenomenon to be used as a mechanistic probe.

The finding of enhanced clonality of T-cell mutants in patients with autoimmune disorders or after organ grafts [above references and Albertini RJ, 2001] supports the dual hypotheses that (i) proliferation underlies function for immunologically reactive T-cells in vivo and (ii) that somatic gene mutations arise preferentially in actively dividing as opposed to quiescent cells. According to these hypotheses, mutant T-cell populations should be enriched for proliferating and, therefore, immunologically active cells. This is the rationale for using selection for T-cell mutants as a surrogate for selecting specific T-cell populations that mediate immunological reactions in individuals in whom such reactions are ongoing. The current study is seeking evidence of such populations in patients with advanced melanoma.

Post-thymic (mature) T-cells possess molecular signatures of their clonal origins. Like all cells of the immune system, T-cells recognize the universe of antigens to which they must respond via surface receptors called T-cell receptors (TCRs). An enormous TCR diversity is generated at the genetic level by somatic rearrangement of relatively few germline-encoded variable (V), diversity (D), junctional (J), and constant (C) region genes, a process that occurs during differentiation in the thymus [Tonegawa, 1983; Alt et al., 1992; Lewis, 1994]. For humans, this is usually during fetal life and childhood, up to mid- to late-adolescense. Once a TCR gene rearrangement has occurred, it remains fixed for the remainder of the life of the now-mature T-cell, being present in all clonal descendants. A T-cell’s rearranged TCR genes, including the hyper-variable complementarity determining region 3 (CDR3), provide an unambiguous molecular signature that defines its clonal relationships.

Although clonality has often been described for HPRT mutants isolated by limiting dilution from blood (i.e., in cloning assays), little is known about its extension to other tissues. In an earlier study, HPRT T-cell mutant frequencies (MFs) were determined for peripheral blood lymphocytes from 48 melanoma patients and 38 normal controls [Albertini MR et al., 2001]. MFs in the patients were significantly higher than in controls, a finding not entirely explained by previous chemotherapy. In nine patients, lymphocytes isolated from melanoma containing lymph nodes (LNs) were also studied for mutations, with the finding that lymph node MFs were slightly but not significantly lower than those of peripheral blood lymphocytes from the same individual. An intriguing finding in one patient was evidence of a MT isolate from the tumor-bearing LN sharing a TCR-β beta chain (TCR-β) gene rearrangement with two mutant isolates from peripheral blood. The rearrangements in this study were characterized by Southern blot analysis, and identity of the rearrangement identified by the sharing of a restriction pattern after multiple enzymatic digests. Nonetheless, this was the first demonstration of clonality of T-cell mutants that encompassed both blood and tissue (LN).

In the present study, we describe a large-scale analysis of the TCR-β gene sequences from WT and HPRT MT T-cells from melanoma patients to investigate the hypothesis that HPRT mutations can be used as biomarkers to study T-cell proliferation and to provide insights into mechanisms underlying the in vivo immune response to human melanoma. We developed a rapid multiplex PCR-based method for sequencing the TCR-β genes from T-cell isolates, and we extended the method to analyses of T-cells from mass cultures. The mass culture system was investigated in lieu of direct cloning to facilitate subsequent functional studies of HPRT- T-cells in melanoma patients. The value of the mass culture is the ability to compensate for cell interactions that may be important for: (a) any functions that are being assessed and (b) growing the cells in vitro. This second benefit may be missed if only the isolates derived from cloning assays are assessed for functional characteristics. In addition, mass culture selection is faster, cheaper, and less time consuming than working with T-cell isolates. Mass culture selection can be applied to blood, to LNs, and to other tissues of interest. We describe these methods here, and their application in demonstrating that mutant T-cell clonal amplifications have representatives in blood, in LNs, and in tumor tissue in melanoma patients. Implications and potential applications of these observations are discussed.

MATERIALS AND METHODS

Human Subjects

The melanoma patients in this study were seen at the William S. Middleton Memorial Veterans Hospital or at the University of Wisconsin Hospital and Clinics between December 2004 and May 2006 and had the diagnosis of biopsy-confirmed melanoma that was either American Joint Committee on Cancer (AJCC) Stage III or IV [Balch et al., 2001]. This study is approved by the Health Sciences Institutional Review Board that serves the William S. Middleton Memorial Veterans Hospital and the University of Wisconsin-Madison. Written informed consent was obtained from the participants in this study.

