Abstract
The human T-cell lymphrotropic virus type 1 (HTLV-1) is causally related to adult T-cell leukemia and lymphoma and the neurodegenerative diseases tropical spastic paraparesis and HTLV-1-associated myelopathy. In the United States the prevalence of infection has been estimated to range from 0.016 to 0.1% on the basis of serologic tests for antibodies to the viral structural proteins. Blood from donors positive for antibodies to HTLV-1 or HTLV-2 is not used for transfusion. However, patients with the cutaneous T-cell lymphoma mycosis fungoides (MF) are HTLV-1 and -2 seronegative yet harbor proviral sequences identical to those that encode the HTLV-1 transactivating and transforming gene product p40tax in their peripheral blood mononuclear cells (PBMCs), and they usually have antibodies to p40tax. Moreover, a study of 250 randomly selected blood donors revealed that approximately 8% of these seronegative individuals also had HTLV-1 tax sequences and antibodies to p40tax, while they lacked sequences and antibodies related to gag, pol, or env. Thus, it seemed important to determine whether the “tax-only” state can be transmitted by transfusion. To this end, PBMCs from HTLV-1 and -2 seronegative tax-only-positive MF patients or from healthy tax-only-positive blood donors were injected into adult rabbits, an established animal model for HTLV-1 infection. The PBMCs of all injected rabbits became tax sequence positive. These observations suggest that HTLV-1 tax can be transmitted by tax-only-positive mononuclear cells.
The human T-cell lymphotropic virus (HTLV) type 1 (HTLV-1) is causally associated with adult T-cell leukemia and lymphoma (27) as well as with the nonneoplastic conditions tropical spastic paraparesis (5) and HTLV-associated myelopathy (22). The vast majority of patients with these diseases as well as healthy carriers of HTLV-1 have antibodies to the structural proteins of this virus. Because infection has been shown to be transmitted by transfusion (18, 21), all blood collected for this purpose has been screened for antibodies to this virus since 1988 (2). However, subsequent studies with patients with the cutaneous T-cell lymphoma mycosis fungoides (MF) revealed that the majority carry the proviral tax sequence of HTLV-1 in their peripheral blood mononuclear cells (PBMCs) and skin-infiltrating lymphocytes, while they have no antibodies to the structural proteins of the virus (14, 24). This raised the question of whether the “tax-only”-positive state may also pertain to some healthy, serologically negative blood donors. Indeed, on the basis of serologic tests for 250 randomly selected donors who presented at the New York University Medical Center blood bank, 8.6% proved to harbor proviral HTLV-1 tax in their PBMCs and also had antibodies to p40tax, the protein encoded by this sequence (36, 39). Therefore, a study to determine whether the tax-only state could be transmitted by transfusion of tax-positive cells seemed to be indicated. It should be recalled here that HTLV-1 tax functions in the transcriptional transactivation of the HTLV-1 long terminal repeat and the transactivation of numerous cellular genes, particularly those involved in inflammation and cell proliferation, such as interleukin-1 (IL-1), IL-2, the α subunit of the IL-2 receptor, IL-6, granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor alpha, the adhesion molecules ICAM-I and LFA-I in addition to several oncogenes, including jun, fos, rel, myc, and ets (1, 6, 35). In vitro, tax has been shown to be taken up by PBMCs, to stimulate proliferation of cultured cells, and to dysregulate immunoglobulin production by B cells (19, 26). Last, but not least, mice made transgenic for HTLV-1 tax develop a variety of malignant neoplasms as well as autoimmune conditions, such as rheumatoid arthritis and Sjögren's syndrome (7, 10, 26). Rabbits were used as recipients of tax-only-positive cells, since this species has proven to be a useful animal model for HTLV-1 infection (3, 17). Cells obtained from patients with MF and/or tax-only-positive healthy blood donors were injected intravenously into this animal species. Positive control animals received cells of the C91PL cell line, a tissue culture cell line infected with prototypic HTLV-1 (28). The PBMCs of all animals became tax sequence positive. As anticipated, the PBMCs of rabbits injected with virus-infected C91PL cells also had gag and env sequences. Neither the animals injected with HTLV-1-infected cells containing proviral sequences spanning the entire viral genome nor the ones injected with cells containing only tax sequences developed antibodies to p40tax, an antibody whose development is known to be late (11). The observations suggest that the tax-only state can be transmitted by cells that harbor only this incomplete, theoretically replication-defective viral sequence, which may, however, have pathophysiologic implications.
MATERIALS AND METHODS
Origins of tax-only-positive donor cells, cell preparation, and animals.
