HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kawamura, N. Articles by Sakiyama, Y. Search for Related Content PubMed PubMed Citation Articles by Kawamura, N. Articles by Sakiyama, Y.

In Vivo Kinetics of Transduced Cells in Peripheral T Cell-Directed Gene Therapy: Role of CD8⁺ Cells in Improved Immunological Function in an Adenosine Deaminase (ADA)^-SCID Patient¹

Nobuaki Kawamura^2,*, Tadashi Ariga^*, Makoto Ohtsu^*, Ichiro Kobayashi^*, Masafumi Yamada^*, Atsushi Tame^*, Hirofumi Furuta^*, Motohiko Okano^*, Masayuki Egashira, Norio Niikawa, Kunihiko Kobayashi^* and Yukio Sakiyama^*

^* Department of Pediatrics, Hokkaido University School of Medicine, Sapporo, Japan; and Department of Human Genetics, Nagasaki University School of Medicine, Nagasaki, Japan

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
We previously reported successful peripheral T cell-directed gene therapy in a boy with adenosine deaminase (ADA)^-SCID. In the present study, to better understand the reconstitutive effect of this gene therapy on his immunological system, we investigated the in vivo kinetics and functional subsets of T cells in PBL. Apparent immunological improvements were obtained after infusion of transduced cells at more than 4 x 10⁸ cells/kg/therapy/3 mo. Frequency of ADAcDNA-integrated cells in PBL, ADA activity in PBL and clinical improvement showed good correlation, even though CD8⁺ cells gradually became predominant in PBL. On the basis that polyethylene glycol (PEG)-ADA was maintained at the same dosage as before gene therapy, we consider that his immunological improvement resulted from the gene therapy itself. Most CD3⁺ cells in PBL after gene therapy expressed TCRß. Analysis of TCR repertoire based on TCR V region usage revealed no expansion of limited clones in his PBL. The T cell subset cells CD8⁺CDw60⁺ and CD8⁺CD27⁺CD45RA^-, which are reported to provide substantial help to B cells, were maintained throughout the gene therapy. Furthermore, his reconstituted peripheral T cells helped normal B cells to produce substantial IgG in vitro. Expression of both Th1- and Th2-type cytokine genes was induced in his reconstituted T cells at the same comparably high level as in normal subjects. Collectively, these results provide evidence of persistent and distinct functions of transduced cells in this patient’s PBL after gene therapy.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Over the past 8 years, a large number of protocols for gene therapy (GT)³ have been instituted for many kinds of diseases (1). However, results with most of these have not met expectations. Stem cell-directed gene therapy, considered the best approach for congenital hemopoietic disorders, has also been tried recently, but again success has been elusive (2, 3, 4). Among the few successful examples has been GT for adenosine deaminase (ADA) deficiency, especially by a peripheral T cell-directed protocol (5, 6, 7). The first patient in the world to undergo GT has maintained her reconstituted immunological function for an unexpectedly long time. A number of problems in the peripheral T cell-directed protocol have been noted: the life of peripheral T cells is limited and CD8⁺ population in PBL increases. The in vivo kinetics and immunological functions of transduced cells have not been reported in detail, and the precise mechanism of effective GT targeting to peripheral T cells remains to be established.

We previously reported a successful series of peripheral T cell-directed GT for a patient with ADA^-SCID (7). In the present study, we present the in vivo kinetics and functions of transduced cells in this patient’s peripheral blood to better understand how peripheral T cell-directed GT has improved his immunological function in vivo.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Case report

The patient and our GT protocol for ADA deficiency have been reported in detail elsewhere (7). Briefly, a 4-yr-old boy who had shown only limited improvement after treatment with polyethylene glycol (PEG)-modified ADA was enrolled in a clinical gene therapy trial. PBMC from the patient were stimulated with anti-CD3 Ab (OKT3; Ortho, Raritan, NJ) and recombinant human IL-2 in AIM-V medium (Life Technologies, Grand Island, NY) supplemented with 5% FCS (Life Technologies) for 72 h. Transduction was performed twice during the next 48 h by exposure to a retroviral vector, LASN, that contained cDNA of human ADA gene. Cultivation was continued for a total of 7 to 11 days before infusion into the patient. No selection procedure for enrichment of gene-transduced cells was performed. Following a total of 11 infusions of transduced cells over 20 mo, a number of immunological improvements were seen, including improved isohemagglutinin titer, delayed-type hypersensitivity (DTH) skin test, serum Ig level, and specific Ab response to specific Ag.

