JOURNAL
OF
Vol. 66, No. 6
VIROLOGY, June 1992, p. 3966-3970
0022-538X/92/063966-05$02.00/0 Copyright © 1992, American Society for Microbiology
Differential Human Immunodeficiency Virus Expression in CD4+ Cloned Lymphocytes: from Viral Latency to Replication A. CHAPEL,"* A. BENSUSSAN,2 E. VILMER,3 AND D. DORMONT"4 Laboratoire de Neuropathologie Experimentale et Neurovirologie, Centre de Recherches du Service de Sante des Armnes, Commissariat a l'Energie Atomique, DSV/DPTE, B.P. 6, 92265 Fontenay aux Roses,1 and Institut National de la Sante et de la Recherche Medicale U93,2 Hopital Robert Debre,3 and Institut Pasteur,4 Pars, France Received 3 June 1991/Accepted 17 March 1992
By using cloning methodology, 13 CD4+, CD8-, CD45RO+, and CD29+ clones, isolated from human immunodeficiency virus (HIV)-negative donors, have been characterized and tested regarding their susceptibility to two strains of HIV type 1 (HIV-1). Infected clones possess integrated provirus. Only six are able to replicate HIV-1, while seven may normally grow without cytopathic effect and without viral replication. These results argue that all CD4+ lymphocyte clones may be infectable but that a heterogeneity exists regarding their abilities to replicate HIV-1.
performed with previously activated cells, this subset delineation is not relevant. Monoclonal CD4+ cells were infected at 50 IU (0.4 x 106 50% tissue culture infective doses) per 106 cells for 24 h (an IU denotes the incorporation of 1 pmol of [33H]TMP per h at 37°C for 1 ml of cell culture supernatant [106 cells]). Two different HIV-1 strains were used, and both were produced only on human peripheral blood mononuclear cells (PBMC). The first, HIV-1 reference strain HIVI-LAVI, was obtained from F. C. Barre-Sinoussi (Institut Pasteur, Paris, France) (2). The second, HIV1-DAS, was isolated in our laboratory during the primary infection of a patient at the time of a spontaneous remission of a neurologic syndrome (5). HIV replication was sequentially studied in supernatants by reverse transcriptase (RT) activity, as previously described (17), and by immunocapture (performed according to Abbott recommendations [8]). Two major HIV replication patterns were identifiable. (i) In "replicating" clones FAS38, C16, FAS75, JAF423, and Dl, an increase in RT activity (31 to 247 IU) and P24 antigen (16,700 to 296,400 pg/ml) occurred between the 6th and the 10th days postinfection, as shown in Fig. la. These viral productions were associated with cytopathic effects (Fig. lb). (ii) In "unreplicating" clones SABl, SAB2, SAB10, JAF410, JAF46, JAF44S, and 20A3, neither RT activity (7.6 to 11.5 IU) (Fig. la) nor P24 antigen (10 to 40 pg/ml [values not significant]) was detectable in supernatants during 21 days of culture. No change in growth rate or decrease in cell viability was observed (Fig. lb). We looked for potential low viral expression or for propagation by cell contact in unreplicating clones. A coculture with umbilical cord lymphocytes and 10-day postinfection monoclonal CD4+ populations was established at a ratio of one monoclonal cell to five umbilical cord cells (3). No significant viral replication was detectable in any of the clones during 21 days of culture (Table 1). In order to investigate the presence of late viral replication in unreplicating clones, a long-term culture (2 months) was performed. No significant viral replication was detected during this 2-month period of experimentation (Fig. la). One clone (JAF48) possesses a different pattern of replication; P24 antigen (2,050 pg/ml; Table 1) was detectable in the absence of significant RT activity (8.9 IU). P24 immunocapture was positive after 14 days of coculture (Table 1).
Cytopathic effect within the CD4+ subset is an important contributor to the state of immunodeficiency (2). In seropositive individuals, quantification of infected cells reveals that only a low proportion of CD4+ T cells are infected with human immunodeficiency virus type 1 (HIV-1) (16). This fact suggests that the loss of CD4+ T cells in infected individuals is not simply the inevitable result of the activation of latent virus or of a spread of productive infection during T-cell expansion.