Peripheral blood was obtained from patients with MF and from healthy blood donors, whose cells were HTLV-1 tax-only positive and who had antibodies to p40tax while being serologically negative for antibodies to HTLV-1 and -2. PBMCs from the tax-only-positive donors had previously been shown by PCR-Southern analysis to harbor tax but not gag-1, gag-2, gag-1 and -2 pol-1, pol-2, or env-1 sequences (24, 25, 39, 40). Donor cells for this study were prepared by Ficoll-Hypaque gradient centrifugation, as routinely carried out in this laboratory (38), after which they were suspended at a concentration of 108 per ml of physiologic saline. The cells were used within 1 to 3 days of collection.
Outbred 3-kg female New Zealand White rabbits were purchased from Charles River Laboratories (Wilmington, Mass.). They were bled from the central ear arteries, and injections were given in marginal ear veins. On days when animals were both bled for testing of blood and injected, bleeding always preceded injection. Control specimens were obtained from all animals before the first injection. Two rabbits (rabbits A and B) received injections of 108 HTLV-1-infected C91PL cells on day 0 and at 3 and 5 months (Table 1). Rabbits C, D, E, F, G, H, and I were injected with 108 PBMCs obtained from patients with MF or healthy blood donors at the following times: rabbit C, on day 0 and at 3, 4, 5, and 7 months; rabbit D, on day 0 and at 3, 4, 5, 7, and 8 months; rabbit E, on day 0 and at 3, 4, 7, and 9 months; rabbit F, on day 0 and at 3, 4, 5, and 7 months; rabbit G, on day 0 and at 3, 4, 5, 7, 8, and 9 months; rabbit H, on day 0 and at 3, 4, and 7 months; and rabbit I, on day 0 and at 9 months. The injection schedule was, to a large extent, contingent on the availability of tax-only-positive patients and/or blood donors.
TABLE 1.
Injection schedule prior to detection of tax sequence positivity
| Rabbit | Day of injection | Day testeda | Resultb |
|---|---|---|---|
| A | 0,c 91c | 62 | − |
| 117 | + | ||
| B | 0,c 91c | 62 | − |
| 117 | + | ||
| C | 0,d 104,e 117e | 62 | − |
| 117 | − | ||
| 150 | + | ||
| D | 0,d 104,e 117e | 62 | − |
| 117 | − | ||
| 150 | + | ||
| E | 0,e 104,e 117e | 62 | − |
| 117 | − | ||
| 150 | + | ||
| F | 0,e 91,e 117e | 62 | − |
| 117 | − | ||
| 150 | + | ||
| G | 0,e 91,e 117e | 62 | − |
| 117 | − | ||
| 150 | + | ||
| H | 0,d 83,e 90e | 41 | − |
| 96 | − | ||
| 129 | + | ||
| I | 0d | 41 | − |
| 96 | − | ||
| 129 | + |
All time points are days after the first injection.
Detection of tax proviral DNA sequences by PCR and Southern analysis of rabbit PBMC lysates.
Injected with HTLV-1-infected (C91PL) cells.
Injected with PBMCs from MF patients.
Injected with PBMCs from healthy blood donors.
Detection of proviral DNA sequences.
Whole-cell lysates were prepared from 105 mononuclear cells isolated by Ficoll-Hypaque gradient centrifugation followed by sonication and boiling in autoclave-sterilized distilled water and incubation in the presence of proteinase K (24). Samples were boiled to inactivate the protease and were then subjected to 30 cycles of PCR amplification (1 min at 94°C, 1 min at 55°C, and 1.5 min at 72°C per cycle), followed by a final incubation for 10 min at 72°C in the buffer and the concentrations of MgCl2 and deoxynucleoside triphosphates described previously (24). Final reaction volumes of 80 μl contained 40 pmol of each of the appropriate primers (see below). Southern analysis following amplification by PCR was necessary in most cases to detect HTLV sequences. As described previously, the 3′ digoxigenin-tailed probes listed below were used for hybridization (24, 40). Detection of bound probe entailed use of Fab′ fragments of antibodies to digoxigenin conjugated with alkaline phosphatase and the alkaline phosphatase substrates 4-nitroblue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolylphosphate (BCIP). The reagents for tailing and detection of bound probes were obtained from Boehringer Mannheim (Indianapolis, Ind.).
The primers and probes used in this study were synthesized in the Oligonucleotide Synthesis and Sequencing Facility at the New York University School of Medicine. The sequences and genome locations of the HTLV gag-1 and env-1 primers and probes were described by Hall et al. (9), and the tax-1 and -2 primers SK43 and SK44 and probe SK45 were described by Kwok et al. (16). The HTLV-1-infected cell line C91PL (28) was used as the positive control, and PBMC lysates from tax sequence-negative volunteers served as negative controls in these assays.
