PLoS Negl Trop DisplosplosntdsPLoS Neglected Tropical Diseases1935-2735Public Library of ScienceSan Francisco, USAPNTD-D-10-0009210.1371/journal.pntd.0001030Research ArticleMedicineClinical immunologyImmune cellsT cellsImmunityImmunity to infectionsImmune responseInfectious diseasesViral diseasesImmunologyInfectious DiseasesHTLV-1 Tax Specific CD8+ T Cells Express Low Levels of Tim-3 in
HTLV-1 Infection: Implications for Progression to Neurological
ComplicationsT Cell Exhaustion Markers in HTLV-1
InfectionNdhlovuLishomwa C.1*LealFabio E.23HasenkrugAaron M.1JhaAashish R.1CarvalhoKarina I.2Eccles-JamesIjeoma G.1BrunoFernanda R.2VieiraRaphaella G. S.1YorkVanessa A.1ChewGlen M.1JonesR. Brad4TanakaYuetsu5NetoWalter K.6SanabaniSabri S.6OstrowskiMario A.4SeguradoAluisio C.3NixonDouglas F.1¶KallasEsper G.23¶Division of Experimental Medicine, Department
of Medicine, University of California San Francisco, San Francisco, California,
United States of AmericaDivision of Clinical Immunology and Allergy,
Department of Infectious Diseases, School of Medicine, University of São
Paulo, São Paulo, BrazilDepartment of Infectious Diseases, School of
Medicine, University of São Paulo, São Paulo, BrazilDepartment of Immunology, University of
Toronto, Toronto, Ontario, CanadaDepartment of Immunology, University of the
Ryukyus, Okinawa, JapanMolecular Biology Laboratory,
Fundação Pró-Sangue, Hemocentro de São Paulo,
São Paulo, BrazilSinghSunit KumarEditorCentre for Cellular and Molecular Biology (CCMB), India* E-mail: lndhlovu@medsfgh.ucsf.edu;lndhlovu@hawaii.edu
Conceived and designed the experiments: LCN FEL DFN EGK. Performed the
experiments: LCN FEL AMH ARJ GMC IGE-J FRB VAY WKN SSS RGSV. Analyzed the
data: LCN FEL AMH ARJ KIC IGE-J VAY. Contributed reagents/materials/analysis
tools: LCN DFN ACS EGK SSS WKN YT. Wrote the paper: LCN FEL. Technical and
scientific input: RBJ MAO. Edited the manuscript: DFN EGK.
¶ These authors also contributed equally to this work.
L.C. Ndhlovu, R. Brad Jones, M.A. Ostrowski and D.F. Nixon are named as inventors
on a patent application related to modulation of Tim-3 in viral infections.
42011264201154e10301910201027220112011Ndhlovu et alThis is an open-access article distributed under the
terms of the Creative Commons Attribution License, which permits unrestricted use,
distribution, and reproduction in any medium, provided the original author and
source are credited.
The T cell immunoglobulin mucin 3 (Tim-3) receptor is highly expressed on
HIV-1-specific T cells, rendering them partially “exhausted” and
unable to contribute to the effective immune mediated control of viral
replication. To elucidate novel mechanisms contributing to the HTLV-1
neurological complex and its classic neurological presentation called HAM/TSP
(HTLV-1 associated myelopathy/tropical spastic paraparesis), we investigated the
expression of the Tim-3 receptor on CD8+ T cells from a cohort
of HTLV-1 seropositive asymptomatic and symptomatic patients. Patients diagnosed
with HAM/TSP down-regulated Tim-3 expression on both CD8+ and
CD4+ T cells compared to asymptomatic patients and HTLV-1
seronegative controls. HTLV-1 Tax-specific, HLA-A*02 restricted
CD8+ T cells among HAM/TSP individuals expressed markedly
lower levels of Tim-3. We observed Tax expressing cells in both
Tim-3+ and Tim-3− fractions. Taken
together, these data indicate that there is a systematic downregulation of Tim-3
levels on T cells in HTLV-1 infection, sustaining a profoundly highly active
population of potentially pathogenic T cells that may allow for the development
of HTLV-1 complications.