Whatever the depletion mechanism is, viral replication to be required. Knowledge of latency and viral expression is crucial to understanding pathogenesis and to designing efficient therapies. It appears that cloned lymphocytes represent an accurate tool in the investigation of these two processes. To study differential HIV replication, human cloned T lymphocytes were isolated from two HIV-negative individuals by cloning methodology (24). Clones Dl, LSO, and 20A3 (AC3) have been described elsewhere (24, 25). Clones C16, FAS75, FAS38, SAB1, SAB2, SAB10, JAF423, JAF44S, JAF48, JAF410, and JAF46 were assayed for their clonality by Southern blot analysis using different restriction enzymes and T-cell receptor ot and P probes as described elsewhere (4). Phenotypic analysis was performed by indirect immunofluorescence on a cytofluorograph (Facstar; Becton-Dickinson, Mountain View, Calif.) (25). Monoclonal antibodies used were OKT3, OKT4, OKT4a, and OKT8 (Ortho Diagnostics, Inc., Raritan, N.J.); UCHL1 (CD45RO) and 4B4 (CD29) (Dakopats, Glostrup, Denmark); and S15 (CD18) and BC96 (CD25), both of which were produced locally. All the clones expressed T-cell receptor at and ,3 probes and reacted similarly with anti-CD3, -CD4, -CD18, -CD25, -CD29, and -CD45 monoclonal antibodies. Negative control LSO was composed of cloned cells CD4-, CD8-, and CD56+ expressing T-cell receptor y and 8 probes, obtained from a seronegative individual. Peripheral blood CD4+, CD45RA-, CD45RO+, and CD29+ T-lymphocyte subsets are preferentially infected by HIV-1 in vitro, and these cells might be related to the main in vivo reservoir within the CD4+ T-cell population in individuals infected with HIV-1 (20). Since our study was seems
*
Corresponding author. 3966
VOL. 66, 1992
NOTES
35 -
3967
b
30
25 -
20
15
-
Iz Uninfected
Replicating
clone C16 -O
10
Infected (LAV I)
Unreplicating clone SABI 0
Uninfected -L-- Infected (LAV I)
60 5
10
15
20
25
30
35
40
45
50
55
60
Days post-infection Days post-infection
FIG. 1. (a) RT activity in cell culture supernatants of clones C16 and SAB10, infected with HIV1-LAV1. Poly(dA) oligo(dT)12 (DNA pol) was used as a cellular DNA polymerase background control. (b) Cellular growth of the replicating clone C16 and of the unreplicating clone SAB10 infected with HIV1-LAV1. 18
not shown). In addition, the CD4+ cloned cells might be distinguished by their ability to mediate cytotoxicity; it is important to note that C16, which replicates HIV, and SAB10, which does not, are two clones derived from the
These results were reproducible over a 3-year period with the two virus isolates HIV1-LAV1 and HIV1-DAS. Similar results have been obtained by others with cell lineages; CEM-E5, T, and Wil-2B cell lines showed diversity in their responses to HIV (9, 19). Either clones replicate early after infection or they do not replicate at all; there is no significant intermediate pattern. This absence of viral expression was not associated with gamma interferon production: no significant difference was observed between replicating and unreplicating clones (data
donor, and both mediate major histocompatibility complex class II allogenic cytotoxic T-lymphocyte activity. As far as we could determine, in CD4+ lymphocyte subpopulations, HIV susceptibility cannot be related to two functionally defined subsets of CD4+ T lymphocytes. Evaluation of viral DNA sequences in host cells by means same
TABLE 1. Phenotypic characterization of cloned CD4+ lymphocytes and virologic results Virologic results Clone or
group'
Phenotype
Unreplicating clones )
CD8-, CD3+
JAF48
CD4+
Replicating clones
J
CD25+, CD45RO+, CD29+
RT" (IU)
P24' (pg)
7.6-11.5 8.9 31-247
10-40
Peak value in cell culture supernatant over a 21-day postinfection period.
'Determined by immunocapture assay.
d
PCRe
P25, +; RT, -
+ + +
31.6 3.6 CD4-, CD8-, CD56+ Replicating clones are FAS38, C16, FAS75, JAF423, Dl, and JAF48. Unreplicating clones are SAB1, SAB2, SAB10, JAF410, JAF46, JAF44S, and 20A3.
LSO (control) h
2,050 16,700-296,400
Cocultured
Viral replication was detected by immunocapture assay and RT activity. Detection of proviral DNA in cloned CD4+ lymphocytes at 10 days postinfection.