Flow cytometric analysis

PBMC was washed with PBS containing 0.5% albumin and stained with FITC or PE-conjugated mAbs anti-CD4 (Leu-3a), anti-CD8 (Leu-2a), anti-HLA-DR, anti-CD45RA (Leu-18), anti-CD27, and anti-CDw60 for 20 min on ice. The stained cells were then washed. All mAbs were purchased from Becton Dickinson Immunocytometry Systems (San Jose, CA) except anti-CDw60, which was purchased from PharMingen (San Diego, CA). A TCRß screening panel (Diversi-T) containing FITC-conjugated mAbs to V2, V12.1, Vß3.1, Vß5.3 + 5.2, Vß5.1, Vß6.7, Vß8 family, Vß12, and Vß13.1 + 13.2 was purchased from T Cell Diagnostics (Woburn, MA) for examination of TCR repertoire. Cell surface phenotype was analyzed on an Epics XL flow cytometer (Coulter, Hialeah, FL).

Purification of CD4⁺ and CD8⁺ cells

PBMC from the patient was reacted with MACS (Magnetic Cell Sorting) CD4 or CD8 microbeads (Miltenyi Biotec, Sunnyvale, CA) for 15 min at 4°C and washed with PBS supplemented with 1% albumin three times. They were applied to a MACS column (Miltenyi Biotec) and rinsed with PBS. After removal of the column from MACS, eluted labeled cells were collected as CD4⁺ or CD8⁺ cells. Purity of each cell was greater than 98% by analysis on FACScan (Becton Dickinson Immunocytometry Systems) after staining with FITC-labeled Abs.

PCR

PCR for ADAcDNA was performed using primer pairs corresponding to the first and second exons of ADA gene, which amplify ADAcDNA but not genomic ADA gene, because the first intron was too long to be amplified. Primers for ß-actin were utilized as an internal control. One microgram of DNA was amplified in a final volume of 50 µl. PCR was conducted in a DNA thermal cycler (Cetus, Emeryville, CA) for 30 cycles: 1 min denaturation at 94°C, 1 min annealing at 58°C, and 1 min extension at 72°C. PCR products were then visualized by being subjected to electrophoresis on 2% agarose in 0.5x TBE buffer containing ethidium bromide.

Semiquantitative PCR was performed as described previously (7). Briefly, primer pairs were designed to amplify the sequence between exon 7 and exon 8, which generated two bands of DNA samples from vector-containing cells by PCR, the large one derived from the endogenous ADA gene containing intron 7 and the smaller one from the LASN provirus. To evaluate the frequency of transduced cells in the patient’s peripheral blood, a standard curve was prepared from a serial dilution of in vitro-transduced and G418-selected B-lymphoblastoid cell lines (LCL) with untransduced cells. The ratio of the amount of amplified ADAcDNA derived from the integrated vector and that of the amplified genomic sequence was calculated after hybridization with an ADAcDNA probe. We have confirmed that this technique could provide quantitative results in samples with a transduction frequency below 50% (7).

To investigate the expression of each cytokine gene, RT-PCR was performed as described elsewhere (8). Briefly, cDNAs were generated from RNA templates in a 15-µl reaction mixture using a First-Strand cDNA synthesis kit (Pharmacia P-L Biochemicals, Piscataway, NJ). Two microliters of cDNA was amplified in a final volume of 50 µl using specific primers for IL-2, IL-4, IL-5, and IFN-. Primers for ß-actin were utilized as an internal control. PCR was conducted for 25 cycles: 1 min denaturation at 94°C, 1 min annealing at 55°C, and 1 min extension at 72°C. PCR products were then visualized by being subjected to electrophoresis on 2% agarose.

Two color fluorescence in situ hybridization

Details of the method of two color fluorescence in situ hybridization (FISH) on interphase nuclei of the PBL have been described elsewhere (9). Briefly, the clone pADA21l containing 1.1-kb human full ADA-cDNA and a 15-kb lambda-genomic ADA clone isolated from the EMBL3 SP6/T7 Lambda Library were used as probes. The cDNA and genomic probes were labeled with digoxigenin-11-dUTP and biotin-16-dUTP, respectively, by nick translation. A probe mixture (cDNA and genomic probes) and DNA of interphase cells on glass slides were denatured. The slides were hybridized at 37°C overnight in a 25-µl mixture containing the denatured probes, human COT-1 DNA, and 25% dextran sulfate, and washed at room temperature. The slides were then incubated at 37°C for 1 h in a 100-µl solution containing FITC avidin and anti-digoxigenin-rhodamin and washed. Interphase nuclei were counterstained with diamidino phenylindole, diazabicyclooctane, and 90% glycerol. Hybridized signals were observed under a fluorescence microscope with dual band pass filters. The sensitivity of the cDNA probe in this FISH system for the transgene has been estimated to be 85% (9).

