Abstract

Although it is widely believed that viral clearance is mediated principally by the destruction of infected cells by cytotoxic T cells, noncytolytic antiviral activity of CD8+ T cells may play a role in preventing the progression to disease in infections with immunodeficiency viruses and hepatitis B virus. We demonstrate here that (1) replication of human T-lymphotropic virus type I (HTLV-I) is more readily detected from CD8+ T-cell–depleted (CD8) peripheral blood mononuclear cells (PBMCs) of healthy HTLV-I carriers than from unfractionated PBMCs, (2) cocultures of CD8 PBMCs with autologous or allogeneic CD8+ T cells suppressed HTLV-I replication, and (3) CD8+ T-cell anti-HTLV-I activity is not abrogated intrans-well cultures in which CD8+ cells are separated from CD8 PBMCs by a permeable membrane filter. These results suggest that class I-unrestricted noncytolytic anti–HTLV-I activity is mediated, at least in part by a soluble factor(s), and may play a role in the pathogenesis of HTLV-I infection.

Introduction

Human T-lymphotropic virus type I (HTLV-I) causes 2 distinct diseases, a T-cell malignancy designated adult T-cell leukemia/lymphoma (ATLL) and a chronic inflammatory nervous system disease designated HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP).1 However, the latency between viral transmission and disease progression is often very long, typically several years to decades; furthermore, the majority (95% or more) of infected individuals are healthy carriers.1 What determines the outcome of HTLV-I infection has not been fully understood; however, CD8+ T-cell response to HTLV-I infection probably plays a role in disease progression. Although it has been widely considered that CD8+ T cells mediate antiviral activity principally by cytolytic mechanisms, noncytolytic antiviral response of CD8+ T cells has been demonstrated in controlling infection with human immunodeficiency virus (HIV),2 simian immunodeficiency virus (SIV),2feline immunodeficiency virus (FIV),3 or hepatitis B virus.4 In this study, we investigated whether MHC class I-unrestricted noncytolytic mechanism is also used by CD8+T cells to control HTLV-I infection.

Study design

Cells

Peripheral blood mononuclear cells (PBMCs) were isolated from HTLV-I seropositive or seronegative healthy individuals (Nagasaki Red Cross Blood Center, Nagasaki, Japan). CD8+ T cells were positively selected, as described previously.5CD8+ T-cell-depleted PBMCs (CD8 PBMCs) contained less than 1.5% CD8+ cells, as determined by flow cytometric analysis (data not shown). In some experiments CD8 PBMCs and autologous or allogeneic CD8+ T cells were cultured together or separated by a semipermeable membrane filter with 0.4-μmol/L pores (Costar, Acton, MA).

Human T-lymphotropic virus type I infection

Replication of HTLV-I was determined by HTLV-I p19 Ag levels in cell-free supernatants from HTLV-I carriers' PBMCs, using a commercially available enzyme-linked immunosorbent assays (ELISA; Cellular Product, Inc, Buffalo, NY).

Results and discussion

Dramatic increase in HTLV-I p19 expression by CD8+ depletion

It has been shown that HIV-1 is more readily isolated from PBMCs of infected individuals when CD8+ T cells are depleted.6 CD8+ T-cell–mediated antiviral activity has also been demonstrated in controlling other lentiviruses such as SIV and FIV.2,3 Furthermore, CD8+depletion in vivo dramatically increased plasma viral loads in SIV-infected macaques.7 These studies suggest that CD8+ T cells play a critical role in the pathogenesis of retroviral infections.

To explore the possibility that CD8+ T cells play a role in preventing replication of HTLV-I, another human retrovirus, CD8+ T cells were depleted from PBMCs of healthy HTLV-I carriers. Depletion of CD8+ T cells from PBMCs resulted in a significant increase in HTLV-I p19 Ag expression, and reconstitution of autologous CD8+ T cells suppressed viral expression in a dose-dependent manner (Table 1).

Table 1.