3968
NOTES
_w *,. _- NF_o~w UNREPLICATING;
J. VIROL. REPLICATING
CONTI'ROL A
"I-
(ONTROL ;I. E(.
SA1BlI
JAF46
fPUS,lI%F4.4 s3AF4101)0DS 1ANA DAS LAVIX
1
N
20A3
SA112
SAI
LS()
\
FAS38 L)1 J1AF423 FA5S75 C16
JAI48
1 1 1f I1T 1 T11 1¶j1
40
asomm
"O'
.11'111W iiiiiiw
MO..Im
11 1
I
f
...
--i'AWIMM,,;a. ....
..
_4
MWIW40
a*
lk ......
N' fl 1614
.44
_
_
_.4.4
-
:;,
hp)
hp
Ta1 4426) p) .n 4)2 21)p
v
_0
P l()I309 h ->>
FIG. 2. Detection by PCR of viral (HIVl-LAVl) penetration in cloned CD4+ lymphocytes at 10 days postinfection. Primer pairs were separately used, and amplified products were then pooled for each infected clone. Control A: CD3-, CD4-, and CD56+ clone (LSO). Controls B: positive, PBMC infected with HIV1-LAV1 at 10 days postinfection; negative, fresh uninfected PBMC.
of the polymerase chain reaction (PCR) was performed as previously described (10, 11, 15, 18, 22). Five pairs of primers were separately used for each infected clone. Amplified sequences were as follows: 1,614 bp in the vif region (positions 4532 to 6146 in HIV1-LAV1 [1]), 853 bp in the first part of the gag gene (positions 321 to 1174), 446 bp in the tat gene (positions 5262 to 5708), 422 bp in the first part of the env gene (positions 5762 to 6184), and 309 bp in the pol gene (positions 2391 to 2700). Amplification products for each clone were pooled and subjected to a comigration. They were then identified by hybridizing a Southern blot with a full-length 32P-labelled HIV probe (specific activity, 5 x 107 cpm/,ug) (6). The influence of a feeder was investigated by infecting mouse spleen cells (C57 B1/6J) with either HIV1LAV1 or HIVl-DAS in the presence of human irradiated PBMC as the feeder (same experimental methodology as for CD4+ clones). In the presence of a feeder, HIV was detected until 7 days postinfection. In the absence of a feeder, no PCR product was detectable in mouse spleen cells 24 h after infection. This result demonstrated that feeder gave rise at PCR background during the first days of infection, whereas virus stock did not (data not shown). Thus, viral penetration and RT were always screened starting at 10 days postinfection. PCR products of all amplified sequences were identifiable in all clones, with no differences in size (Fig. 2); therefore, no deletion was detectable in amplified sequences. Nevertheless, DNA extraction and PCR methods do not allow us to know whether or not viral DNA is integrated into the genome of the host cell. Identification of viral DNA in clones at different times from 14 to 60 days postinfection confirmed the HIV infection of both replicating and unreplicating clones. Detection of PCR products from 21 to 60 days postinfection allowed us to discard the hypothesis that cloned infected cells died early (until 10 days postinfection), before the propagation of infection. Although the proportion of virus-infected cells for each culture is unknown, a long-
term culture apparently does not modify this balance. Thus, cells which harbor the viral genome grow as uninfected cells. This result demonstrates that, in culture, the possibility exists for HIV-1-infected human CD4+ T lymphocytes to grow normally and to produce interleukin 2 (data not shown) without cytopathic effect and without viral replication. Penetration of the two viral strains occurs in all clones. Nevertheless, modification of a CD4 molecule into the site of fixation of gpl20, the requirement of an accessory molecule (for instance, LFA1), a difference in membrane fluidity, or a block in infection at a stage after virus entry might be thrown back since the two subpopulations of CD4+ clones were infected. Preincubation of human cloned lymphocytes with monoclonal antibody anti-Leu3a (at 1:200, as previously described [13]), prior to HIV infection, inhibited viral infection, since neither viral replication (Fig. 3) nor viral penetration as assessed by PCR (data not shown) was detectable in the two types of clones with the two strains of HIV-1. These results demonstrate the requirement of HIV-1 fixation to the CD4 molecule in all tested clones and argue in favor of the unity of the path of infection in CD4+ lymphocyte subtypes. HIV does not replicate in resting T cells; therefore, we expected that activation of T cells in culture would initiate viral replication. Lack of HIV replication in CD4+ cloned lymphocytes might at least be explained by an intracytoplasmic (or nuclear) regulation. Specific products of T-cell activation might stimulate transcription of proviral DNA (14): transcription factors, such as NF-KB (21), act directly on the HIV long terminal repeat (23). These HIV enhancerbinding proteins, which are constitutive or inducible, are specific for cell type (7, 12). These enhancer-binding proteins could act differently in each of our clones that depend on interleukin 2 for growth. A clear understanding of the lack of viral replication necessitates, first, an exhaustive study of unreplicating
VOL. 66, 1992
1100o
NOTES
AwlA
5.