ADA activity

ADA activity was measured as described previously (7). Briefly, PBMC were washed twice with PBS to remove FCS and then suspended in 100 mmol/l Tris (pH 7.4) containing 1% BSA. Cell lysates were obtained by five rapid freeze-thaw cycles. Cellular debris was removed by centrifugation, and the lysates were stored at -80°C until use. ADA enzyme activity was assayed by measurement of the conversion of [¹⁴C]adenosine (Amersham Life Science, Arlington Heights, IL) to [¹⁴C]inosine and [¹⁴C]hypoxanthine followed by TLC separation of the reaction products. The results were expressed as nanomoles of inosine and hypoxanthine produced per min by 10⁸ cells (nmol/min/10⁸ cells).

In vitro IgG production

A total of 1 x 10⁵ peripheral B cells from a normal individual was cultured with the same number of peripheral T cells from the patient or a normal individual in round-bottom microtiter plates. Cells were stimulated with PWM or Staphylococcus aureus Cowan I (SAC) to produce IgG in triplicate. Culture supernatants were harvested after 6 days for determination of IgG secretion by an IgG-specific ELISA.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
PBL count after infusion of transduced cells

The dose of transduced cells for infusion was first increased step-wise to check the safety of the GT procedure (Fig. 1). PBL count increased to a peak level within 1 wk after each infusion and decreased gradually to near the pre-GT level over the next several weeks. PBL count just before each GT increased constantly from the 7th infusion on, suggesting an accumulation of transduced cells. Maintenance of the patient’s PBL count at a high level seemed to require infusion of more than 4 x 10⁸ cells/kg/therapy/3 mo. The change in CD8⁺ cell count was parallel to that in total PBL count, whereas CD4⁺ cell count remained low over the entire course. These findings indicate that most of the transduced cells were of the CD8⁺ population.

View larger version (38K): [in this window] [in a new window]   FIGURE 1. Kinetics of PBL and T cell subset during gene therapy. Open circle, open triangle, and filled triangle indicate the number of PBL, CD8+ cells, and CD4+ cells, respectively. A filled arrow at the bottom represents an infusion of transduced cells in each GT. Increase in PBL means the cell number increased during the week after each infusion.

The number of CD4⁺ and CD8⁺ cells in both infused cells and patient’s PBL were examined after the 7th GT. The number of infused CD4⁺ cells was quite small, because more than 90% of cells were CD8⁺ cells after stimulation with anti-CD3 Ab for a gene transduction and subsequent in vitro culture with IL-2 (Table I). This may be the major reason why the increase in CD4⁺ cell number in PBL after each infusion was so small compared with that in CD8⁺ cell number.

Integration and expression of ADAcDNA in PBL

The integration of ADAcDNA in CD4⁺ and CD8⁺ cells purified from the patient’s PBL just before the 11th GT was examined (Fig. 2). ADAcDNA was apparently integrated in both CD4⁺ and CD8⁺ cells, but integration in CD4⁺ cells was less than that in CD8⁺ cells. These data indicate that both CD8⁺ and CD4⁺ cells carrying ADAcDNA were maintained for at least several months in the patient’s PBL, although the increase in CD4⁺ cells was less satisfactory.

View larger version (27K): [in this window] [in a new window]   FIGURE 2. Integration of transduced ADAcDNA in peripheral T cell subset. Vector-derived ADAcDNA and ß-actin gene were amplified from DNA sample purified from each type of cell just before the 11th GT and detected after electrophoresis on agarose gel. P, N, and M indicate positive control (LASN-transduced LCL which contained human ADAcDNA), negative control (nontransduced LCL), and DNA marker, respectively. Lanes 1 to 3 represent PCR products from total PBL, CD4+, and CD8+ cells, respectively.

View larger version (27K): [in this window] [in a new window]   FIGURE 3. Integration and expression of transduced ADAcDNA in PBL. An open circle indicates ADA activity in PBL. A filled or a hatched bar represents the frequency of transduced cells in PBL detected by semiquantitative PCR or FISH.