Author Summary
The retrovirus, Human T lymphotropic virus type 1 (HTLV-1) infects 10–20
million people worldwide. The majority of infected individuals are asymptomatic;
however, approximately 3% develop the debilitating neurological disease,
HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). There is
also currently no cure, vaccine or effective therapy for HTLV-1 infection. The
precise role of CD8+ killer T cells in the control or contribution of
HTLV-1 disease progression remains unclear. The T-cell immunoglobulin mucin
domain-containing (Tim) proteins are type 1 transmembrane proteins. Three human
Tim proteins (Tim-1, -3, and -4) exist and display markedly diverse expression
patterns and functions. Tim-3 is upregulated on CD8+ T cells
during chronic viral infections leading to a population of poorly functioning T
cells. We investigated the expression of Tim-3 on T cells from patients with
asymptomatic and symptomatic HTLV-1 infection and compared this with HTLV-1
uninfected donors. Patients diagnosed with HAM/TSP down-regulated Tim-3
expression on T cells when compared to asymptomatic patients and uninfected
controls. Our study provides evidence of a novel mechanism for the persistent
inflammation observed in HTLV-1 infected patients with neurological deficits and
significantly advances our understanding of how the Tim-3 pathway functions.
Support for this work was provided by funds from the National Institutes of
Health (NIH), University of California, San Francisco-Gladstone Institute of
Virology & Immunology Center for AIDS Research (P30 AI027763). Additional
support was provided by the Brazilian Program for STD and AIDS, Ministry of
Health (914/BRA/3014 - UNESCO/Kallas), the São Paulo City Health
Department (2004-0.168.922-7/Kallas), Fundação de Amparo a
Pesquisa do Estado de São Paulo (04/15856-9/Kallas and
2010/05845-0/Kallas and Nixon), the John E. Fogarty International Center (D43
TW00003). The project described was also supported by Award Number R56AI083112
from the National Institute of Allergy and Infectious Diseases (LCN). Additional
support was provided by the Montana State Complex Biological Systems Research
Scholars Program, funded by the Howard Hughes Medical Institute to AH. The
funders had no role in study design, data collection and analysis, decision to
publish, or preparation of the manuscript.Introduction
The vast majority of HTLV-1-infected individuals with low and stable HTLV-1 proviral
load levels are clinically asymptomatic for life [1]. However, 1–3% of
subjects develop progressive neurological complications related to HTLV-1 infection,
classically denominated as HTLV-1 associated myelopathy/tropical spastic paraparesis
(HAM/TSP) [2],
[3], [4]. The infection
can also lead to a debilitating malignancy, known as HTLV-1 associated adult T cell
leukemia (ATL) in approximately 2–5% of infected individuals [4], [5], [6], [7].
The immune response, and in particular the cellular immune response, plays an
important role in the control of HTLV-1 infection [8], [9], [10], [11], [12]. In vitro
studies further demonstrate that CD8+ T cell responses are able to
directly lyse HTLV-1-infected CD4+ T cells [9], [11], [13]. In patients with HAM/TSP,
CD8+ T cells are capable of producing multi-cytokine responses
and are able to release cytotoxic molecules [14], [15]. Recent studies have selected
out patients with HLA-A*02 and HLA-Cw08 genes as being associated with lower
HTLV-1 proviral load and a reduced risk of progression to HAM/TSP [16], [17].
While these data support an important protective role for the CD8+ T
cell immune response with the potential for viral control, other studies suggest
that HTLV-1-specific CD8+ T cells may paradoxically contribute to
the neuromuscular immunopathology through autoimmune mechanisms, leading to the
clinical manifestation of HAM/TSP [18]. Furthermore, patients with HAM/TSP also present with
high numbers of HTLV-1 Tax-specific CD8+ T cells in the cerebrospinal fluid
[15], [19], [20], [21], [22] that are
thought to play a immunopathogenic role, either by release of neurotoxic cytokines,
such as TNF-α and IFN-γ [23], [24], or by direct cytotoxicity. It is evident from these
studies that the precise role of CD8+ T cells in the control or
pathogenesis of HTLV-1 disease progression remain unclear. Further knowledge of the
mechanisms leading to T cell induced immunopathology in HTLV-1 infection will be
important in determining successful immune-based therapies and provide insights for
effective vaccine designs.