Nomenclature

The TCR-β gene nomenclature is in accordance with the IMGT gene-segment names that were approved by the HUGO Gene Nomenclature Committee in 1999 [Lefranc et al., 2005; Lefranc MP, and Lefranc G, 2001]. For brevity, we have defined the CDR3 amino acid sequence as the non-templated N-region and the D-region amino acid sequence between the last amino acid encoded by the V-region genomic sequence and the first amino acid encoded by the J-region genomic sequence. The term TCR-β gene pattern refers to TCR-β V-region, CDR3, and J-region (V-D-J). All members of a TCR-β defined clone have identical TCR-β gene patterns.

PBMC Cloning Conditions

Peripheral blood mononuclear cells (PBMCs) were obtained from peripheral blood by standard techniques. PBMCs from subjects in this study were cryopreserved in medium containing 90% fetal calf serum (FCS) (HyClone, Logan, UT) and 10% dimethylsulfoxide (DMSO) for subsequent analysis or were incubated immediately in the cloning assay. To accommodate for cell death that may occur following thawing of cells, all PBMC, whether used fresh or cryopreserved, were cultured at 1 × 106 cells/ml for 16–18 hours in modified RPMI-1640 supplemented with 20% HL-1 medium (Mediatech, Herndon, VA), and 5% FCS, prior to cloning and set-up of mass cultures. We used this approach for all cells, whether fresh or cryopreserved, to maintain consistency of culture conditions for all samples. Following this rest period, the number of viable cells was determined and the cells used in cloning assays. Methods for the T-cell cloning assay for this study have been previously described [O’Neill et al., 1987; Albertini MR et al., 2001]. Briefly, PBMCs were plated by limiting dilution into 96-well microtiter plates to establish cloning plates. PBMCs were plated at 1 to 5 cells/well in the absence of 6-thioguanine (TG) and at 1 × 104 cells/well in the presence of TG (10−5 M) in cloning medium containing RPMI-1640 supplemented with 10% lymphokine activated killer cell supernatant, 20% HL-1, 5% FCS and 0.25 μg/ml PHA (Sigma, St Louis, MO: Patients 1 and 2; Remel, Lenexa, KS: Patients 3, 5 and 9). Accessory feeder cells used were a derivative of WIL-2 lymphoblastoid cells designated as TK6, and these cells were grown in RPMI-1640 containing 10% FCS. Irradiated TK6 cells (9 × 103 cGy) were plated at an initial cell density of 1 × 104 cells/well in both selection and non-selection plates. An inverted phase-contrast microscope was used to score wells for visible colony growth on Day 14 of incubation. The calculations for cloning efficiency (CE) and MF determinations are described in the Statistical Analysis Section and are based on the colony growth on Day 14 of incubation. Single-cell-derived T-cell isolates (SCD-T) were then expanded in cloning medium containing 2.0 × 105 irradiated TK6 cells/ml either with or without TG (10−5 M). SCD-T were split and advanced into larger wells every 3–4 days and maintained in culture until a final lymphocyte count of 1–30 × 106 cells/isolate was obtained. SCD-T cells were then cryopreserved to allow for subsequent molecular analyses.

Mass Culture Conditions

PBMCs were cultured at a cell density of 1 × 106 cells/ml in T25 flasks in a total volume of 20 ml of modified RPMI-1640 (i.e., 2 × 107 cells/flask) supplemented with 20% HL-1 medium and 5% FCS for 16–18 hours. For MT mass cultures, PBMCs were centrifuged and suspended at approximately 1 × 106 viable cells/ml and cultured in the presence of TG (10−5 M) in cloning medium (HPRT selection cultures). WT (non-selection) mass cultures were expanded in an analogous fashion, but with a lower starting number of cells (5 × 103 cells/flask) and without TG. Irradiated TK6 were added to the T25 flask at a density of 2.5 × 105 cells/cm2 surface area. Cultures were incubated and sub-cultured every 4 days, or as needed, using these methods until the required number of viable cells was obtained.

