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Human Cancer Biology |
Authors' Affiliations: 1 Harvard Skin Disease Research Center, Department of Dermatology, Brigham and Women's Hospital, and 2 Biostatistics Core Facility, Dana-Farber Harvard Cancer Center, Department of Biostatistics, Dana-Farber Cancer Institute, Boston, Massachusetts
Requests for reprints: Thomas S. Kupper, Harvard Skin Disease Research Center, Department of Dermatology, Brigham and Women's Hospital, Harvard Institutes of Medicine, 77 Avenue Louis Pasteur, Boston, MA, 02115. Phone: 617-525-5550; Fax: 617-525-5571; E-mail: tskupper{at}rics.bwh.harvard.edu.
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Abstract |
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Experimental Design: We analyzed TRECs from unfractionated peripheral blood T cells from 108 CTCL patients by quantitative PCR. In patients with obvious peripheral blood involvement, we also analyzed TRECs from clonal and nonclonal T cells.
Results: We found a decrease in the number of TRECs in peripheral blood of patients with CTCL at all stages of disease, and this decrease was proportional to the loss of complexity of the T cell repertoire as measured by complementarity-determining region 3 spectratyping. In patients with leukemic CTCL and a numerically expanded clone, we also found a significantly lower-than-expected number of TRECs in the nonclonal normal T cells.
Conclusions: We hypothesize that the nonmalignant T cells have proliferated to fill the empty T cell repertoire space left by the loss of other T cells, leading to diminished TRECs and loss of T-cell receptor diversity.
Recently, a new biomarker, known as T-cell receptor excision circles (TREC), has been reported to reflect recent thymic emigrants in human peripheral blood (11, 12). Signal joint 
Rec-J 
(Rec-J) TRECs in the T-cell receptor locus are excised late in the course of thymic T cell development by the TCRA locus recombination process, which generates the repertoire of antigen-specific TCR /ß T cells (11). TRECs exist in cells as stable intracellular extrachromosomal circular excision products. These episomes do not replicate during mitosis and are thus diluted during subsequent T cell proliferation. The concentration of TRECs therefore measures the ratio of unexpanded naïve T cells relative to T cells that have undergone clonal expansion. Measurement of TRECs in human peripheral blood and thymus with the quantitative PCR can offer a tool to identify recent thymic emigrants and thus to estimate thymic output. The two other major known biological parameters affecting TREC levels are longevity of naïve T cells and the dilution of TRECs by cell division (13).
If our hypothesis is correct regarding the deletion of nonmalignant T cells in CTCL, with the clonal expansion of surviving nonmalignant T cells to fill the T cell compartment, then we would predict that TREC levels should be decreased in the peripheral blood of patients with CTCL. In the present study, we analyzed TRECs in peripheral blood T cells from 108 patients with CTCL. We found a decreased copy number of TRECs in the blood of both early- and late-stage CTCL patients. The decrease in TREC levels was proportional to the degree of loss in complexity of the T cell repertoire, as measured by complementarity-determining region 3 (CDR3) spectratyping. We also found that patients with advanced disease and a dominant malignant clone had the lowest TREC levels. In these patients, even the nonmalignant T cell population showed reduced levels of TREC. The cause of the expansion of these nonmalignant apparently normal T cells is unknown, but we hypothesize that, in these patients, the remaining T cells have expanded to fill the empty "space" in the T cell compartment created by the loss of normal T cells and of their attendant diversity.
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Materials and Methods |
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Preparation of purified CD3+ T cells. Peripheral blood mononuclear cells were isolated from heparinized venous blood obtained from patients with CTCL and healthy volunteers by density gradient centrifugation over Ficoll (Histopaque, Sigma, St. Louis, MO). CD3+ T cell populations were separated with immunomagnetic beads following the manufacturer's protocols (Miltenyi Biotec, Auburn, CA). Briefly, for CD3+ T cell selection, after 10 minutes of incubation with 20 µL of an antibody cocktail mixture, peripheral blood mononuclear cells were incubated for 15 minutes with 20 µL of magnetic beads per 107 cells. CD3+ T cells were then isolated from peripheral blood mononuclear cells by negative selection over MiniMACS separation LS columns. Sorted populations were analyzed by flow cytometry, and purity ranged between 96% and 99%.
Genomic DNA extraction. Genomic DNA was extracted from CD3+T cells by means of the Wizard Genomic DNA Purification Kit (Promega, Madison, WI) according to the manufacturer's instructions. Prior to PCR amplification, DNA concentration in all samples was determined by UV spectrophotometry (Smartspec TM3000, Bio-Rad, Hercules, CA), and all samples typically had A260/A280 ratios >1.6.