During chronic viral infections, virus-specific CD8+ T cells undergo
an altered pattern of differentiation and can become exhausted [25], [26]. CD8+ T cell
exhaustion is a transcriptionally altered state of T cell differentiation distinct
from functional effector or memory CD8+ T cells [27].
CD8+ T cell exhaustion leads to profound T cell dysfunction and
the inability of the T cells to control retroviral replication [28], [29], [30]. Conversely, downregulation
of exhaustion markers could lead to a highly functional population of T cells. T
cell immunoglobulin and mucin domain-containing protein 3 (Tim-3), is upregulated on
CD8+ T cells during chronic viral infections [29], [30], [31], [32], [33], [34], [35], [36].
Programmed death receptor-1 (PD-1) is also known as another immune exhaustion
biomarker expressed in chronic viral infections [28], [37], [38], [39], [40], [41], [42], [43]. High levels of PD-1 and Tim-3 on
virus-specific T cells have been shown to lead to poor proliferative capacity and,
in some cases, ineffective Th1 cytokine production [29], [39], [44]. A sustained downregulation
of these receptors would lead to an exacerbated constitutively active T cell
population. The phenotypic profile of immune exhaustion markers on T cells is
unknown in seropositive HTLV-1 individuals. In this study, we show for the first
time that HTLV-1 associated complications may be related to the highly responsive
inflammatory Tax-specific T cells in HTLV-1-infected individuals. These results
support the idea that HTLV-1 infection induces mechanisms resulting in a limited T
cell exhaustion profile, leading potentially to neuro-immunopathology and disease
complications.
Materials and MethodsEthics Statement
The research involving human participants reported in this study was approved by
the institutional review board of the University of Sao Paulo (IRB #0855/08) Sao
Paulo, Brazil. Informed consent was obtained for all subjects. All clinical
investigation were conducted according to the principles expressed in the
Declaration of Helsinki (http://www.wma.net/en/30publications/10policies/b3/index.html).
Humans Subjects
Patients were serially recruited in the HTLV-1 Outpatient Clinic at the
University of Sao Paulo, Brazil in two stages with written informed consent
approved by the University of Sao Paulo's Institutional Review Board
(#0855/08). The diagnosis of HAM/TSP based on criteria outlined by the WHO [45] (Table 1). The majority of
the patients were female (63%) with a median age of 48 (IQR: 22–66)
years. We enrolled age and sex matched healthy uninfected volunteers without
clinical and laboratory evidence of HTLV-1-associated disease, from the same
demographics as the infected subjects. All HTLV -1 seropositive subjects tested
negative for Hepatitis B, Hepatitis C, and HIV infections. No other inflammatory
diseases or disorders were present in any of the participants. Blood samples
were processed with Ficoll-Paque PLUS (Amersham Pharmacia Biotech, Uppsala,
Sweden) gradient centrifugation, and peripheral-blood mononuclear cells (PBMC)
were isolated and cyropreserved in fetal bovine serum (FBS) containing
10% DMSO in liquid nitrogen.
10.1371/journal.pntd.0001030.t001
Patients description.
ID Number
Gender
Age
Clinical Presentation
PBMC
HLA-A*02
(years)
(cps/1000)
Status
237
M
39
asymptomatic
20
pos
410
F
43
asymptomatic
14
pos
411
F
47
asymptomatic
84
pos
405
F
22
asymptomatic
15
pos
403
F
53
asymptomatic
604
pos
240
F
N/A
asymptomatic
0
pos
425
M
29
asymptomatic
43
416
M
48
asymptomatic
140
pos
221
M
N/A
asymptomatic
9
pos
424
M
46
asymptomatic
106
418
M
66
asymptomatic
<1
419
F
33
asymptomatic
72
421
M
54
asymptomatic
23
423
F
42
asymptomatic
72
218
F
46
HAM/TSP
2
pos
402
F
50
HAM/TSP
152
pos
224
F
57
HAM/TSP
1923
pos
412
F
53
HAM/TSP
117
pos
312
F
N/A
HAM/TSP
161
pos
413
F
61
HAM/TSP
1510
pos
420
M
64
HAM/TSP
12
422
F
64
HAM/TSP
ND
HD1
N/A
N/A
Healthy
HD2
F
46
Healthy
HD3
F
39
Healthy
HD4
F
29
Healthy
HD5
F
60
Healthy
HD6
M
37
Healthy
HD7
F
45
Healthy
ND = not detected, N/A
= not available.