Tumor-Infiltrated Lymph Node (TILN) and Tumor-Infiltrating Lymphocyte (TIL) Mass Culture Conditions

Surgical specimens from TILN from regional LNs or TIL from any metastasis histologically positive for melanoma were initially rinsed in RPMI-1640 medium and then finely minced into less than 1-mm × 1-mm pieces with surgical iris scissors. The mechanically disaggregated specimens were enzymatically digested with Pronase (type XIV Protease, Sigma) by performing 4 to 6 sequential 20-min incubations of the mechanically disaggregated specimen in RPMI-1640 containing 20 U/ml Pronase. The cells in suspension were added to an equal volume of RPMI-1640 with 16% FCS after each incubation, while the mechanically disaggregated specimen received an additional incubation with 20 U/ml Pronase. Following the disaggregation procedure, cells were washed with Phosphate Buffered Saline (PBS) and processed as described above for PBMC mass cultures.

Preparation and Sequencing of cDNAs Encoding the TCR-β from SCD-T

Total RNA was extracted from each individual expanded SCD-T using an RNeasy Mini Kit (Qiagen Inc., Valencia, CA). Total RNA was reversed transcribed into cDNA in a reaction primed by oligo-deoxythymidine (oligo-dT) using SuperScript III Reverse Transcriptase (Invitrogen, Carlsbad, CA). TCR-β transcripts were subsequently amplified for each SCD-T in a single PCR reaction using Taq DNA Polymerase in Storage Buffer A and a TCR-β constant region primer (Cβ Internal, Table I) at a final concentration of 0.2 pmol/ul together with a complete mixture of 26 different human variable TCR-β primers (HUV Beta, Table I) corresponding to the different variable region beta chain (Vβ) gene families, each at a final concentration of 0.2 pmol/ul, according to the manufacturer’s instructions (Promega, Madison, WI). The design of these primers and development of these methods were adapted from the doctoral dissertation of one of us (unpublished data; Judice, S.A., 2001, Human and mouse T-cell trafficking, mutation, and kinetics, University of Vermont, Burlington, VT). The PCR cycling consisted of 30 cycles (95°C for 45 sec; 60°C for 1 min; 72°C for 2 min) followed by a final extension (72°C for 5 min) and a cool-down hold (4°C hold). The TCR-β PCR products were gel purified and sequenced at the University of Wisconsin Biotechnology Center. Analysis of the cDNA sequence and identification of the TCR-β variable, CDR3, and joining regions of the TCR-β mRNA was performed using the DNA sequence analysis tools on the National Center for Biotechnology Information website (http://www.ncbi.nlm.nih.gov). By convention, TCR-β CDR3 motifs are presented below as the converted amino acid sequence, i.e., as the phenotype, which is the antigen reactive product of the rearrangement.

TABLE I
DNA Oligonucleotides Used for Amplification of Human TCR-β.

Preparation and Characterization of cDNAs Encoding the TCR-β from Mass Culture T-cells

Total RNA from mass culture T-cells was prepared as described in the above section. 5′ RACE cDNA synthesis of the TCR-β transcripts from total RNA was conducted using GeneRacer RLM-RACE kit (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions using a gene-specific 3′ oligonucleotide complementary to the constant region of the TCR-β mRNA (24TRBC, Table I). TCR-β transcripts were subsequently amplified using BD Advantage 2 PCR Enzyme System (BD Biosciences Clontech, Palo Alto, CA), a TCR-β constant region primer (24TRBC2, Table I), and the GeneRacer 5′ Nested primer at a final concentration of 0.2 pmol/μl, each according to the manufacturer’s instructions. The 24TRBC and 24TRBC2 oligo sequences were obtained through a personal communication and are published [Lefranc, 2004; Lefranc et al., 2005; IMGT website (http://imgt.cines.fr)]. The PCR cycling consisted of 20 cycles (95°C for 15 sec; 65°C for 30 sec; 68°C for 2 min) followed by a final extension (68°C for 5 min) and a cool-down hold (4°C hold). These PCR conditions were performed to avoid PCR bias inherent in the system [Butler et al., 2005]. TCR-β cDNA was cloned into PCR®4TOPO® (Invitrogen, Carlsbad, CA) using topoisomerase according to manufacturer’s instructions. Plasmids containing TCR-β cDNA were sequenced and analyzed as described in the above section.

