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Department of Internal Medicine II and Department of Laboratory Medicine, Faculty of Medicine, University of Miyazaki, Kiyotake, Miyazaki
Laboratory of Tumor Cell Biology, Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Minato-ku, Tokyo, Japan
Department of Epidemiology, Boston University School of Public Health, Department of Epidemiology, Harvard School of Public Health
Division of Biostatistics and Epidemiology, University of Massachusetts Medical School Cancer Center, Boston
Background.
The clonal expansion of human T lymphotropic virus type 1 (HTLV-1)infected T cells is considered to be important for the maintenance of infection. However, the process by which the clonality of HTLV-1infected T cells is established is not understood.
Methods.
HTLV-1 clonality in 4 adult seroconverters was analyzed by inverse long polymerase chain reaction (PCR) followed by cloning of the PCR products and evaluation of restriction fragmentlength polymorphism. The results were compared with those for 8 long-term HTLV-1 carriers.
Results.
The clonality of HTLV-1infected T cells in the seroconverters arose stochastically and was variable 35 years after seroconversion. On the basis of the frequency with which clones of cells infected with unique HTLV-1 provirus integration sites appeared, it was clear that the seroconverters had a greater number of unique clones with fewer infected cells than did the long-term carriers.
Conclusions.
The clonality of the HTLV-1infected T cells in the adult seroconverters, who had been newly infected via HTLV-1carrier spouses, was more heterogeneous and less stable than that of the HTLV-1infected T cells in long-term carriers, who were more likely to have been infected during infancy. The mechanism for the selective maintenance of certain clones in asymptomatic HTLV-1 carriers likely plays a role in the initiation of leukemogenesis.
Human T lymphotropic virus type 1 (HTLV-1) is the causative agent of adult T cell leukemia/lymphoma (ATL), but only a small proportion of HTLV-1 carriers develop this disease [13]. A high number of HTLV-1infected T cells is considered to be a risk factor for the development of ATL [4]. When an individual is infected by HTLV-1, the virus randomly integrates into the genome of affected T cells in the form of a provirus. T cells with identical integration sites for the HTLV-1 provirus are considered to have originated from the same infected cell and, thus, to belong to a unique clone. Southern-blot hybridization is currently used to investigate the integration sites of the provirus, but the sensitivity of this assay is limited [5, 6].
Recently, novel assays based on polymerase chain reaction (PCR) methodssuch as inverse PCR, inverse long PCR, and linker-mediated PCRhave been applied in the analysis of the clonality of HTLV-1infected T cells in carriers [68]. By amplifying the regions adjacent to the HTLV-1 provirus integrated into the genome of infected T cells, these assays allow the clonality of HTLV-1infected T cells to be identified even in asymptomatic carriers. If a clone has a large number of cells, it will be detected consistently in every analysis that uses these PCR-based assays and will be considered a major clone [8, 9]. However, if a clone has only a small number of cells, it will be detected in a stochastic mannerthat is, inconsistentlyand will be considered a minor clone [8, 9]. When the profile of the clonality of HTLV-1infected T cells from a carrier is examined over time, the major clones are found to persist for at least several years [8, 10]. The proviral DNA loads in a carrier appear to be maintained primarily by the clonal expansion of HTLV-1infected T cells [7, 8, 1012]; however, there has been no evaluation to date of how the clonality of infected cells is established after initial infection.
We previously described 23 HTLV-1 seroconverters from the Miyazaki Cohort Study, a community-based follow-up study in Japan of the natural history of HTLV-1 infection [1315]. In the present study, we analyze the clonality of HTLV-1infected T cells in 4 adult seroconverters during the first year after seroconversion and again 35 years later. We then compare the clonality of their HTLV-1infected T cells with that of cells in 8 long-term carriers from the same cohort.
SUBJECTS, MATERIALS, AND METHODS
Subjects.
The Miyazaki Cohort Study was established in 1984 and involves residents of 2 small villages in Miyazaki Prefecture, Japan, who attended government-supported annual health examinations [16]. Informed consent was obtained from all of the study subjects, and the study protocol was approved by the human-subjects committees of the University of Miyazaki and the Harvard School of Public Health. Twenty-three seroconverters were identified in this cohort, and the characteristics of these subjects have been described elsewhere [13, 14]. Samples of peripheral-blood mononuclear cells (PBMCs) from 4 seroconverters were available for the present study. It was confirmed that 3 of these seroconverters were infected via their HTLV-1carrier spouses [15]. Samples obtained during the first year after seroconversion and 35 years later were analyzed. Eight HTLV-1 carriers, matched to the seroconverters by sex and age (within 4 years), were selected as control subjects; these subjects had been antiHTLV-1 positive since they first began participating in the cohort study, and their spouses were either antiHTLV-1 negative or seroconverters. Therefore, the control subjects were likely to have been infected perinatally and were considered to be long-term carriers (>50 years). Table 1 describes the HTLV-1 status of each subject's spouse as well as the sex of each subject and his or her age and proviral DNA load at the time when samples of PBMCs were obtained. Because of insufficient sample size, proviral DNA loads were measured at 1 time point only for seroconverters A and B and long-term carrier E. The median proviral DNA loads in the first available samples from the 4 seroconverters and the 8 long-term carriers were 2640 and 2440 copies/100,000 PBMCs, respectively; the difference between these 2 groups was not significant (P = .497).