Pentamers, Peptides and Cytokines
Conjugated Pentamers were obtained commercially from Proimmune (Oxford, UK). The
HLA-A*02 restricted HTLV-1 Tax (LLFGYPVYV) and CMV (NLVPMVATV) peptides were
obtained from New England Peptide (Gardner, MA). In some experiments rIL-2
[80 IU/ml] (Roche Diagnostics, Mannheim, Germany) and rIL-15 [50
ng/ml] (R&D Systems, Minneapolis, MN) were used during in
vitro culture studies.
Flow Cytometry Assessment
Cryopreserved PBMC were rapidly thawed in warm RPMI 1640 with 10% FBS,
washed in FACS buffer (PBS, with 0.5% bovine serum albumin, 2 mM EDTA
(Sigma-Aldrich, St. Louis, MO)). For staining, 5×105 cells were
incubated with conjugated antibodies against Tim-3 (R&D Systems,
Minneapolis, MN), PD-1 (Biolegend, San Diego, CA), CD4, CD8, CD3 (all from BD
Biosciences, San Jose, CA) for 30 min on ice. In some experiments, PMBC were
then fixed and permeabilized prior to staining with conjugated anti-Tax (clone
Lt-4) antibodies [46] or a control labeled IgG. Fluorescence minus one
(FMO) samples were prepared for each fluorochrome to facilitate gating as well
as conjugated isotype control antibodies. Anti-mouse IgG-coated beads were
stained with each fluorochrome separately and used for software-based
compensation. Analysis was performed using a FACSCanto instrument (BD
Biosciences) and at least 100,000 events were collected and analyzed with FlowJo
software (TreeStar, Ashland, OR).
To define pentamer positive cells: staining was initially performed immediately
after thawing with biotin-labeled HLA-A2 Tax or CMV epitope specific pentamer
fluorotags followed a secondary staining step with fluorophore conjugated
antibodies against CD8 (BD), Tim-3 (R&D Systems), PD-1 (Biolegend) and CD3
(BD), and with labeled streptavidin. Cells were washed twice with PBS containing
1% FBS, then fixed in 2% paraformaldehyde and run on a customized
BD FACSCanto within 12 hours.
Viral Load Assessment
HTLV-1 proviral DNA was extracted from PBMC using a commercial kit (Qiagen GmbH,
Hilden Germany) and according to the manufacturer's instructions. The
extracted DNA was used as a template to amplify a fragment of 158 bp from the
viral tax region using previously published primers[47]. The SYBR green
real-time PCR assay was carried out in 25 µl PCR mixture containing
10× Tris (pH 8.3; Invitrogen, Brazil), 1.5 mM MgCl2, 0.2
µM of each primer, 0.2 mM of each dNTPs, SYBR Green (18.75
Units/r×n; Cambrex Bio Science, Rockland, ME) and 1 unit of platinum Taq
polymerase (Invitrogen, Brazil). The amplification was performed in the Bio-Rad
iCycler iQ system using an initial denaturation step at 95°C for 2 minutes,
followed by 50 cycles of 95°C for 30 seconds, 57°C for 30 seconds and
72°C for 30 seconds. The human housekeeping β globin gene primers GH20
and PC04[48]
were used as an internal control calibrator. For each run, standard curves for
the value of HTLV-1 tax were generated from MT-2 cells of log10
dilutions (from 105 to 100 copies). The threshold cycle
for each clinical sample was calculated by defining the point at which the
fluorescence exceeded a threshold limit. Each sample was assayed in duplicate
and the mean of the two values was considered as the copy number of the sample.