oooa)0 A~~~~~~~~~~ 6.
9000 ' 800C DO
'
700(DO -
600( 0
7.
|''-'ffi''- Without anti-Leu 3a
'
With anti-Leu 3a
8.
.
; .40.
DO' ;~~~~~~~~~
i
9.
500C
00-.''
C.
10.
4004
3004
00 2 4 6'.0 12 00-.''
2004
0.'' !
;
1
6
8
2
11.
;~~~~~~~~~~~~~~~~~~......
0
12
6
18
2
12.
1004
13.
Days post-infection
FIG. 3. Detection of P24 antigen in cell culture supernatant of clone C16, with or without preincubation with anti-Leu3a monoclonal antibody, before infection with HIV1-LAV1.
clones (metabolism, secretions, membrane antigens, and functions) and, second, an investigation of the molecular mechanisms of viral expression in our clones; such an investigation is now in progress. We thank Magali Fasseu and Jean Franqois Bourge for technical assistance. This work has been partly supported by IBM and Caisse Nationale de l'Assurance Maladie des Travailleurs Salaries (no. 872392E).
REFERENCES 1. Alizon, M. A., P. Sonigo, F. Barre-Sinoussi, J. C. Chermann, P. Tiollais, L. Montagnier, and S. Wain-Hobson. 1984. Molecular cloning of lymphadenopathy-associated virus. Nature (London) 312:753-756. 2. Barre-Sinoussi, F. C., J. C. Chermann, F. Rey, M. T. Nugeyre, S. Chamaret, J. Gruest, C. Dauget, C. Axler-Blinc, F. VezinetBrun, C. Rouzioux, W. Rozenbaum, and L. Montagnier. 1984. Isolation of a T-lymphotropic retrovirus from a patient at risk of acquired immune deficiency syndrome (AIDS). Science 224: 497-500. 3. Barre-Sinoussi, F. C., U. Mathur-Wagh, F. Rey, F. BrunVezinet, S. R. Yancovitz, C. Rouzioux, L. Montagnier, D. Muildvan, and J. C. Chermann. 1985. Isolation of LAV antibodies from US patients with AIDS. JAMA 253:1737-1739. 4. Bensussan, A., S. K. Meuer, S. F. Schzossman, and E. L. Reinherz. 1984. Delineation of immunoregulatory amplified pop-
14. 15.
16.
17.
18.
19.
20.
21. 22.