HTLV-I replication in unfractionated PBMCs, CD8 PBMCs, and CD8 PBMCs reconstituted with autologous or allogeneic CD8+ T cells

Carrier p19 Ag level in culture supernatant (pg/mL)  
Unfract PBMCs CD8PBMCs CD8 PBMCs plus* 
auto CD8+ cells allo CD8+ cells 
(9:1) (4:1) (9:1) (4:1) 
47 212 115  (46%) 25  (88%) 180  (15%) 77  (64%) 
36 465 190  (59%) 28  (94%) 358  (23%) 228  (51%)  
52 1120 512  (54%) 60  (95%) 2540  (—) 602  (46%)  
<25 240 152  (37%) 35  (85%) 140  (42%) 70  (71%) 
207 526 529  (—) 272  (48%) 393  (25%) 312  (41%)  
91 493 480  (3%) 111  (78%) 530  (—) 242  (51%) 
Carrier p19 Ag level in culture supernatant (pg/mL)  
Unfract PBMCs CD8PBMCs CD8 PBMCs plus* 
auto CD8+ cells allo CD8+ cells 
(9:1) (4:1) (9:1) (4:1) 
47 212 115  (46%) 25  (88%) 180  (15%) 77  (64%) 
36 465 190  (59%) 28  (94%) 358  (23%) 228  (51%)  
52 1120 512  (54%) 60  (95%) 2540  (—) 602  (46%)  
<25 240 152  (37%) 35  (85%) 140  (42%) 70  (71%) 
207 526 529  (—) 272  (48%) 393  (25%) 312  (41%)  
91 493 480  (3%) 111  (78%) 530  (—) 242  (51%) 

HTLV-I = human T-cell lymphotropic virus type I; PBMCs = peripheral blood mononuclear cells; unfract = unfractionated. PBMCs were obtained from 3 pairs of HTLV-I carriers (1–6). Three million unfractionated PBMCs or CD8 PBMCs were propagated in RPMI 1640 medium supplemented with 10% fetal bovine serum. Cell-free culture supernatants were collected on day 7 of culture, and p19 levels were determined by ELISA.

*

Where indicated, CD8 PBMCs were reconstituted with autologous or allogeneic CD8+ T cells at the indicated ratio.

Allogeneic cultures were performed between carriers 1 and 2, carriers 3 and 4, and carriers 5 and 6.

Percentage suppression is expressed as: 100-(p19 Ag level of CD8 PBMC reconstituted with CD8+ T cells/p19 Ag level of CD8 PBMC alone).

Class I-unrestricted noncytolytic response of CD8+T cells

Next, we investigated whether CD8+ T-cell–mediated anti–HTLV-I activity resulted principally from HTLV-I-specific class I-restricted cytotoxic T-lymphocyte response (CTL). To do so, we first performed coculture experiments in which CD8 PBMCs of HTLV-I carriers were reconstituted with autologous or allogeneic CD8+ T cells, and p19 Ag levels in cell-free supernatants were compared. As shown in Table 1, autologous CD8+ T cells markedly, and allogeneic CD8+ T cells modestly suppressed HTLV-I replication. Allogeneic CD8+ T cells derived from HTLV-I seronegative healthy individuals had little activity against HTLV-I (data not shown); however, it was not possible in this study to determine whether Ag-specific stimulation is essential for CD8+ T-cell anti–HTLV-I activity. These results suggest that, although CTL response plays a critical role in controlling HTLV-I replication, there exists another antiviral activity that does not require MHC class I compatibility.

Because it is possible that anti–HTLV-I activity of allogeneic CD8+ T cells was attributed to CTL response mediated by partially matched class I molecules, CD8+ T cells were separated from CD8 PBMCs by a semipermeable membrane filter (trans-well cultures). Although less efficient than CD8+ T cells in cocultures, these cells could suppress HTLV-I replication even when direct cell-to-cell contact, which is essential for class I-restricted CTL response, was prohibited (Table2). Furthermore, the percentage viabilities of the control cells and the cells in trans-well cultures were similar (88% ± 8% and 90% ± 9%, respectively), and HTLV-I proviral DNA was detected in trans-well cultures at levels comparable to CD8 PBMC cultures that yielded substantial amounts of p19 Ag (data not shown). These results suggest that class I-unrestricted noncytolytic response of CD8+ T cells may play a role in controlling HTLV-I infection.

Table 2.