The amount of HTLV-1 proviral load was calculated as follows: copy number of
HTLV-1 (tax) per 1,000 cells = (copy number of HTLV-1
tax)/(copy number of β globin/2) ×1,000 cells. The method could detect
1 copy per 103 PBMC.
Elispot Assays
MAIPS4510 Elispot plates (Millipore, Danvers, MA) were coated with anti-IFN-γ
(10 µg/ml) (Mabtech, Nacka Strand, Sweden) in PBS, 50 µl/well,
either overnight at 4°C or for one hour at room temperature. After three
washes with PBS, PBMC (1×105 cells/well) and the appropriate
antigens were added (Tax peptide and CMV peptide), with a final volume of 200
µl/well. Plates were incubated at 37°C in 5% CO2 for
16–20 hours. After washing with phosphate-buffered saline (PBS) plus
0.1% Tween 20 (PBST), biotinylated anti-IFN-γ 1 µg/ml)
(Mabtech), antibodies were added to the appropriate wells in PBS 0.1%
tween 1% BSA (PBSTB) for 30 minutes at room temperature. Plates were
washed again three times with PBST, and alkaline phosphatase-conjugated
streptavidin (Jackson Immunoresearch, West Grove, PA) was added (50 µl of
1∶1,000 dilution in PBSTB) and incubated for 30 min at room temperature.
Plates were washed in PBSTB, soaked for 1 hour in PBSTB and incubated with blue
substrate (Vector Labs, Burlingame, CA) until spots were clearly visible, then
rinsed with tap water. When plates were dry, spots were counted using an
automated ELISPOT reader.
Statistical Analysis
Statistical analysis was performed by using GraphPad Prism statistical software
(GraphPad Software, San Diego, CA). Non-parametric statistical tests were used.
The Mann-Whitney U was used for comparison tests and the Spearman rank test were
used for correlation analyses.
ResultsSubjects
Peripheral venous blood was drawn from 22 HTLV-1 seropositive patients and 7
HTLV-1 seronegative matched donors, all screened for the presence of
HLA-A*02 alleles, and peripheral blood mononuclear cells (PBMC) were
extracted and cryopreserved.
Tim-3 and PD-1 Expression on CD8+ and CD4+ T Cells in Patients with
HTLV-1 Infection
Tim-3 and PD-1 are two cellular molecules expressed on T cells implicated in
immune exhaustion. We evaluated the expression and co-expression of Tim-3 and
PD-1 on T cells derived from HTLV-1 seropositive (both asymptomatic carriers and
patients with the diagnosis of HAM/TSP) and seronegative controls to determine
whether they were modulated in HTLV-1 infection. We observed a significant
decrease in the frequency of Tim-3+ PD-1−
expressing CD8+ and CD4+ T cells among HTLV-1
seropositive subjects (CD8+: median 8.01%, IQR
5.42–10.50; CD4+: median 4.3%, IQR 3.50–5.99)
compared to HTLV-1 seronegative controls (CD8+ median
15.10%, IQR 10.50–17.60; CD4+: median 6.84%,
IQR 5.74–7.85) (Figure 1A and
B). Patients with HAM/TSP (red circles) had significantly lower
levels of Tim-3+ PD-1− expressing
CD8+ (p = 0.002) and
CD4+ (p = 0.004) T cells compared to
healthy uninfected controls (open circles). In contrast, the frequency of
Tim-3− PD1+ T cells trended to an increase
in subjects with HTLV-1 infection (CD8+: median 18.80%,
IQR 10.42–24.90; CD4+: median 20.70%, IQR
13.6–25.35) compared to healthy uninfected controls (CD8+:
median 9.22%, IQR 8.97–15.50; CD4+: median
13.60%, IQR 12.7–18.6) (Figure 1A and B). Only a few T cells
co-expressed both Tim-3 and PD-1, and no differences were observed between
uninfected subjects and those with HTLV-1 asymptomatic infection or HAM/TSP
patients. Using linear regression analysis we observed no association between
the frequency of Tim-3 or PD-1 expression on CD8+ T cells in
HTLV-1 infected subjects and proviral load. (p = 0.68;
r = 0.1043; or p = 0.89;
r = −0.03202, respectively).