3969
ulation recognizing autologous Ia molecules analysis with human T cell clones. J. Exp. Med. 159:559-576. Boussin, F., D. Dormont, B. Spire, H. Fleury, F. Rey, D. Bequet, J. Goasguen, and P. Brunet. 1988. Isolation of HIV in a seronegative demented patient without symptoms of immune deficiency. Cancer Detect. Prev. 12:257-265.8. Cunningham, M. 1982. Spot blot: a hybridation assay for specific DNA sequences in multiple samples. Anal. Biochem. 128:415-421. Franza, B. R., S. F. Joseph, M. Z. Gilman, W. Ryan, and B. Clarkson. 1987. Characterization of cellular proteins recognizing the HIV enhancer using a microscale DNA-affinity. Nature (London) 330:391-395. Goudsmit, J., F. De Wolf, D. A. Paul, L. G. Epstein, J. M. A. Lange, W. J. A. Krone, H. Speelman, E. C. Wolters, J. Van der Noordaa, J. M. Olesk, H. J. Van der Helm, and R. A. Coutinho. 1986. Expression of HIV antigen (HIV-Ag) in serum and cerebrospinal fluid during acute and chronic infection. Lancet ii: 177-180. Klatzman, D., E. Champagne, S. Chamaret, J. Gruest, D. Guetard, T. Hercend, J. C. Gluckman, and L. Montagnier. 1984. T-lymphocyte T4 molecule behaves as the receptor for human retrovirus LAV. Nature (London) 312:767-768. Laure, F., C. Rouzioux, F. Veber, C. Jacomet, V. Courgnaud, S. Blanche, S. Burgard, C. Griscelli, and C. Brechot. 1988. Detection of HIV1 DNA in infants and children by means of polymerase chain reaction. Lancet i:538-541. Loche, M., and B. Mach. 1988. Identification of HIV-infected seronegative individuals by a direct diagnostic test based on hybridisation to amplified viral DNA. Lancet i:418-421. Lubon, H., P. Ghazal, J. A. Nelson, and L. Hennighausen. 1988. Cell-specific activity of the human immunodeficiency virusenhancer repeat in vitro. AIDS Res. Hum. Retroviruses 4:381391. McDougall, J. S., A. Mawle, S. P. Cort, J. K. A. Nicholson, G. D. Cross, J. A. Scheppler Campbell, D. Hicks, and J. Sligh. 1985. Cellular tropism of the human retrovirus HTLVIII/LAV. J. Immunol. 135:3151-3152. Nabel, G., and D. Baltimore. 1987. An inducible transcription factor activates expression of human immunodeficiency virus in T cells. Nature (London) 326:711-713. Ou, C.-Y., S. Kwok, S. W. Mitchell, D. H. Mack, J. J. Sninsky, J. W. Krebs, P. Feorino, D. Warfield, and G. Schochetman. 1988. DNA amplification for direct detection of HIV1 in DNA of peripheral blood mononuclear cells. Science 239:295-297. Psallidopoulos, M. C., S. M. Schnittman, L. M. Thompson III, M. Baseler, A. S. Fauci, H. C. Lane, and N. P. Salzman. 1989. Integrated proviral human immunodeficiency virus type 1 is present in CD4+ peripheral blood lymphocytes in healthy seropositive individuals. J. Virol. 63:4626-4631. Rey, M. A., B. Spire, D. Dormont, F. Barre-Sinoussi, L. Montagnier, and J. C. Chermann. 1984. Characterization of RNA dependant DNA polymerase of a new human T lymphotropic retrovirus (lymphadenopathy associated virus). Biochem. Biophys. Res. Commun. 121:126-133. Saiki, R. K., D. H. Gelfand, S. Stoffel, S. J. Scharf, R. Higuchi, G. T. Horn, K. B. Mullis, and H. A. Herlich. 1987. Primerdirected enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239:487-491. Scheppler, J. A., A. C. Mawle, and McDougall. 1988. Evaluation of target cells for HIV-1-specific antibody dependant cellular cytotoxicity. J. Immunol. Methods 115:247-253. Schnittman, S. M., H. C. Lane, J. Greenhouse, J. S. Justement, M. Baselerand, and A. S. Fauci. 1990. Preferential infection of CD4+ memory T cells by human immunodeficiency virus type 1: evidence for a role in the selective T-functional defects observed in infected individuals. Proc. Natl. Acad. Sci. USA 87:6058-6062. Sen, R., and D. Baltimore. 1986. Multiple nuclear factors interact with the immunoglobulin enhancer sequences. Cell 46:705-716. Sharf, J. S., G. T. Horn, and H. A. Erlich. 1986. Direct cloning analysis of enzymatically amplified genomic sequences. Science
3970
NOTES
233:1076-1078. 23. Tong-Starksen, S. E., P. A. Luciw, and B. M. Peterlin. 1987. Human immunodeficiency virus long terminal repeat responds to T-cell activation signal. Proc. Natl. Acad. Sci. USA 84:68456849. 24. Vilmer, E., P. Gugliemi, V. David, G. Leca, C. Rabian, L. Degos, M. Boiron, and A. Bensussan. 1988. Predominant expression of circulating CD3+ lymphocytes bearing y T cell receptor in a
J. VIROL.
prolonged immunodeficiency after allogeneic bone marrow transplantation. J. Clin. Invest. 82:755-761. 25. Vilmer, E., F. Triebel, C. Rabian, V. David, M. Schump, G. Leca, L. Degos, T. Hercend, F. Sigaux, and A. Bensussan. 1988. Predominant expansion of circulating lymphocytes bearing y T cell receptor with preferential expression of variable gamma genes, after allogeneic bone marrow transplantation. Blood 72:841-849.