Most of the increased CD3⁺ cells in the patient’s PBL after GT expressed TCRß, even though TCR⁺ cells were slightly decreased during that time (Table III). Analysis of TCR repertoire based on TCR V region usage revealed that no limited clones were expanded in his PBL. These results suggest that the transduction procedure did not promote preferentially the growth of T cells bearing specific TCR V regions. On this basis, the increased CD8⁺ cells in his PBL may be considered to have contained functionally diverse populations, as seen in normal individuals.

Human CD8⁺ T cells are functionally heterogeneous and reported to contain a distinct population that possesses helper activity for B cells (10, 11, 12). We then analyzed T cell subset expressing CD8⁺CDw60⁺ or CD8⁺CD27⁺CD45RA^-, both of which have been reported to provide substantial help to B cells (13, 14). CD8⁺CDw60⁺ cells were maintained in his PBL throughout the GT, whereas the CD8⁺CD27⁺CD45RA^- cell population decreased (Table IV). However, this did not mean a decrease in the number of CD8⁺CD27⁺CD45RA^- cells in his peripheral blood, because the total number of peripheral CD8⁺ cells after GT was almost ten times than that before. In contrast, CD8⁺CD27^-CD45RA⁺ cells were significantly increased after GT. This population has been reported to include cytotoxic effector T cells (14).

View larger version (26K): [in this window] [in a new window]   FIGURE 4. Helper function of patient’s T cells in in vitro IgG production. IgG production from normal B cells with combination of normal or patient’s T cells was measured by IgG-specific ELISA assay after 6 days’ culture with stimulation of PWM or SAC.

View larger version (48K): [in this window] [in a new window]   FIGURE 5. Cytokine gene expression in PBL. Expression of each cytokine gene was analyzed by RT-PCR using RNA samples from patient’s PBL just before the 11th GT. Lane 1 and 2 indicate RT-PCR products from PBL without and with stimulation by PHA, respectively.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Cheever et al. (16) reported that cultured T cells could proliferate in vivo in response to stimulation by Ag, distribute widely, and survive long term to provide effector function and immunologic memory. Tough et al. (17) reported that long-lived T cells have multiple phenotypes and contain a mixture of memory cells, naive cells, and memory cells masquerading as naive cells. In humans, chromosome marker studies on irradiated patients indicate that CD45RA^low cells divide more frequently than CD45RA^high cells (18). However, the turnover of CD45RA^low cells is quite slow, because most can remain in interphase for months. All these previous reports suggest the relatively long-term survival of transferred T cells and are consistent with our observation that PBL count just before each GT had constantly increased since the 7th infusion, and that most of these were CD45RA⁺ cells including not only naive but also memory cells.

The median survival time of transferred cells in adoptive T cell therapy is reported to be proportionally increased, as the dose of effector T cells is increased (16). Our results indicate that at least 4 x 10⁸ cells/kg/therapy/3 mo were required to increase and maintain our patient’s PBL count. In two patients receiving GT at the National Institutes of Health (5), both were infused a substantial number of transduced cells, but only the first case has shown an apparent clinical improvement. Satisfactory GT is therefore additionally dependent on other factors, including differences in gene mutation, while the number of transduced cells may be an essential factor in peripheral T cell-directed GT.

In serial infusion, CD8⁺ cells gradually increase to become a major population of the patient’s PBL. This imbalance in T cell subset may be a characteristic problem in peripheral T cell-directed GT, on the basis of the fact that the major regulatory T cells are considered to be in the CD4⁺ population. In our patient, however, the frequency of transduced cells in PBL, ADA activity in PBL, and clinical improvement correlated well, even though CD8⁺ cells were predominant. Moreover, because the patient continued to receive the same dose of PEG-ADA during GT as before, his clinical improvement should have resulted from the GT itself.