Quantification of T-cell receptor excision circles. Real-time PCR analysis was done to quantify the signal-joint Rec-J (Rec-J) TRECs. Rec-J rearrangements occur late during T cell differentiation in the thymus, mostly after thymocyte expansion. Rec-J TRECs are considered to be a valid marker for these young T cells (14). We used a fluoresceinated probe that hybridizes between the PCR primers. For PCR, the following primers and probes were used: sense, 5'-CGT GAG AAC GGT GAA TGA AGA GCA GAC A-3'; antisense, 5'-CAT CCC TTT CAACCA TGC TGA CAC CTC T-3'; and detection probe sequence, 5'-VIC-TTT TTG TAA AGG TGC CCA CTC CTG TGC ACG GTG A-TAMRA-3' (15, 16).
The VIC fluorophore emits at a wavelength of 340 nm once it has been liberated from the proximity of the TAMRA quencher element by the action of the DNA polymerase. Each PCR reaction was done in a 50 µL reaction mixture containing 0.075 µg of DNA and the following concentrations of other components: 25 µL of TaqMan buffer A (ABI, Foster City, CA), 3 mmol/L MgCl2, 300 nmol each primer, 100 nmol probe, 200 nmol dATP/dCTP/dGTP, 400 nmol dUTP, 17 units of uracil-N-glycosylase, and 2 units of AmpliTaq Gold DNA polymerase (ABI). PCR was done under the following conditions: 50°C for 2 minutes followed by 95°C for 10 minutes, after which 50 cycles of amplification were carried out (94°C for 30 seconds, 60°C for 30 seconds). PCR was done with a spectrofluorometric thermal cycler (ABI Prism 7700 Sequence Detector System) that measures the independent fluorescent spectrum of each well. A series of standard dilutions of a plasmid containing the signal-joint break point was used to quantify TRECs. The plasmid was obtained after amplification of human cord blood genomic DNA with the primers: sense, 5'-AAA GAG GGC AGC CCT CTC CAA GGC AAA A-3'; and antisense, 5'-ACT TCC ATC GCA ATT CAG GAC TCA CTT-3' (17). Each patient and control DNA sample was run in duplicate on the same 96-well plate along with the dilution series of the TRECs plasmid and a dilution series of a rRNA plasmid and repeated twice. The established primers and probes for rRNA were purchased from Clontech (Palo Alto, CA). The rRNA copy number served to standardize for the amount of genomic DNA content. For each sample, the Ct value threshold cycle, defined as the minimal number of cycles necessary to exceed threshold values, was measured and applied to the standardization curve created from the dilution standard curve of known plasmid concentrations. Results were extrapolated to TRECs copy numbers per 1 µg DNA.
CDR3 spectratyping to identify contracted profiles and monoclonal peaks. For spectratyping analysis, total RNA was extracted from 3 x 106 CD3+ T cells using the Clontech RNA purification kit. Two to 5 µg of total RNA (A260/A280 = 1.7-2.0) was reverse-transcribed using oligo-dT primers and Powerscript Reverse Transcriptase (Clontech). TCR ß-variable (BV) segments were amplified with 1 of 26 BV subfamily-specific primers, as well as with CB primers recognizing both CB1 and CB2 regions. Sequences of BV 1 to 9, 11, 13 to 16, 18, and 20 primers were as in Choi et al. (18); those of CB and BV 10 primers, as in Genevee et al. (19); those of BV 22 and 24, as in Moss et al. (20); those of BV 12, as in Hall and Finn (21); those of BV 17 and 19, as in Bragado et al. (22); and those of BV 21 and 23, as in Hand et al. (23). BV subfamilies are numbered as in Wei et al. (24). PCR products were applied to a 5% polyacrylamide sequencing gel, and the size distribution of each fluorescent PCR product was determined by electrophoresis on an automated 377 DNA sequencer (ABI). With this technique, an amplified TCR BV subfamily migrates as a series of bands. Data were analyzed with GeneScan software (ABI) that assigns a size and peak area to the different PCR products.
Scoring of CDR3 profiles was done by determining the number of contracted BV CDR3 size profiles in each subject's T cell CDR3 repertoire. Contracted profiles were defined as follows: oligoclonal (two to four peaks), monoclonal (one peak), or absent (no peaks detectable). The analysis was done by two different investigators in a blinded fashion (8).