OF
Vol. 66, No. 6
VIROLOGY, June 1992, p. 3966-3970
0022-538X/92/063966-05$02.00/0 Copyright © 1992, American Society for Microbiology
Differential Human Immunodeficiency Virus Expression in CD4+ Cloned Lymphocytes: from Viral Latency to Replication A. CHAPEL,"* A. BENSUSSAN,2 E. VILMER,3 AND D. DORMONT"4 Laboratoire de Neuropathologie Experimentale et Neurovirologie, Centre de Recherches du Service de Sante des Armnes, Commissariat a l'Energie Atomique, DSV/DPTE, B.P. 6, 92265 Fontenay aux Roses,1 and Institut National de la Sante et de la Recherche Medicale U93,2 Hopital Robert Debre,3 and Institut Pasteur,4 Pars, France Received 3 June 1991/Accepted 17 March 1992
By using cloning methodology, 13 CD4+, CD8-, CD45RO+, and CD29+ clones, isolated from human immunodeficiency virus (HIV)-negative donors, have been characterized and tested regarding their susceptibility to two strains of HIV type 1 (HIV-1). Infected clones possess integrated provirus. Only six are able to replicate HIV-1, while seven may normally grow without cytopathic effect and without viral replication. These results argue that all CD4+ lymphocyte clones may be infectable but that a heterogeneity exists regarding their abilities to replicate HIV-1.
performed with previously activated cells, this subset delineation is not relevant. Monoclonal CD4+ cells were infected at 50 IU (0.4 x 106 50% tissue culture infective doses) per 106 cells for 24 h (an IU denotes the incorporation of 1 pmol of [33H]TMP per h at 37°C for 1 ml of cell culture supernatant [106 cells]). Two different HIV-1 strains were used, and both were produced only on human peripheral blood mononuclear cells (PBMC). The first, HIV-1 reference strain HIVI-LAVI, was obtained from F. C. Barre-Sinoussi (Institut Pasteur, Paris, France) (2). The second, HIV1-DAS, was isolated in our laboratory during the primary infection of a patient at the time of a spontaneous remission of a neurologic syndrome (5). HIV replication was sequentially studied in supernatants by reverse transcriptase (RT) activity, as previously described (17), and by immunocapture (performed according to Abbott recommendations [8]). Two major HIV replication patterns were identifiable. (i) In "replicating" clones FAS38, C16, FAS75, JAF423, and Dl, an increase in RT activity (31 to 247 IU) and P24 antigen (16,700 to 296,400 pg/ml) occurred between the 6th and the 10th days postinfection, as shown in Fig. la. These viral productions were associated with cytopathic effects (Fig. lb). (ii) In "unreplicating" clones SABl, SAB2, SAB10, JAF410, JAF46, JAF44S, and 20A3, neither RT activity (7.6 to 11.5 IU) (Fig. la) nor P24 antigen (10 to 40 pg/ml [values not significant]) was detectable in supernatants during 21 days of culture. No change in growth rate or decrease in cell viability was observed (Fig. lb). We looked for potential low viral expression or for propagation by cell contact in unreplicating clones. A coculture with umbilical cord lymphocytes and 10-day postinfection monoclonal CD4+ populations was established at a ratio of one monoclonal cell to five umbilical cord cells (3). No significant viral replication was detectable in any of the clones during 21 days of culture (Table 1). In order to investigate the presence of late viral replication in unreplicating clones, a long-term culture (2 months) was performed. No significant viral replication was detected during this 2-month period of experimentation (Fig. la). One clone (JAF48) possesses a different pattern of replication; P24 antigen (2,050 pg/ml; Table 1) was detectable in the absence of significant RT activity (8.9 IU). P24 immunocapture was positive after 14 days of coculture (Table 1).
Cytopathic effect within the CD4+ subset is an important contributor to the state of immunodeficiency (2). In seropositive individuals, quantification of infected cells reveals that only a low proportion of CD4+ T cells are infected with human immunodeficiency virus type 1 (HIV-1) (16). This fact suggests that the loss of CD4+ T cells in infected individuals is not simply the inevitable result of the activation of latent virus or of a spread of productive infection during T-cell expansion.