Statistical Analysis

HPRT clonal assay data were processed by initially calculating a CE for each assay and a PBMC MF for each individual. To determine standard estimates for CE and MF, CE was calculated by use of the Poisson relationship, P0 = exp(−x), where P0 is the fraction of wells without colony growth and x is the average number of clonable cells per well. The value of x divided by the number of cells added to each well (n0) defines the cloning efficiency. The HPRT MF with this calculation is the ratio of the mean CE in the presence (selection) and absence (non-selection) of TG. Averaging yielded CE and MF values in cases with multiple dilutions.

The comparison of TCR-β V-D-J usage between WT T-cells and MT T-cells involves an evaluation of clustering patterns compared with what would be expected in a sample if the HPRT selection process were not enriching for amplifying T-cell clones. On that null hypothesis, the population sampled in WT and that sampled in MT are equivalent, and observed differences are attributable to stochastic variation alone. Fisher’s exact test for comparing two multinomial samples is therefore appropriate [Agresti, 2002]. This test utilizes data on the frequency of every distinct CDR3 sequence, and allows that even in the absence of enrichment by the selection to MT, there may be variation in the in vivo size of T-cell clones. It is sensitive to violations of the null hypothesis that would make TCR-β V-D-J usage patterns distinct in the selected population, such as excessive clustering on one or two particular sequences. We implemented Fisher’s test using independent Monte Carlo sampling with B=50000 samples per comparison.

We developed an extension to Fisher’s exact test in order to accommodate the possibility that in vitro growth after selection in the MT mass culture cells could create excessive clustering, even if selection is not enriching for amplifying T-cell clones (a population bottleneck could occur if initial cell frequency is low compared with MF). Recall that Fisher’s test may be derived by modeling the number Xi of occurrences in the WT of a TCR-β V-D-J sequence i as a Poisson variable with rate i, where n is the number of clones sequenced and θi is the abundance of i in vivo. Similarly Yi, the analogous count for the MT cells has the same Poisson distribution, on the null hypothesis that selection is not enriching for amplified clones. Fisher’s test emerges by conditioning on totals ΣXi, ΣYi, and Xi + Yi for all i. Our extended method followed this logic, but we replaced the Poisson model for the sample abundance Yi in the MT with a Negative Binomial model having the same mean, but having a variance inflated by an amount determined by MF and cell density. The conditional probability mass function for TCR-β V-D-J counts is intractable analytically but is amenable to Monte Carlo simulation, which we used to derive a p-value measuring the significance of the most frequent MT TCR-β V-D-J (details in statistics manuscript in preparation). P-values <0.05 were considered significant.

RESULTS

Cloning Assays: In vivo Clonality of SCD-T from Peripheral Blood

Two-hundred ninety-eight MT and 133 WT SCD-T, isolated from cloning assays in five patients in which non-selected cloning efficiencies ranged from 0.08 to 0.15 and MFs ranged from 17.6 × 10−6 to 97.3 × 10−6 (Table II), were analyzed for TCR-β V-region, CDR3, and J-region sequences (Table III). Using the criterion that an identical TCR-β gene pattern in two or more independent SCD-T from a given individual reflects their in vivo clonal relationship, MT SCD-T from Patient 1 showed evidence of four expanding in vivo clones, with 3, 9, 14, and 2 representatives of each among the 69 MT SCD-T studied. Unexpectedly, evidence of a single in vivo expanding clone reflected in 2 WT SCD-T sharing a TCR-β gene pattern without a counterpart among the mutants was found among the 36 WT SCD-T studied (see below and Table VI for MT-WT sharing of TCR-β gene patterns). In this Patient, 41 MT and 34 WT SCD-T showed unique TCR-β gene patterns, ie., specific TCR-β V-D-J sequences in only a single isolate. The sequence analysis of the TCR-β V-D-J for MT and WT SCD-T from Patients 1,2,3,5 and 9 is shown in Table III.