Flow cytometric analyses. The presence or absence of a dominant clone in CTCL patients was determined by three-color flow cytometric analysis. Analysis was done on peripheral blood mononuclear cells using the following monoclonal antibodies to the TCR BV chain: PE conjugated antibodies to BV 1, BV 2, BV 5.1, BV 5.2, BV 5.3, BV 7, BV 9, BV 11, BV 12, BV 13.1, BV 13.6, BV 14, BV 16, BV 17, BV 18, BV 20, BV 21.3, BV 22 (Immunotech/Beckman Coulter, Brea, CA); PE-conjugated antibodies to BV 3, BV 8, BV 23 (BD Bioscience, San Diego, CA); CD7 FITC (BD Bioscience), and CD4 PerCp (BD Bioscience). The isotype controls used were IgG1PE, IgG2a PE, IgG2b PE, rat IgG1 PE (Immunotech/Beckman Coulter), IgG1 FITC, and IgG1 PerCp (Becton Dickinson, Mansfield, MA). Cells were washed with PBS and then fixed with 1% paraformaldehyde; antibodies were used at a 1:100 dilution. Immunophenotypic analysis of cells was done with a CellQuest flow cytometer (Becton Dickinson).
Dilution experiments. Normal CD3+ T cells from healthy volunteers were mixed with Jurkat cells (which do not have TRECs) in 11 combinations from 0% to 100% to simulate a hypothetical patient who had a mixture of clonal CTCL cells that are assumed to have expanded greatly and nonmalignant T cells. DNA was extracted and TREC levels were measured as described above.
Comparison of dilution experiment with patient data. TREC levels were compared between dominant clone–positive patients' actual data and predicted data extrapolated from dilution experiments. The predicted data was derived from the patient's percentage of clonal cells based on BV antibody flow cytometry measurements.
Purification of dominant clones and nonclonal cells. Dominant clones from CTCL patients were selected with antibodies to the appropriate TCR BV region as follows. First, CD3+ T cells were negatively selected with magnetic beads as described above. These cells were then incubated with PE-conjugated antibody to TCR BV targeted to the clonal BV (described above) and selected with anti-PE microbeads (Miltenyi Biotec). Nonclonal populations were passed through, and clonal cells were positively collected. The purity of the sorted population was analyzed by flow cytometry and ranged between 96% and 99%.
Statistical analysis. Linear regression models were fitted to the TREC data from all patients. The models included logarithmic values (to the base 10) of the TREC data as the dependent variable and age, gender, stage, number of monoclonal peaks, and contracted profiles as the independent variables. We also examined the correlations between log TRECs and the number of monoclonal peaks and contracted profiles. The Wilcoxon-Mann-Whitney test was used to evaluate differences in TREC data between the normal cohort and cohorts with various mixtures of Jurkat and normal cells. The Bonferroni correction was used for multiple comparisons. We constructed 95% confidence intervals on the predicted data from simulated experiments and checked if the actual TREC data was contained within them. We also constructed 95% confidence intervals on the CD3 T cells from 33 normal controls and checked if TREC data in nonclonal T cell population from 4 patients was contained within them.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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The correlation between log TRECs and the number of contracted profiles and monoclonal peaks was –0.47 and –0.46, respectively (Fig. 2A and B). There was a high positive correlation between the number of contracted BV spectratype profiles and monoclonal BV spectratype peaks (0.82), suggesting colinearity. Therefore, a linear model was fitted to the log TREC data, with the number of contracted BV spectratype profiles as the independent variable (Table 2). The number of contracted profiles was a significant covariate. In this analysis, patients with five or greater contracted BV family profiles had TREC levels that were >100-fold lower than normal levels. Patients with three to five abnormal BV spectratypes had TREC values that were intermediate but still reduced. Patients with more than five monoclonal peaks had TREC values >100-fold lower on average than patients with one or no monoclonal peaks (Fig. 2B).
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Discussion |
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TREC levels are measured by PCR and are expressed as a ratio of TREC-specific DNA to 1 µg of T cell DNA. High TREC levels suggest that a T cell population contains a significant fraction of unexpanded naïve T cells, whereas lower TREC levels suggest a smaller fraction of unexpanded cells. A lower number of naïve cells could be due either to the preferential loss of such cells or to their clonal expansion, a process that would tend to dilute the TREC DNA as a fraction of total DNA in a cell population. We measured this dilution of TREC experimentally and showed that mitogenic stimulation of a T cell population produced a 10-fold decrease in TREC levels in 7 days and a 100-fold decrease in 14 days (data not shown). In patients with a dominant malignant clone that comprised >70% of total T cells,TREC levels would be expected to be lower than normal. This was indeed the case, but our data suggest that this cannot be accounted for simply by the presence of a large number of TREC-negative CTCL cells. Indeed, the nonmalignant cells have fewer TREC than would be expected from the analysis of a comparable number of normal T cells.