Whatever the depletion mechanism is, viral replication to be required. Knowledge of latency and viral expression is crucial to understanding pathogenesis and to designing efficient therapies. It appears that cloned lymphocytes represent an accurate tool in the investigation of these two processes. To study differential HIV replication, human cloned T lymphocytes were isolated from two HIV-negative individuals by cloning methodology (24). Clones Dl, LSO, and 20A3 (AC3) have been described elsewhere (24, 25). Clones C16, FAS75, FAS38, SAB1, SAB2, SAB10, JAF423, JAF44S, JAF48, JAF410, and JAF46 were assayed for their clonality by Southern blot analysis using different restriction enzymes and T-cell receptor ot and P probes as described elsewhere (4). Phenotypic analysis was performed by indirect immunofluorescence on a cytofluorograph (Facstar; Becton-Dickinson, Mountain View, Calif.) (25). Monoclonal antibodies used were OKT3, OKT4, OKT4a, and OKT8 (Ortho Diagnostics, Inc., Raritan, N.J.); UCHL1 (CD45RO) and 4B4 (CD29) (Dakopats, Glostrup, Denmark); and S15 (CD18) and BC96 (CD25), both of which were produced locally. All the clones expressed T-cell receptor at and ,3 probes and reacted similarly with anti-CD3, -CD4, -CD18, -CD25, -CD29, and -CD45 monoclonal antibodies. Negative control LSO was composed of cloned cells CD4-, CD8-, and CD56+ expressing T-cell receptor y and 8 probes, obtained from a seronegative individual. Peripheral blood CD4+, CD45RA-, CD45RO+, and CD29+ T-lymphocyte subsets are preferentially infected by HIV-1 in vitro, and these cells might be related to the main in vivo reservoir within the CD4+ T-cell population in individuals infected with HIV-1 (20). Since our study was seems
*
Corresponding author. 3966
VOL. 66, 1992
NOTES
35 -
3967
b
30
25 -
20
15
-
Iz Uninfected
Replicating
clone C16 -O
10
Infected (LAV I)
Unreplicating clone SABI 0
Uninfected -L-- Infected (LAV I)
60 5
10
15
20
25
30
35
40
45
50
55
60
Days post-infection Days post-infection
FIG. 1. (a) RT activity in cell culture supernatants of clones C16 and SAB10, infected with HIV1-LAV1. Poly(dA) oligo(dT)12 (DNA pol) was used as a cellular DNA polymerase background control. (b) Cellular growth of the replicating clone C16 and of the unreplicating clone SAB10 infected with HIV1-LAV1. 18
not shown). In addition, the CD4+ cloned cells might be distinguished by their ability to mediate cytotoxicity; it is important to note that C16, which replicates HIV, and SAB10, which does not, are two clones derived from the
These results were reproducible over a 3-year period with the two virus isolates HIV1-LAV1 and HIV1-DAS. Similar results have been obtained by others with cell lineages; CEM-E5, T, and Wil-2B cell lines showed diversity in their responses to HIV (9, 19). Either clones replicate early after infection or they do not replicate at all; there is no significant intermediate pattern. This absence of viral expression was not associated with gamma interferon production: no significant difference was observed between replicating and unreplicating clones (data
donor, and both mediate major histocompatibility complex class II allogenic cytotoxic T-lymphocyte activity. As far as we could determine, in CD4+ lymphocyte subpopulations, HIV susceptibility cannot be related to two functionally defined subsets of CD4+ T lymphocytes. Evaluation of viral DNA sequences in host cells by means same
TABLE 1. Phenotypic characterization of cloned CD4+ lymphocytes and virologic results Virologic results Clone or
group'
Phenotype
Unreplicating clones )
CD8-, CD3+
JAF48
CD4+
Replicating clones
J
CD25+, CD45RO+, CD29+
RT" (IU)
P24' (pg)
7.6-11.5 8.9 31-247
10-40
Peak value in cell culture supernatant over a 21-day postinfection period.
'Determined by immunocapture assay.
d
PCRe
P25, +; RT, -
+ + +
31.6 3.6 CD4-, CD8-, CD56+ Replicating clones are FAS38, C16, FAS75, JAF423, Dl, and JAF48. Unreplicating clones are SAB1, SAB2, SAB10, JAF410, JAF46, JAF44S, and 20A3.
LSO (control) h
2,050 16,700-296,400
Cocultured
Viral replication was detected by immunocapture assay and RT activity. Detection of proviral DNA in cloned CD4+ lymphocytes at 10 days postinfection.