TABLE II
Patients in the Study.
TABLE III
Clonality Analysis of Mutant (MT) and Wild Type (WT) Single-Cell-Derived T-cells (SCD-T) from Peripheral Blood Mononuclear Cells (PBMC).
TABLE VI
Analysisa of in vivo Expanded Mutant (MT) and Wild Type (WT) T-cell Clones Present in Peripheral Blood Mononuclear Cells (PBMC) and Tumor-Infiltrated lymph Node (TILN) or Tumor-Infiltrating Lymphocytes (TIL) from Patients 1, 5, and 9b,c.

The Fisher’s test was used to examine the differences in clonality distributions between MT and WT T-cell SCD-T for this set of patients. This difference was significant for Patient 1 (p<0.0001) and Patient 9 (p = 0.002), but not for Patient 2 (p = 0.068) or Patient 3 (p = 0.10). There was no in vivo clonality identified in either the MT or WT SCD-T from Patient 5.

Mass cultures: In vivo Clonality of Mass Cultures Derived from Peripheral Blood

Peripheral blood T-cells from each of the five patients were propagated in mass in vitro cultures, with or without TG selection, as described above, to obtain populations of MT and WT T-cells, respectively. The duration of cultures differed among the patients to obtain the requisite numbers of cells for analyses, but the selection and non-selection culture durations for each individual were the same. These were 25, 13, 18, 19, and 17 days for patients 1, 2, 3, 5, and 9, respectively. Total RNA from these mass populations were then obtained and analyzed as described.

A total of 107 MT cDNAs and 67 cDNAs from WT cDNAs were analyzed for Patient 1 (Table IV). Seven clonal clusters were detected among the cDNAs from the MT T-cells, with 6, 16, 3, 55, 3, 7 and 14 copies of each defining TCR-β V-D-J sequences (Table IV). There were only three unique TCR-β gene patterns among these cDNAs from the MT T-cells. However, all 67 cDNAs from the WT T-cells derived from this patient showed unique TCR-β gene patterns. The sequence analysis of the TCR-β V-D-J for MT and WT PBMC mass cultures from Patients 1,2,3,5 and 9 is shown in Table IV. The generalized Fisher’s test was used to examine the differences in clustering as an indicator of clonality distributions between the cDNAs derived from MT and those derived from WT T-cells. The differences between cDNAs derived from MT and WT T-cells was highly significant for Patient 1 (p<0.0001), Patient 2 (p = 0.007), Patient 3 (p <0.0001), Patient 5 (p = 0.0006), and Patient 9 (p < 0.0001).

TABLE IV
Clonality Analysis of Mutant (MT) and Wild Type (WT) Mass Cultures from Peripheral Blood Mononuclear Cells (PBMC).

In both Patient 1 and Patient 9, identical TCR-β gene patterns were found among the MT SCD-T derived from cloning assays, and in the cDNAs derived from mass cultures of MT T-cells, indicating that mass cultures of peripheral blood T-cells can identify the same in vivo proliferating MT T-cell clones (Tables III, ,IV,IV, and summarized in Table VI).

Mass cultures: In vivo Clonality of Mass Cultures Derived from TILN and TIL

T-cells derived from tumor-containing LNs (Patient 1) or metastatic melanoma tissue (Patients 2, 5 and 9) were propagated in vitro to obtain sufficient cells for analysis. Two anatomically separated subcutaneous nodules were harvested from Patient 9, from a back nodule (9A) and from a neck nodule (9B). Due to low numbers of cells in some of these samples, T-cells from Patients 1, 2, and the 9A tumor sample required growth in vitro for 12, 22, or 10 days, respectively, prior to their culture with or without TG selection for an additional 40, 25, or 21 days, respectively. Adequate numbers of T-cells were obtained directly from Patient 5 and from the Patient 9B tumor sample allowing direct in vitro culture with or without TG selection for a total of 29 or 19 days, respectively. Insufficient cells for tissue analyses were obtained from Patient 3.