To better understand the reconstitutive effect of GT on our patient’s immunological system, we investigated the repertoire and functional subset of T cells in his peripheral blood. Analysis of TCR repertoire in PBL after GT showed no limit in TCR V region usage, after CD8⁺ cells became predominant in his PBL. CD8⁺ cells in his PBL should therefore have contained functionally diverse populations as in normal individuals. Recent reports have shown increasing evidence of the existence of regulatory CD8⁺ cells, even though the main regulatory T cells should be in CD4⁺ population. Rieber et al. (13) reported that the CD8⁺CDw60⁺ subset provided substantial help to B lymphocytes. T cells with this phenotype were maintained in our patient throughout the GT. Hamann et al. (14) also reported that CD8⁺CD27⁺CD45RA^- cells had helper activity. CD8⁺ cells with this phenotype were also maintained in number, although the relative number of these cells in his PBL decreased after GT. It is thus conceivable that these two CD8⁺ T cell subsets may have played a role in the improvement of his immunological function. On the other hand, CD8⁺CD27^-CD45RA⁺ cells, which are considered to be CTL-type effector cells, significantly increased after the GT. These may also play a role in host defense against some pathogens. In addition, on the basis that low numbers of highly purified CD4⁺ cells, but not CD8⁺ cells, were successfully engrafted in the spleen of congenic SCID mice (19), a small but distinct population of CD4⁺ cells with transduced ADAcDNA likely had a longer life span and were involved in his immunological function.

Presently, peripheral T cell-directed GT is the most effective method available in GT for ADA^-SCID. Its fundamental drawback is its limited duration of effect. Technical improvements and modifications such as increased transduction efficiency with new vectors and increased CD4⁺ population during transduction procedures may improve efficiency. These approaches will improve the efficacy of this type of GT in providing an immediate but relatively short-term effect, but the final goal of GT for hemopoietic disorders must remain stem cell-directed GT.

	Acknowledgments

 
We thank Drs. R. M. Blaese and G. J. McGarity for providing the LASN retrovirus vector. We also thank Dr. G. Harris for his review of the manuscript.

	Footnotes

 
¹ This paper was supported by a Health Science Research grant from the Ministry of Health and Welfare of Japan.

² Address correspondence and reprint requests to Dr. Nobuaki Kawamura, Department of Pediatrics, Hokkaido University School of Medicine, North 15, West 7, Kita-ku, Sapporo, 060-8638, Japan. E-mail address:

³ Abbreviations used in this paper: GT, gene therapy; ADA, adenosine deaminase; FISH, fluorescence in situ hybridization; SAC, Staphylococcus aureus Cowan I; PEG, polyethylene glycol; LCL, lymphoblastoid cell line.