3968
NOTES
_w *,. _- NF_o~w UNREPLICATING;
J. VIROL. REPLICATING
CONTI'ROL A
"I-
(ONTROL ;I. E(.
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of the polymerase chain reaction (PCR) was performed as previously described (10, 11, 15, 18, 22). Five pairs of primers were separately used for each infected clone. Amplified sequences were as follows: 1,614 bp in the vif region (positions 4532 to 6146 in HIV1-LAV1 [1]), 853 bp in the first part of the gag gene (positions 321 to 1174), 446 bp in the tat gene (positions 5262 to 5708), 422 bp in the first part of the env gene (positions 5762 to 6184), and 309 bp in the pol gene (positions 2391 to 2700). Amplification products for each clone were pooled and subjected to a comigration. They were then identified by hybridizing a Southern blot with a full-length 32P-labelled HIV probe (specific activity, 5 x 107 cpm/,ug) (6). The influence of a feeder was investigated by infecting mouse spleen cells (C57 B1/6J) with either HIV1LAV1 or HIVl-DAS in the presence of human irradiated PBMC as the feeder (same experimental methodology as for CD4+ clones). In the presence of a feeder, HIV was detected until 7 days postinfection. In the absence of a feeder, no PCR product was detectable in mouse spleen cells 24 h after infection. This result demonstrated that feeder gave rise at PCR background during the first days of infection, whereas virus stock did not (data not shown). Thus, viral penetration and RT were always screened starting at 10 days postinfection. PCR products of all amplified sequences were identifiable in all clones, with no differences in size (Fig. 2); therefore, no deletion was detectable in amplified sequences. Nevertheless, DNA extraction and PCR methods do not allow us to know whether or not viral DNA is integrated into the genome of the host cell. Identification of viral DNA in clones at different times from 14 to 60 days postinfection confirmed the HIV infection of both replicating and unreplicating clones. Detection of PCR products from 21 to 60 days postinfection allowed us to discard the hypothesis that cloned infected cells died early (until 10 days postinfection), before the propagation of infection. Although the proportion of virus-infected cells for each culture is unknown, a long-
term culture apparently does not modify this balance. Thus, cells which harbor the viral genome grow as uninfected cells. This result demonstrates that, in culture, the possibility exists for HIV-1-infected human CD4+ T lymphocytes to grow normally and to produce interleukin 2 (data not shown) without cytopathic effect and without viral replication. Penetration of the two viral strains occurs in all clones. Nevertheless, modification of a CD4 molecule into the site of fixation of gpl20, the requirement of an accessory molecule (for instance, LFA1), a difference in membrane fluidity, or a block in infection at a stage after virus entry might be thrown back since the two subpopulations of CD4+ clones were infected. Preincubation of human cloned lymphocytes with monoclonal antibody anti-Leu3a (at 1:200, as previously described [13]), prior to HIV infection, inhibited viral infection, since neither viral replication (Fig. 3) nor viral penetration as assessed by PCR (data not shown) was detectable in the two types of clones with the two strains of HIV-1. These results demonstrate the requirement of HIV-1 fixation to the CD4 molecule in all tested clones and argue in favor of the unity of the path of infection in CD4+ lymphocyte subtypes. HIV does not replicate in resting T cells; therefore, we expected that activation of T cells in culture would initiate viral replication. Lack of HIV replication in CD4+ cloned lymphocytes might at least be explained by an intracytoplasmic (or nuclear) regulation. Specific products of T-cell activation might stimulate transcription of proviral DNA (14): transcription factors, such as NF-KB (21), act directly on the HIV long terminal repeat (23). These HIV enhancerbinding proteins, which are constitutive or inducible, are specific for cell type (7, 12). These enhancer-binding proteins could act differently in each of our clones that depend on interleukin 2 for growth. A clear understanding of the lack of viral replication necessitates, first, an exhaustive study of unreplicating
VOL. 66, 1992
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clones (metabolism, secretions, membrane antigens, and functions) and, second, an investigation of the molecular mechanisms of viral expression in our clones; such an investigation is now in progress. We thank Magali Fasseu and Jean Franqois Bourge for technical assistance. This work has been partly supported by IBM and Caisse Nationale de l'Assurance Maladie des Travailleurs Salaries (no. 872392E).
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