Quantitation of HTLV-1 proviral DNA.
Provirus copy numbers (i.e., provirus DNA load) were measured by real-time PCR; the ABI PRISM 7000 sequence detection system (Applied Biosystems) was used. Chromosomal DNA was isolated by SDSprotease K digestion of PBMCs, followed by phenol-chloroform extraction and ethanol precipitation of DNA. Quantitative real-time PCR was performed by multiplex PCR with 2 sets of primers, 1 for the HTLV-1 provirus and 1 for the human gene encoding the RNase P enzyme. The primers and the probe for the gene encoding RNase P were purchased from Applied Biosystems; those for the pX region of the HTLV-1 provirus were the forward primer pX2-S (5-CGGATACCCAGTCTACGTGTT-3), the reverse primer pX2-AS (5-CAGTAGGGCGTGACGATGTA-3), and the FAM-labeled pX2 probe (5-CTGTGTACAAGGCGACTGGTGCC-3). The control templates used were as follows: genomic DNA of the TLOM1 cell line that harbors a single copy provirus and that of normal control PBMCs mixed with a plasmid DNA that contains almost the whole genome of the HTLV-1 provirus (SacI site of 5-LTR to SacI site of 3-LTR). The copy number of the plasmid DNA was calculated on the basis of the size and weight of the plasmid DNA, as measured by spectrophotometry.
Analysis of HTLV-1infected T cells by inverse long PCR.
Inverse long PCR was used to amplify the genomic DNA adjacent to the integration sites of the HTLV-1 provirus [8]. In brief, genomic DNA was digested with EcoRI, self-ligated by T4 ligase, and digested again with MluI. Long PCR amplification of the resultant DNA was performed by use of the XL PCR Kit (Applied Biosystems). The primers used in this assay were primer 1 in the U5 region of the long-terminal repeat (LTR) (5-TGCCTGACCCTGCTTGCTCAACTCTACGTCTTTG-3; positions 556589) and primer 2 in the U3 region of the LTR (5-AGTCTGGGCCCTGACCTTTTCAGACTTCTGTTTC-3; positions 83458378) [8]. PCR products were electrophoresed on 0.8%1.5% agarose gel that contained ethidium bromide. All assays were performed in triplicate.
Assessment of clonality.
To differentiate the clones of HTLV-1infected T cells in each subject, the amplified regions adjacent to the integration site of the HTLV-1 provirus were cloned, after which restriction fragmentlength polymorphism (RFLP) analysis was performed. The PCR products were ligated by use of the TOPO Cloning Reaction Kit and the pCR-XL-TOPO vector (Invitrogen). The assays were performed in accordance with the protocol provided by the manufacturer. In brief, after transformation into TOP10 Escherichia coli cells, the reaction mixture was spread on an Luria-Bertani plate that contained kanamycin and was incubated overnight at 37°C. From each subject, 2530 colonies were randomly picked and cultured overnight at 37°C. Plasmid DNA extracted from the cultured cells was then digested with EcoRI and electrophoresed on 1.5%2.0% agarose gel that contained ethidium bromide. If the genomic DNA amplified from regions adjacent to the integration sites of the HTLV-1 provirus was correctly integrated into the plasmid, 3 bands would appear: 1 band (3.5 kb) derived from the pCR-XL-TOPO vector and 2 bands of different lengths derived from the genomic DNA adjacent to the 3 and 5 LTRs of the HTLV-1 provirus. Because the lengths of the 2 bands derived from the regions adjacent to the HTLV-1 provirus were unique to T cells whose provirus had an identical integration site, this method was able to identify the HTLV-1infected T cells belonging to an identical clone. To ensure the accuracy of the RFLP analysis, the DNA sequences of the cloned PCR products were analyzed by use of the ABI PRISM Big Dye Terminator Cycle Sequencing Ready Reaction Kit and an ABI PRISM 310 genetic analyzer (Applied Biosystems), when necessary.
Statistical analysis.
The Mann-Whitney U test and the 2 test were used to compare proviral DNA loads and frequencies of clones, respectively, between the group of 4 seroconverters and the group of 8 long-term carriers.