We previously reported a "collapse" of the T cell repertoire in patients with CTCL, and whereas this was most easily shown in patients with stage III and IV disease, it could be seen in a certain proportion of patients with stage I disease. In reviewing the spectratypes of these patients, we found that many BV families did not show the normal Gaussian distribution of CDR3 lengths. We routinely saw a reduction in the number of peaks (two to four, oligoclonal), single peaks (monoclonal), and no peaks (absent), sometimes in the same patient. Because the absolute number of T cells, as measured by fluorescence-activated cell sorting analysis with BV antibodies, was not reduced in these BV families, we hypothesized that normal T cells were being lost and that the remaining T cells in the same BV family were expanding clonally, a process that should result in such a spectratype profile. If this is true, the TREC levels in these BV families should also be markedly reduced. Although it is impractical to do a large number of TREC analyses on T cell subsets isolated with BV antibody, it is reasonable to predict that, in aggregate, TREC levels in CD3+ T cells in the peripheral blood of such patients would be lower than normal through simple dilution of TREC DNA after cell proliferation and that TREC levels would be even lower in patients with a large number of contracted profiles. A corollary to this would be that patients with multiple monoclonal peaks would have reduced TREC levels.
When we analyzed TREC levels as a function of contracted BV spectratypes in 108 patients with CTCL, we found a striking and statistically significant correlation between the number of contracted profiles and TREC levels, and patients with multiple contracted profiles often had very low TREC levels. Furthermore, because of a high positive correlation between the number of contracted profiles and monoclonal peaks (r = 0.82), TREC levels were strikingly correlated with the number of monoclonal peaks. These data support the hypothesis that patients with highly abnormal spectratypes have lost normal T cells and that other nonmalignant T cells have expanded clonally to fill the empty "space" in the T cell compartment created by this loss of T cells. Patients with poorly controlled HIV have both abnormal spectratypes and lower levels of TRECs, and both seem to normalize after highly active antiretroviral therapy (27, 28). This has been attributed to recovery of thymic function as viral loads decrease. We cannot rule out the possibility that thymic output is diminished in patients with CTCL. However, because the median age of our population was 61, baseline thymic function was already stable and diminished (25, 26). A further decrement in thymic function would not be expected to have such a profound effect on TREC levels and would not explain the correlation between TREC levels and spectratype findings.
In a subset of patients, we separated the expanded malignant clone from the putatively nonmalignant normal Tcell population. Although the sample size was limited, the TREC level was clearly reduced in this nonmalignant normal Tcell population (Fig. 5), again consistent with the interpretation that normal T cells had been lost and remaining cells had expanded clonally to fill the empty space. Neither the mechanism by which this occurs nor the level at which the "set point" of T cell numbers is clear. One possibility is that the T cell compartment is homeostatically maintained at a certain size, a well-established phenomenon (29, 30). Because many patients with CTCL, even those with expanded monoclonal populations of Sezary cells, have normal absolute T lymphocyte counts, this implies that both normal and malignant T cells are subject to the same regulatory growth controls with regard to homeostasis as are the T cells in normal patients.
The cytokine responsible for maintaining homeostasis may well be interleukin-7. With regard to HIV infected patients, a low TREC number was reported to be associated with elevated serum interleukin-7 concentration (31, 32). We have analyzed plasma interleukin-7 levels in a larger number of CTCL patients and normal controls, and plasma interleukin-7 levels are significantly higher in CTCL patients than in normal controls.3 We cannot exclude the possibility that our observations are the result of polyclonal activation by an unknown antigen; however, if this is true, the normal mechanisms to limit the expansion of the T cell compartment after antigen activation must be impaired in this disease. Finally activation and proliferation of T cells by superantigen is also possible (33, 34). It should be noted that these possibilities are not mutually exclusive, and that each may contribute to the findings we have observed.
In summary, our data indicate that CTCL is not simply a malignancy of a skin-homing lymphocyte, but is associated with a profound global alteration in the T cell repertoire. The apparent loss of normal T cells, with compensatory expansion of remaining nonmalignant cells, is poorly understood but seems to occur very early in the course of disease. There is some evidence that spectratype abnormalities can resolve to some extent after the successful treatment of CTCL (6). It will be important to determine whether TREC levels normalize after successful treatment and, if so, whether the extent of normalization is the same in younger and older patients. Finally, the prognostic importance of TREC measurements and BV spectratypes needs to be determined. It is worth asking whether patients with early-stage CTCL who have severely abnormal TREC values and spectratypes are at greater risk for progression of disease than are those who have normal values. Given the uncommon nature of this disease, a multicenter trial will probably be critical to addressing this question.
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Acknowledgments |
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Footnotes |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 12/ 8/04; revised 3/30/05; accepted 4/28/05.
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1-w29/V ß1-w24) for the study of human T cell receptor variable V gene segment usage by polymerase chain reaction. Eur J Immunol 1992;22:1261–9.[Medline]
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