The primers and probe used to detect human DNA sequences in PBMCs from tax sequence-positive rabbits were designed to amplify and identify a 260-bp portion of the coding sequence for human CD24 (13). This region encodes a human Alu sequence not found among rabbit genomic sequences (4, 13). The primer and probe sequences used consisted of 5′-CCACGACTAATTTCAAAATGCT-3′ (sense primer), 5′-AAGCCTGTAATCCCAGCACTTTGGG-3′ (antisense primer), and 5′-GTGCGATCTCGATCATGTACCATTTG-3′ probe. PCR and Southern analyses were performed as described before with lysates of PBMCs (24). Positive and negative controls for the assays for detection of human Alu sequences consisted of PBMC lysates from three human blood donors and two rabbits which had never been injected with human cells, respectively.
Sequence analysis.
The tax sequences amplified from the donor cells used for inoculation and from PBMCs obtained from five rabbits injected with tax-only-positive human cells were subjected to oligonucleotide sequence analysis, as described previously (39).
The sequences detected in the samples were compared with those published for HTLV-1 and -2 (29, 30). For the 87-bp tax proviral DNA sequence being analyzed, HTLV-1 and -2 differ by 16 bp.
Detection of HTLV-1 tax mRNA in rabbit PBMCs.
Reverse transcription-PCR and Southern analyses were performed with PBMCs obtained from all nine rabbits 5 months after injection as described previously (25). Briefly, total cellular RNA was isolated from ∼105 PBMCs and was treated with DNase I, followed by reverse transcription with Moloney murine leukemia virus reverse transcriptase (GIBCO-BRL, Gaithersburg, Md.) in the presence of 50 pmol of HTLV-1 and -2 tax primer SK43. PCR and Southern analyses were subsequently carried out with tax primer SK44 and digoxigenin-tailed tax probe SK45 as described above. Positive and negative controls for these assays consisted of cells of the HTLV-1-infected cell line C91PL (28) and lysates of PBMCs from HTLV tax sequence-negative volunteers, respectively.
Detection of HTLV-1 and -2 antibodies.
Plasma specimens from all nine rabbits were tested for antibodies to the HTLV-1 and -2 structural proteins gag and env with the HTLV Blot 2.3 kit obtained from Cellular Products (Buffalo, N.Y.), but with goat anti-rabbit immunoglobulin G (heavy and light chains) conjugated with alkaline phosphatase (Pierce Chemical Co., Rockford, Ill.) as the secondary antibody and the alkaline phosphatase substrates NBT and BCIP as described before (40).
Detection of p40tax antibodies by western blot analysis.
Since the p40tax antigen is not commerically available, it was prepared by cloning the proviral DNA sequences that encode the full-length p40tax-I open reading frame from the prototypic HTLV-1-infected cell line C91PL (28) into the glutathione S-transferase fusion protein expression vector pGEX-2T essentially as described before (25). Western blot assays were carried out with thrombin-cleaved p40tax antigens, and plasma specimens were diluted 1:10 for testing. Blots were developed by using secondary antibodies (goat-anti rabbit immunoglobulin G; heavy and light chains conjugated with alkaline phosphatase) (Pierce Chemical Co.) and the substrates NBT and BCIP.
RESULTS
All rabbits were negative for HTLV sequences and antibodies prior to injection of human cells.
Rabbits A and B failed to convert to HTLV sequence and antibody positivity during the first 3 months after injection with HTLV-1-infected C91PL cells. Therefore, these animals were reinjected with C91PL cells at 3 months, and both animals then converted to being both HTLV sequence and antibody positivity within 1 month after the second injections (Tables 1 to 3). Although PBMCs from both rabbits (A and B) were shown to be positive for tax mRNA (Table 3), neither animal developed antibodies to p40tax during the 12 months of observation.
TABLE 3.
| Rabbit | Inoculum | mRNA or antibody detection at the indicated time (mo) after first injection:
|
|||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| tax mRNA (5) | Anti-gag (5) | Anti-env (5) | Anti-p40tax
|
||||||||||||
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 10 | 11 | 12 | |||||
| A | C91PLc | + | + | + | − | − | − | − | − | − | − | ||||
| B | C91PL | + | + | + | − | − | − | − | − | − | |||||
| C | MFd + Ne | + | − | − | − | − | − | − | − | − | − | ||||
| D | MF + N | + | − | − | − | − | − | − | − | − | − | ||||
| E | N | − | − | − | − | − | − | − | − | − | − | − | − | ||
| F | N | + | − | − | − | − | − | − | − | − | − | ||||
| G | N | + | − | − | − | − | − | − | − | − | − | − | − | ||
| H | MF + N | + | − | − | − | − | − | − | − | − | − | ||||
| I | MF | + | − | − | − | − | − | − | − | − | − | − | − | ||
Detected by reverse transcription-PCR and Southern analysis of PBMC lysates (25).