RESULTS
We first examined whether the clonality of HTLV-1infected T cells as reconstituted by inverse long PCR/cloning/RFLP analysis would reflect the frequency of clones in the original sample. Genomic DNA derived from leukemic cells with 1 copy of HTLV-1 provirus from a patient with ATL (who was not one of the subjects of the present study) was serially diluted (1 : 10) in the genomic DNA derived from PBMCs from an asymptomatic HTLV-1 carrier (proviral DNA load, 1800 copies/100,000 PBMCs), who also was not one of the subjects of the present study. The DNA was then analyzed by inverse long PCR. The ATL clone was demonstrated to be major, because it was detected in every triplicate inverse long PCR assay in which the dilution of the total number of HTLV-1infected T cells was 5% (figure 1A). However, when the sample was diluted to 0.6% of the total number of HTLV-1infected T cells, the ATL clone appeared in only 2 of the 3 assays, and the number of additional bands of different sizes, which were the inverse long PCR products of the genomic DNA from the HTLV-1 carrier used for the dilution, increased in the triplicate analysis.
The frequencies of the PCR products derived from the ATL cells were assessed by cloning those products and then conducting the RFLP analysis (figure 1B). Each lane revealed one 3.5-kb band derived from the vector and 2 bands derived from the regions adjacent to the integration site of the HTLV-1 provirus. When the sizes of the latter 2 bands were identical among the different PCR products analyzed, the PCR products were considered to belong to an identical clone (figure 1B; identical numbers indicate identical clones). The frequencies of detection of the ATL clone among the tested PCR products were 18 (64%) of 28, 3 (12%) of 25, and 1 (4%) of 24 in the dilutions of 1%, 0.1%, and 0.01%, respectively, when the DNA from the original ATL cells among the carrier's PBMCs was used. The expected provirus copies derived from the ATL clone among the total copies of HTLV-1 provirus in the dilutions of 1%, 0.1%, and 0.01% of the DNA used were 1500 (36%) of 4173, 150 (5%) of 2847, and 15 (0.6%) of 2715, respectively. Therefore, the frequency of detection of the ATL clone among the tested PCR products correlated with the proportion of provirus derived from the ATL clone among the total copies of HTLV-1 provirus, although the former was higher than the latter. Thus, the clonality of the HTLV-1infected T cells as reconstituted by inverse long PCR/cloning/RFLP analysis seemed to reflect the clonality in the original sample. The reason for the preferential detection of ATL clones after the reconstitution may be that the size of the PCR product of this clone was favorably detected by the inverse long PCR/cloning assay. The frequencies of the PCR products of the HTLV-1infected T cells with unique HTLV-1 provirus integration sites were also examined by RFLP analysis and were found to be 8 (29%) of 28, 14 (56%) of 25, and 17 (71%) of 24 in the dilutions of 36%, 5%, and 0.6%, respectively. Accordingly, if the original PBMCs contained a large number of cells belonging to a major clone, such as the ATL clone in this analysis, then the frequency of unique clones was considered to be low. In contrast, the frequency of unique clones was considered to be high when the clonality of the HTLV-1infected T cells consisted of a variety of clones with a lower total proportion than the major clone.
Seroconverters BD, as well as long-term carriers FL, were also examined for the frequency of unique clones by inverse long PCR/cloning/RFLP analysis, and the results are summarized in table 2. For the 4 seroconverters combined, 56 of 90 cloned PCR products during the first year after seroconversion and 66 of 88 cloned PCR products 35 years later had unique provirus integration sites. When the 2 time points were combined, 114 clones (64%) were found to be unique among 178 cloned PCR products. Among the 8 long-term carriers, 113 (57%) of 199 cloned PCR products and 109 (59%) of 184 cloned PCR products were considered to be unique at the 2 time points. When these 2 time points were combined, 180 clones (47%) were unique among 383 cloned PCR products. The proportions of unique clones in the group of 4 seroconverters were higher than those in the group of 8 long-term carriers at both time points. The difference between the seroconverters and the long-term carriers was especially evident when their respective proportions of unique clones were compared (64% vs. 47%; P = .0002). Because there was no marked difference in the level of proviral DNA load between the seroconverters and the long-term carriers, these results suggest that the frequency of unique clones of HTLV-1infected T cells in the long-term carriers was less than that in the seroconverters. In other words, the clonality of HTLV-1infected T cells in the seroconverters was found to be more heterogeneous than that in the long-term carriers.
In addition, when we compared the frequency of clones that were consistently detected at both time points, we found that the seroconverters had only 8 (7%) of the 114 unique clones, whereas the long-term carriers had 42 (23%) of the 180 unique clones (table 2)a highly significant difference (P = .0003). Sequencing of the cloned PCR products confirmed that the clones consistently detected at both time points were identical (data not shown). This result suggests that the clonality of HTLV-1infected T cells is less stable in seroconverters than in long-term carriers, at least for several years after seroconversion.