In Patient 1, 45 cDNAs from the MT T-cells were analyzed for TCR-β V-D-J gene sequences, and all showed the identical gene patterns (Table V). Therefore, this single clonal cluster accounted for all MT T-cells propagated from a tumor-bearing LN in this patient. By contrast, seven clusters of TCR-β gene patterns, represented by 9, 3, 2, 5, 2, 3 and 2 repeats, were observed among the 36 cDNAs obtained from the WT T-cells grown from this lymph node, while 10 showed unique TCR-β gene patterns. The sequence analysis of the TCR-β V-D-J for MT and WT TIL and TILN mass cultures from Patients 1,2,5 and 9 is shown in Table V. Note that sequence analysis of TIL from a back nodule (9A) and from a neck nodule (9B) is provided for Patient 9. As for the studies for the mass cultures obtained from peripheral blood, a generalized Fisher’s test was used to examine the differences in cluster distributions between MT and WT and to accommodate the likelihood that in vitro growth after selection of mutant cells would create clustering. Again, the difference between cDNAs derived from MT T-cells and those derived from WT T-cells were highly significant for Patients 1, 2, 5, and the Patient 9A tumor sample (p<0.0001) as well as for the Patient 9B tumor sample (p = 0.004).

TABLE V
Clonality Analysis of Mutant (MT) and Wild Type (WT) Mass Cultures from Tumor-Infiltrated Lymph Node (TILN) and Tumor-Infiltrating Lymphocytes (TIL).

It is noteworthy that in Patient 1 and in Patient 9, identical TCR-β gene patterns were seen for MT T-cells derived from peripheral blood as SCD-T and/or mass cultures and from mass cultures derived from TILN and/or TIL (Table VI). This indicates not only that proliferating MT T-cell clones can be identified in mass cultures of blood, but that they are also present at tumor sites in melanoma patients.

Shared TCR-β Gene Patterns Between MT and WT T-cell Fractions

In addition to clonality, as manifest by TCR-β gene patterns in the MT and WT fractions from peripheral blood and TIL/TILN from patients with melanoma, the unique TCR-β gene patterns within each fraction were analyzed for MT-WT sharing. A single MT isolate obtained as a SCD-T from peripheral blood of Patient 5 showed an identical gene pattern with that seen in WT T-cells obtained from TIL in that same patient (Table VI). The possibility that this in vivo clone grown in mass culture without TG might be an HPRT mutant has not been formally excluded. However, this finding reflects clonality, with representatives in both the MT and WT fractions of T-cells.

DISCUSSION

T-cell cloning for HPRT mutations is arguably the most frequently used assay for assessing somatic gene mutations arising in vivo in humans. Although its major use is in population monitoring for environmental mutagens, there are other applications of the assay. One is in characterizing the target cells in which the mutations arise, i.e., the T-cells. That use is illustrated by the results presented here. The goal of HPRT T-cell selection in melanoma patients is to identify a probe for the in vivo T cell response to human melanoma that provides mechanistic insights and helps direct subsequent monitoring and/or treatment strategies for patients with melanoma.

Malignant melanoma, a malignancy present in all five patients reported here, is expected to elicit a T-cell immunological response against tumor antigens, with resultant T-cell proliferation. To provide the rationale for studying surrogate selection in the context of melanoma, additional background information about T-cell responses to melanoma will be provided. Definitive evidence that melanoma-associated antigens (MAA) can stimulate cytotoxic and regulatory T-cell responses against human melanoma as well as serve as tumor-rejection antigens for T-cells in model systems has been present for many years [Mukherji et al., 1989; Van der Bruggen et al., 1991]. The characterized MAA can effectively stimulate cellular immune responses in vitro, and immunodominant epitopes have been identified and characterized [Kawakami et al., 1995; Kawakami et al., 1994; Kierstead et al., 2001; Topalian et al., 1994, Zarour et al., 2002]. Ongoing investigations are attempting to translate this understanding of MAA into effective in vivo therapeutic strategies [Pardoll, 2002]. However, clinical responses remain infrequent. While tumor-rejection antigens on human melanomas would provide an opportunity for effective targeted immune-based therapy, the MAA associated with effective in vivo anti-melanoma T-cell activity still need to be identified. It is possible that study of in vivo expanding T-cells in melanoma patients will provide insights into the in vivo immune response to this disease.