Detected by Western blot assays of plasmas. HTLV-1 structural protein antibodies were assayed with the Diagnostic Biotechnology HTLV Blot 2.3 kit obtained from Cellular Products (40). HTLV p40tax antibodies were detected with recombinant tax antigen originally cloned from the HTLV-1-infected cell line C91PL (25, 28).
C91PL, an HTLV-1-infected cell line (28).
MF, PBMCs from MF patients.
N, PBMCs from healthy tax-only-positive blood donors.
tax sequences were detected in rabbits H and I 3 months after the first injection with PBMCs from MF patients and/or healthy blood donors (Tables 1 and 2). tax-specific sequences were not detected in rabbits C, D, E, F, and G until 4 months after injection of tax-only-positive human cells. A representative Southern blot of the tax sequences detected in rabbit PBMCs is shown in Fig. 1.
TABLE 2.
Detection of HTLV-1 proviral DNA sequences in rabbits
| Rabbit | Inoculum | Sequences detected at the indicated times (mo) after first injectiona:
|
|||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| gag (5) | env (5) |
tax
|
|||||||||
| 1 | 3 | 4 | 5 | 6 | 7 | 9 | 10 | ||||
| A | C91PLb | + | + | − | + | + | + | + | |||
| B | C91PL | + | + | − | + | + | + | + | |||
| C | MFc + Nd | − | − | − | − | + | + | + | + | + | |
| D | MF + N | − | − | − | − | + | + | + | + | + | |
| E | N | − | − | − | − | + | + | + | + | + | |
| F | N | − | − | − | − | + | − | + | + | + | |
| G | N | − | − | − | − | + | + | − | + | + | |
| H | MF + N | − | − | − | + | + | + | + | + | ||
| I | MF | − | − | − | + | + | + | + | + | + | |
FIG. 1.
Southern blot of 159-bp HTLV-1 tax proviral DNA sequences amplified by PCR from whole-cell lysates of PBMCs from rabbits. The tax probe, SK45, used for hybridization was tailed at the 3′ end with digoxigenin, and bound probe was detected by using Fab′ fragments of antibodies to digoxigenin conjugated with alkaline phosphatase and the alkaline phosphatase substrates NBT and BCIP. Lanes: 1, HTLV-1-infected cell line C91PL; 2 and 3, plasma from rabbits A and B, respectively, injected with C91PL cells; 4 to 10, plasma from rabbits C to I, respectively, injected with tax-only sequence-positive human PBMCs. The arrowhead indicates the 159-bp tax proviral DNA amplification product.
Although tax mRNA was demonstrated in PBMC lysates from all rabbits except rabbit E (Table 3), none of the rabbits (rabbits C to I) injected one to seven times with tax-only-positive PBMCs from MF patients and/or healthy blood donors developed antibodies to p40tax (Table 3). It is noteworthy that this also pertained to rabbits A and B, which had been inoculated with C91PL cells that contained the whole virus.
As expected, rabbits A and B became positive for sequences and antibodies to HTLV-1 structural proteins gag and env (Tables 2 and 3).
Sequence analyses demonstrated that the same HTLV-1 tax proviral sequences detected in cells used for injection were present in PBMCs obtained from rabbits (Fig. 2).
FIG. 2.
Representative HTLV-1 (HTLV-I) tax sequence detected in PBMC lysates from five rabbits injected with tax sequence-only PBMCs from patients with MF or tax-only-positive blood donors. The tax sequences for prototypic HTLV-1 and -2 (HTLV-II) are those published by Seiki et al. (29), and Shimotohno et al. (30), respectively. Dashed lines indicate sequence identity with the prototypic HTLV-1 sequence.
Human CD24-encoded Alu sequences were not detected in PBMCs from any of the nine tax sequence-positive rabbits tested 6 months after they were first injected with HTLV-1-infected cells (rabbits A and B) or PBMCs from tax-only-positive donors (rabbits C to I). Thus, it is unlikely that any human cells survived and proliferated in the rabbits.