DISCUSSION
In the present study, inverse long PCR analysis of the seroconverters showed the existence of many clones of HTLV-1infected T cells; these clones appeared in a stochastic manner. The clonality of HTLV-1infected T cells was more heterogeneous in the seroconverters than in the long-term carriers. In addition, major clones were rarely detected in the seroconverters. These observations might have been due to the fact that the clones in the seroconverters consisted of a small number of HTLV-1infected T cells that could only be detected by inverse long PCR in a stochastic manner.
The clonality of HTLV-1infected T cells in the seroconverters was also found to be unstable for at least several years after seroconversion. Although this instability might have been due to stochastic detection of those clones, an alternative explanation is that turnover of the infected cells may occur for several years after initial infection in the seroconverters. In fact, the proportion of unique clones was somewhat higher in the samples obtained 35 years after seroconversion (75%) than in the samples obtained during the first year after seroconversion (62%). Because the inverse long PCR assay may not detect clones that have small numbers of infected cells, it is possible that many infected cells had not developed into clones of a detectable number at the time of seroconversion. The clonal expansion of certain HTLV-1infected T cells may have occurred thereafter, with the number of cells belonging to those clones reaching a level detectable by inverse long PCR later. At the same time, some clones might have been eliminated by the host immune response [17], given that the level of proviral DNA load in the peripheral blood of the seroconverters did not appear to increase during this period.
The clonality of HTLV-1infected T cells in the long-term carriers was shown to be less heterogeneous and more stable than that in the seroconverters. This finding suggests that major clones (i.e., those that have large numbers of infected cells) are maintained in the peripheral blood of long-term carriers. It is of interest to know why, among the many clones of infected cells, only some of these clones develop into major ones. This question is especially important, because these major clones were shown to be maintained for many years and may be related to the development of ATL. HTLV-1 infection is considered to persist in conjunction with the balance between the replication of HTLV-1infected T cells and the host immune response to these cells [7, 8, 12, 17]. One of the most important factors related to this balance is thought to be the HTLV-1 Tax protein, which has been shown to promote the proliferation of infected cells [1821]. The HTLV-1 Tax protein is also reported to be a good target for the host cellular immune response to the virus [17]. Therefore, the balance between these 2 different attributes of Tax might influence the ability of HTLV-1infected T cells to survive and develop into major clones [7, 8, 12, 22].
The differences between adult seroconverters and long-term carriers include not only the length of the infection period (several years vs. >50 years) but also the time and manner of infection (sexual transmission in adulthood vs. transmission through breast-feeding in infancy). Because long-term carriers are assumed to have been infected as infants, it is natural to suppose that, during childhood, they experienced many bacterial and viral infections, which stimulated their T cells to replicate. It has been reported that some infections, such as infection with strongyloides, promote the clonal expansion of HTLV-1infected T cells [2325]. Therefore, it is possible that HTLV-1 infection during infancy is important for the development of major, persistent clones of HTLV-1infected T cells, with ATL developing from one of those clones many decades after infection.
There are several limitations to the present study. In addition to the small number of subjects evaluated, the amount of genomic DNA used in the analyses (3 g) was derived from 500,000 PBMCs for each subject, and only 1%5% of the PBMCs were actually HTLV-1infected T cells. Also, smaller products of inverse long PCR tend to be more easily cloned into plasmid DNA in the cloning/RFLP assay. Therefore, the results of the present experiments may reflect only part of the clonality of HTLV-1infected T cells for each subject, although this condition would be the same for both the seroconverters and the long-term carriers. It also has been reported that the genomic DNA adjacent to the integration sites of the provirus in HTLV-1infected T cells cannot always be amplified by inverse long PCR, because of variation in the viral DNA sequence [8, 26]. It is necessary to investigate whether a provirus with variation in viral DNA would be detected more frequently in long-term carriers than in seroconverters.
In conclusion, the present study showed that the clonality of HTLV-1infected T cells in 4 adult seroconverters, who had been newly infected via their spouses, was more heterogeneous and less stable than that of HTLV-1infected cells in 8 long-term carriers, who likely had been infected during infancy. Moreover, major clones, which consist of many infected cells and persist for a long time, were rarely detectable in the seroconverters but were common in the long-term carriers. It is possible that the ATL clone develops as a subclone of such major clones. The mechanism by which certain clones are selectively maintained should be clarified, because it may be related to the initiation of leukemogenesis.
Acknowledgments
We thank Yuko Nakamura and Yuka Takahama (University of Miyazaki, Kiyotake, Miyazaki, Japan), for providing technical assistance.
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