Several methods have been utilized to investigate in vivo clonally amplified T-cells including TCR-β analysis by Southern blot [Albertini MR et al., 2001], MaGiK method [Killian et al., 2002], RT in situ PCR [Nuovo et al., 2001], padlock probes and microarrays [Baner et al., 2005] and quantitative RT-PCR with spectratyping [Degauque et al., 2004]. These methods can provide a global look at the TCR-β repertoire in melanoma patients and have identified CDR3 length alterations [Degauque et al., 2004; Speiser et al., 2006] over-expression of TCR-β V-region subfamilies [Degauque et al., 2004; Sensi et al., 1997; Speiser et al., 2006; Willhauck et al., 2003] and clonal expansion of T-cells after melanoma vaccination or cytokine immunotherapy [Degauque et al., 2004; Meidenbauer et al., 2004; Sensi et al., 1997; Speiser et al., 2006; Willhauck et al, 1996]. Additional insights into the detailed antigen specificity of in vivo T-cell responses to melanoma could be provided by identifying T cells undergoing ongoing or repetitive in vivo cell division and using a multiplex PCR-based method and subsequent analysis of TCR-β V-D-J gene sequences.

Elsewhere, we have suggested that a preferential occurrence of clonality among MT T-cells compared to WT T-cells is a result of an increased likelihood of spontaneous mutations in rapidly proliferating cells as opposed to quiescent G0 cells — the condition of the vast majority of mature T-cells in vivo [O’Neill et al., 1994; Falta et al., 1999; Albertini RJ, 2001]. It may be that the rapidly proliferating cells may have more opportunity to make replication errors or less time to repair DNA damage. In part, preferential clonality in the MT fractions of T-cells may be a reflection of the need for amplification of HPRT mutations in order to be recovered in cloning assays. In any case, we have suggested that this phenomenon could be exploited for isolation of T-cells of immunological relevance — a form of surrogate selection. This strategy has been used to recognize such cells in autoimmune disorders and in organ transplantation [Albertini RJ, 2001 and references therein]. The study reported here suggests its application in cancer patients.

It may be asked —as was done in this study — the degree to which T-cell clonality identified by cloning assays represents the broader in vivo situation. Does this clonality reflect what is present in peripheral blood? More importantly for cancer patients, does it reflect what might be occurring within the tumors themselves?

In vivo clonality, as defined here, is a characteristic of the cells used to detect HPRT mutations, and not necessarily of the HPRT mutations themselves. Cellular clonality for the post-thymic, mature T-cells originates when the TCR genes undergo rearrangement, usually during thymic differentiation. HPRT mutational clonality begins with the occurrence of that mutation, which may be before, during, or after thymic differentiation. The timing of HPRT mutations relative to T-cell maturation has implications as to the anatomical sites of mutation [Albertini RJ, 2001]. Post-thymic mutation implies origination in the peripheral lymphoid tissue, i.e., LNs, spleen, peripheral blood, or other tissues where T-cells circulate; pre-thymic mutation implies origination in bone marrow or in the thymus itself.

In this regard, the finding of an identical TCR-β V-D-J sequence in a MT SCD-T clone and in the WT fraction of TIL derived mass culture cells from Patient 5 (Table VI) is of note. We have only rarely observed MT–WT sharing among clonal SCD-T derived from HPRT cloning assays, i.e., as SCD-T clones [O’Neill et al., 1994]. However, similar analyses of MT and WT fractions obtained from mass cultures of PBMCs or tissue-derived lymphocytes (TILN and TIL in the current study) have not previously been undertaken. The studies reported here are being extended to examine potential preferential usage of V-regions as well as potential clonal relationships between patients, with the addition of more patients and analyses of restricted TCR-β gene usage among and within patients. Indeed, we have observed MT–WT sharing of TCR-β gene patterns in mass cultures derived from blood and TILN/TIL (data not shown). The MT-WT sharing data at the mass culture level suggests that the preferential recovery of expanding MT T-cell clones is not simply due to dilution of clonality of WT T-cells because clonality, in this fraction, can be observed if it is extensive enough. However, when it is this extensive, an increased frequency of somatic mutations occur, as reflected in the presence of HPRT mutations, with the finding that these expanding in vivo clones usually have MT representatives. Importantly, the presence of identical TCR-β gene patterns in both the MT and WT fractions of expanding T-cell clones indicates that the HPRT mutation has occurred in mature T-cells in the periphery, i.e., after the TCR gene rearrangement has occurred.