DISCUSSION
It is well established, that transmission of HTLV-1 infection may occur by transfusion of cellular blood products obtained from HTLV-1-positive donors. Because carriers of the virus usually have antibodies to it, testing for such antibodies, which has been in place in the United States since 1988 (2), seemed to preclude infection by this route. However, the existence of seronegative virus carriers among inhabitants of areas where the virus is endemic has been recognized. In a study conducted in Japan, it was believed to be responsible for seroconversion in 3 of 600 blood recipients (20). Moreover, healthy carriers of HTLV-1 as well as patients with HTLV-1-associated diseases may lose some proviral sequences while they retain others. In such instances, it is not unusual for the tax sequence to be retained (12). Because of the low prevalence of HTLV infection in the United States, this issue has received little attention. However, patients with MF, as well as some of their healthy relatives, have deleted HTLV-1 sequences. These individuals commonly retain HTLV-1 tax, while they test serologically negative for antibodies to the structural proteins of the virus by methods in current use (24, 40). The possible significance of this observation may have been underscored by observations of a 7-year-old patient with MF whose skin and blood lymphocytes harbored only tax and pol sequences but who was found to be seronegative for antibodies to HTLV-1. The patient's healthy mother was serologically positive and a proven carrier of the complete virus (37). Thus, there is no doubt that seronegative carriers of viruses from which some proviral sequences are deleted exist and that this state may be associated with disease.
Recognition that a fairly high percentage of healthy HTLV antibody-negative U.S. blood donors may also carry deleted HTLV-1 sequences and that it is the tax sequence and its gene product, p40tax, which are most often retained (36, 39) should raise concern. It has been established that p40tax is the transcriptional transactivator of the long terminal repeat of HTLV-1. In addition, it is instrumental in the upregulation of innumerable cellular growth factors, cytokines, and oncogenes (for reviews, see references 1, 6, 31, and 35). Whether HTLV-1 tax can be transmitted by tax-only-containing cells is an important question which needed to be addressed. The protocol used to transfer human tax-only-positive PBMCs to rabbits was the same as the one used by others to transfer human cells containing the complete virus to this animal species (3, 17). It is assumed that the injected virus-containing cells are taken up by rabbit phagocytes and that they or their progeny are capable of proliferating with the viral genome. The same theory would be applicable to the transfer of the tax-only state to rabbits by cells that harbor only the tax sequence. This seems to have occurred in the experiments reported here. It should be mentioned that since our studies were completed, a report by Koya et al. (15) showed that rats inoculated with allogeneic HTLV-1 tax-containing FPMI cells retained detectable HTLV-1 tax sequences for more than a year, while no antibodies to the structural proteins of the virus developed.
The mechanism whereby the tax-only state is maintained in the absence of replicating virus has not yet been fully explained even in individuals who were initially infected with the whole virus. However, because of its strong transactivating function, as well as the observation that transformed cells do not necessarily contain viral sequences, and the fact that HTLV tax is usually lost in preparations designed to isolate high-molecular-weight DNA (23), it is likely that the tax sequence is episomal. Indeed, since this paper was first submitted, the authors have obtained data to support this possibility (41). Episomal tax may replicate autonomously under the same controls as chromosomal DNA, i.e., once per cell cycle. This has been demonstrated for other episomal viral sequences, such as those of latent Epstein-Barr virus (34; for a review, see reference 8). In individuals who harbor the complete virus, the increase in the number of positive cells over time has been attributed to clonal expansion of HTLV-bearing cells rather than independent replication of the virus (33). This is believed to account not only for the long latent period before HTLV-associated diseases become manifest but also for the remarkable genomic stability of this virus.
Another question raised by the present observations was whether the tax-only sequence positivity of injected rabbits was due to expansion of tax-only-positive rabbit cells or whether xenotransplantation, i.e., proliferation of tax-positive human cells, had occurred. Although the latter possibility was considered remote, tax-harboring cells have a proliferation advantage (33), and on the basis of studies reviewed in detail elsewhere (32, 33), it could not be entirely excluded. However, a search for human sequences in lysates prepared from tax-only-positive rabbit PBMCs yielded negative results, thus permitting the conclusion that rabbit cells which had taken up HTLV-1 tax had proliferated to the level at which the sequence could be detected. That none of the animals, even those injected with C91PL cells, generated antibodies to p40tax during the period of observation (12 months) is not surprising since antibodies to p40tax are late in developing, even in humans, following primary infection with HTLV-1 (18, 20). Be that as it may, the studies reported here have provided clear evidence that HTLV-1 tax can be transmitted by cells which do not carry intact virus or sequences that encode the structural proteins of replication-competent HTLV-1. Since the transforming and transactivating properties of p40tax have been abundantly demonstrated in vivo as well as in vitro, these observations warrant consideration in the context of transfusion medicine.
ACKNOWLEDGMENTS
These studies were supported in part by a grant from the National Blood Foundation and by the Mendik Foundation.
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