Anti-HTLV-I response of CD8+ T cells that does not require direct cell-to-cell contact

Carrier p19 Ag level in culture supernatant (pg/mL)  
CD8PBMCs CD8 PBMCs plus:  
auto CD8+ (trans-well) auto CD8+ (coculture) 
1280 100  (92%) 71  (95%)  
4080 2200  (46%) 360  (91%) 
4930 2020  (59%) 1110  (79%) 
Carrier p19 Ag level in culture supernatant (pg/mL)  
CD8PBMCs CD8 PBMCs plus:  
auto CD8+ (trans-well) auto CD8+ (coculture) 
1280 100  (92%) 71  (95%)  
4080 2200  (46%) 360  (91%) 
4930 2020  (59%) 1110  (79%) 

See Table 1 for abbreviations.

Two million CD8 PBMCs derived from healthy HTLV-I carriers were cultured alone, reconstituted with a half million autologous CD8+ T cells (coculture), or separated from one million of autologous CD8+ T cells by a semipermeable membrane filter (trans-well).

A previous study demonstrated the inverse relationship between numbers of peripheral CD8+ T cells and levels of gamma interferon production and HTLV-I expression.8 Furthermore, it has also been reported that persons with asymptomatic HTLV-I infection have heightened immune reactivity, whereas those with ATLL do not,9 suggesting that there may be immune regulation of HTLV-I infection. Recently, HTLV-I–specific CTLs have been found in healthy carriers or patients with HAM/TSP,10 and it has been described that HTLV-I is not latent and is actively replicating in infected individuals, and that the chief determinants of the equilibrium viral load of HTLV-I are virus-specific CTL response.11 In this study, we identified anti–HTLV-I activity other than CTL response. The significance of the non-CTL response in vivo warrants further investigation.

Supported in part by grants from The Japan Leukemia Research Foundation and The Mother and Child Health Foundation.

The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 U.S.C. section 1734.

References

References
1
Uchiyama
T
Human T cell leukemia virus type I (HTLV-I) and human diseases.
Annu Rev Immunol.
15
1997
15
37
2
Levy
JA
Mackewicz
CE
Barker
E
Controlling HIV pathogenesis: the role of the noncytotoxic anti-HIV response of CD8+ T cells.
Immunol Today.
17
1996
217
224
3
Jeng
CR
English
RV
Childers
T
Tompkins
MB
Tompkins
WAF
Evidence for CD8+ antiviral activity in cats infected with feline immunodeficiency virus.
J Virol.
70
1996
2474
2480
4
Guidotti
LG
Ando
K
Hobbs
MV
et al
Cytotoxic T lymphocytes inhibit hepatitis B virus gene expression by a noncytolytic mechanism in transgenic mice.
Proc Natl Acad Sci U S A.
91
1994
3764
3768
5
Moriuchi
H
Moriuchi
M
Combadiere
C
Murphy
PM
Fauci
AS
CD8+ T-cell-derived factor(s), but not β-chemokines RANTES, MIP-1α, and MIP-1β, suppress HIV-1 replication in monocytes/macrophages.
Proc Natl Acad Sci U S A.
93
1996
15431
15435
6
Walker
CM
Moody
JM
Stites
DP
Levy
JA
CD8+ lymphocytes can control HIV infection in vitro by suppressing virus replication.
Science.
234
1986
1563
1566
7
Jin
X
Bauer
DE
Tuttleton
SE
et al
Dramatic rise in plasma viremia after CD8+ T cell depletion in simian immunodeficiency virus-infected Macaques.
J Exp Med.
189
1999
991
998
8
Moore
JL
Poiesz
BJ
Zamkoff
KW
et al
Inverse relationship between constitutive gamma interferon production and human T-cell lymphoma/leukemia virus expression in cultured T lymphocytes.
J Virol.
53
1985
440
446
9
Tendler
CL
Greenberg
SJ
Burton
JD
et al
Cytokine induction in HTLV-I associated myelopathy and adult T-cell leukemia: alternate molecular mechanisms underlying retroviral pathogenesis.
J Cell Biochem.
46
1991
302
311
10
Bangham
CRM
Kermode
AG
Hall
SE
Daenke
S
The cytotoxic T-lymphocyte response to HTLV-I: the main determinant of disease?
Semin Virol.
7
1996
41
48
11
Wodarz
D
Nowak
MA
Bangham
CRM
The dynamics of HTLV-I and the CTL response.
Immunol Today.
20
1999
220
227

Author notes

Hiroyuki Moriuchi, Department of Pediatrics, Nagasaki University School of Medicine, 1-7-1 Sakamoto, Nagasaki 852-8501, Japan; e-mail: hiromori@net.nagasaki-u.ac.jp.