As shown in Tables IV, ,VV and andVI,VI, preferential clusterings of TCR-β gene patterns were clearly reflected in the cDNAs derived from mass cultures of T-cells in peripheral blood and in tumors (in LN or other tissue). There were more clusters, and clusters of larger sizes, in the cDNAs derived from MT cultures than in those derived from WT cultures. These differences were statistically significant for all patients. Although some preferential clustering may have been expected because of size differences in starter population between the selected and non-selected in vitro cultures (the MT populations were much smaller based on calculations involving starting numbers of cells and MF) and/or differences in in vitro outgrowth of selected cells, allowances in the statistical analyses were made for that consideration.

The most compelling demonstration that MT T-cell clonality as reflected in cloning assays represents the broader in vivo situation is the identity of the TCR-β gene rearrangements in MT SCD-T to the rearrangement clusters of TCR-β cDNAs derived from both PBMC and TILN/TIL MT mass cultures in Patients 1 and 9 (Table VI). The finding of identical MT T-cells both in the peripheral blood and at sites of tumor suggests trafficking of expanding MT in vivo T-cells clones between the peripheral blood and sites of tumor. These findings validate analysis of MT isolates from blood when the target organ is not accessible, and these analyses can be used to investigate T-cells potentially involved in the in vivo T-cell response to human melanoma. Members of these expanding MT T-cell clones are expected to be present at frequencies difficult to detect within the WT fraction of T-cells from the same patient.

In conclusion, in vivo clonal amplifications of MT T-cells are present in the peripheral blood and at sites of tumors of melanoma patients. These expanding in vivo T-cell clones can be identified by analysis of TCR-β gene usage of TG-selected clonal SCD-T and of TG-selected mass cultures. In vivo MT T-cells in melanoma patients provide candidate probes to investigate the in vivo T-cell response to human melanoma.

Acknowledgments

Grant Sponsors: This material is based on work supported by the Office of Research and Development, Biomedical Laboratory Research and Development Service, Department of Veterans Affairs; the U.S. Department of Energy; the Gretchen and Andrew Dawes Melanoma Research Fund; Ann’s Hope Foundation; the Jay Van Sloan Memorial from the Steve Leuthold Family; and the Tim Eagle Memorial.

We thank Drs. David Mahvi, Sharon Weber, and Tracey Weigel for identifying appropriate surgical specimens; Ms. Kathy Schell, Ms. Terri Messier, and Mr. Adam Breunig for technical assistance; Ms. Melinda Baker and Ms. Susi Nehls for assistance with manuscript preparation; and Drs. J. Patrick O’Neill, Barry Finette, and Mark Allegretta for stimulating discussions.

Abbreviations

AJCC
American Joint Committee on Cancer
C
constant
cDNA
complementary DNA
CDR3
third complementarity determining region
CE
cloning efficiency
D
diversity
DMSO
dimethylsulfoxide
DNA
deoxyribonucleic acid
FCS
fetal calf serum
HL-1
type of tissue culture media
HPRT
hypoxanthine guanine phosphoribosyltransferase
HPRT-
HPRT deficient
IMGT
Immunogenetics
J
junctional
LNs
lymph nodes
MAA
melanoma-associated antigen
MF
mutant frequencies
mRNA
messenger RNA
MT
mutant
NIH
National Institute of Health
NLM
National Library of Medicine
Oligo-dT
oligo-deoxythymidine
PBMCs
peripheral blood mononuclear cells
PBS
phosphate buffered saline
PCR
polymerase chain reaction
PHA
phytohemagglutin
RACE
rapid amplification of cDNA ends
RNA
ribonucleic acid
RPMI-1640
type of tissue culture media
SCD-T
single-cell-derived T-cell
TCR
T-cell receptor
TCR-β
T-cell receptor beta
TG
6-thioguanine
TIL
tumor-infiltrating lymphocyte
TILN
tumor-infiltrated lymph node
TK6
lymphoblastoid cell line (derivative of WIL-2)
V
variable
WIL-2
lymphoblastoid cell line
WT
wild type

Footnotes

1Running title: T cell mutant clonal expansions in human melanoma

The authors have no financial or other conflicts of interests to disclose related to this publication.

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