Received for publication February 8, 1999. Accepted for publication May 26, 1999.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Marcel, T., J. D. Grausz. 1997. The TMC worldwide gene therapy enrollment report, End 1996. Hum. Gene Ther. 8:775.[Medline]
2. Kohn, D. B., K. I. Weinberg, J. A. Nolta, L. N. Heiss, C. Lenarsky, G. M. Crooks, M. E. Hanley, G. Annett, J. S. Brooks, A. El-Khoureiy, K. Lawrence, S. Wells, R. C. Moen, J. Bastian, D. E. Williams-Herman, M. Elder, D. Wara, T. Bowen, M. S. Hershfield, C. A. Mullen, R. M. Blaese, R. Parkman. 1995. Engraftment of gene-modified umbilical cord blood cells in neonates with adenosine deaminase deficiency. Nat. Med. 1:1017.[Medline]
3. Hoogerbrugge, P. M., V. W. van Beusechem, A. Fischer, M. Debree, F. le Deist, J. L. Perignon, G. Morgan, B. Gaspar, L. D. Fairbanks, C. H. Skeoch, A. Moseley, M. Harvey, R. J. Levinsky, D. Valerio. 1996. Bone marrow gene transfer in three patients with adenosine deaminase deficiency. Gene Ther. 3:179.[Medline]
4. Kohn, D. B., M. S. Hershfield, D. Carbonaro, A. Shigeoka, J. Brooks, E. M. Smogorzewska, L. W. Barsky, R. Chan, F. Burotto, G. Annett, et al 1998. T lymphocytes with a normal ADA gene accumulate after transplantation of transduced autologous umbilical cord blood CD34⁺ cells in ADA-deficient SCID neonates. Nat. Med. 4:775.[Medline]
5. Blaese, R. M., K. W. Culver, A. D. Miller, C. S. Carter, T. Fleisher, M. Cleric, G. Shearer, L. Chang, Y. Chiang, P. Tolstoshev, et al 1995. T lymphocyte-directed gene therapy for ADA^-SCID: initial trial results after 4 years. Science 270:475.[Abstract/Free Full Text]
6. Bordignon, C., L. D. Notarangelo, N. Nobili, G. Ferrari, G. Casorati, P. Panina, E. Mazzolari, D. Maggioni, C. Rossi, P. Servida, A. G. Ugazio, F. Mavilio. 1995. Gene therapy in peripheral blood lymphocytes and bone marrow for ADA-immunodeficient patients. Science 270:470.[Abstract/Free Full Text]
7. Onodera, M., T. Ariga, N. Kawamura, I. Kobayashi, M. Ohtsu, M. Yamada, A. Tame, H. Furuta, M. Okano, S. Matsumoto, et al 1998. Successful peripheral T-lymphocyte-directed gene transfer for a patient with severe combined immune deficiency caused by adenosine deaminase deficiency. Blood 91:30.[Abstract/Free Full Text]
8. Kawamura, N., H. Furuta, A. Tame, I. Kobayashi, T. Ariga, M. Okano, Y. Sakiyama. 1997. Extremely high serum level of IgE during immunosuppressive therapy: paradoxical effect of cyclosporine A and tacrolimus. Int. Arch. Allergy Immunol. 112:422.[Medline]
9. Egashira, M., T. Ariga, N. Kawamura, O. Miyoshi, N. Niikawa, Y. Sakiyama. 1998. Visible integration of the adenosine deaminase (ADA) gene into the recipient genome after gene therapy. Am. J. Med. Genet. 75:314.[Medline]
10. Massague, J.. 1990. The transforming growth factor-ß family. Annu. Rev. Cell Biol. 6:597.
11. Maggi, E., M. G. Giudizi, R. Biagiotti, F. Annunziato, R. Manetti, M. P. Piccinni, S. P. Parronchi, L. Sampognaro, G. Giannarini, G. Zuccati, S. Romagnani. 1994. Th2-like CD8⁺ T cells showing B cell helper function and reduced cytolytic activity in human immunodeficiency virus type 1 infection. J. Exp. Med. 180:489.[Abstract/Free Full Text]
12. Paganelli, R, E. Scala, I. J. Ansotegui, C. M. Ausiello, E. Halapi, E. Fanales-Belasio, G. D’Offizi, I. Mezzaroma, F. Pandolfi, M. Fiorilli, A. Cassone, F. Aiuti. 1995. CD8⁺ T lymphocytes provide helper activity for IgE synthesis in human immunodeficiency virus-infected patients with hyper-IgE. J. Exp. Med. 181:423.[Abstract/Free Full Text]
13. Rieber, E. P., G. Rank. 1994. CDw60: a marker for human CD8⁺ T helper cells. J. Exp. Med. 179:1385.[Abstract/Free Full Text]
14. Hamann, D, P. A. Baars, M. H. G. Rep, B. Hooibrink, S. R. Kerkhof-Garde, M. R. Klein, R. A. W. van Lier. 1997. Phenotypic and functional separation of memory and effector human CD8⁺ T cells. J. Exp. Med. 186:1407.[Abstract/Free Full Text]
15. McDonagh, M., E. B. Bell. 1995. The survival and turnover of mature and immature CD8 T cells. Immunology. 84:514.[Medline]
16. Cheever, M. A., W. Chen. 1997. Therapy with cultured T cells: principles revisited. Immunol. Rev. 157:177.[Medline]
17. Tough, D. F., J. Sprent. 1994. Turnover of naive- and memory-phenotype T cells. J. Exp. Med. 179:1127.[Abstract/Free Full Text]
18. Michie, C. A., A. McLean, C. Alcock, P. C. Beverley. 1992. Life span of human lymphocytes subsets defined by CD45 isoforms. Nature 360:264.[Medline]
19. Rudolphi, A., S. Spieß, P. Conradt, M. H. Claesson, J. Reimann. 1991. CD3⁺ T cells in severe combined immune deficiency (scid) mice. I. Transferred purified CD4⁺ T cells, but not CD8⁺ T cells, are engrafted in the spleen of congenic scid mice. Eur. J. Immunol. 21:523.[Medline]

This article has been cited by other articles:

				M. B. Aldrich, W. Chen, M. R. Blackburn, H. Martinez-Valdez, S. K. Datta, and R. E. Kellems Impaired Germinal Center Maturation in Adenosine Deaminase Deficiency J. Immunol., November 15, 2003; 171(10): 5562 - 5570. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kawamura, N. Articles by Sakiyama, Y. Search for Related Content PubMed PubMed Citation Articles by Kawamura, N. Articles by Sakiyama, Y.