10.1371/journal.pntd.0001030.g001
Tim-3 expression on T cells in HTLV-1 infection.
Graphs show the frequencies of co-expression of Tim-3 and PD-1 on (A)
CD8+ (left), and (B) CD4+ (right), T cells as assessed by
multiparametric flow cytometry from PBMCs derived 18 HTLV-1 seropositive
(12 asymptomatic and 6 with diagnosis of HAM/TSP) infected subjects and
7 HTLV-1 seronegative healthy uninfected donors from our initial
recruitment. Statistically significant differences are reported as
p<0.05.
Distribution of Tim-3 Expression on HTLV-1-Specific T Cells
HLA- A*02 positive HTLV-1-infected patients have high amounts of circulating
CD8+ T cells specific for an immunodominant HLA- A*02
-restricted epitope, HTLV-1 Tax 11–19 [20], [49], [50]. In HAM/TSP patients, these
HTLV-1's Tax-specific CD8+ T cells correlate with HTLV-1
proviral load [23]. Among this cohort, we identified 15 HLA-A2 positive
subjects (asymptomatic carriers, n = 9 and HAM/TSP,
n = 6; Table
1), and evaluated the Tim-3 and PD-1 receptor expression on
Tax-specific CD8+ T cells. Eight patients had Tax-specific
CD8+ T cells (median 2.45%, IQR 1.11–5.31) as
determined by specific pentamers. Among these patients we also observed
HLA-A*02 -restricted CMVpp65 CD8+ T cells (median 2.49%, IQR
1.87–11.37). Interestingly, Tim-3 levels were dramatically reduced on
CD8+ Tax 11–19-specific T cells (median 24.77%,
IQR 15.2–39.54) compared to the expression of PD-1 (median 48.06%,
IQR 36.81–65) (Figure 2A,C
and E). We also evaluated Tim-3 expression on HLA- A*02 CMV
specific T cells and found a similar pattern of expression with Tim-3 levels
reduced on CD8+ CMV-specific T cells (median 27.62%, IQR
21.48–43.19) compared to PD-1 (median 47.70%, IQR
40.45–51.16) (Figure 2B,D and
F).
10.1371/journal.pntd.0001030.g002
Tim-3 expression on HTLV-1-specific CD8+ T cells in HTLV-1
infection.
PBMC from HLA-A*02+ chronically HTLV-1 infected individuals were
stained with matched HLA pentamers presenting CMV and HTLV-1 epitopes,
and with an anti-Tim-3 antibody. Shown are representative flow cytometry
data from one HTLV-1-infected person using HLA-A*02 pentamers
presenting the (A) HTLV-I-Tax 11–19 epitope and, (B) CMV-pp65
epitope ‘NLVPMVATV’. (C, D) Plots show co-expression of
Tim-3 (upper panel) and PD-1 (lower panel) with the respective
HLA-A*02 pentamers (Tax (left) and CMVpp65 (right)) from the gated
CD8+ T population depicted in Fig 2 A, B. The percentages of cells
in the upper left and right quadrants of the flow plots demonstrated in
Figure 2C, D
reflect only the percentage of pentamer expressing cells. The compiled
expression data of the frequency of Tax (E) and CMVpp65 (F) pentamer
cells on either Tim-3+ or Tim-3- and PD-1+ or PD-1- CD8+
T cells from 8 subjects are shown in Figure 2 E and F. Statistical
analyses comparing pooled responses were performed using the
Mann-Whitney test.
Relationship between the Functionality of Tax 11-19-Specific
CD8+ T Cells and Tim-3 Levels
To determine whether there was an association with Tim-3 or PD-1 levels on Tax
11–19-specific CD8+ T cells and their functionality, we
evaluated the production of IFN-γ in response to the HLA-A*02-restricted
Tax 11–19 immuno-dominant epitope and in comparison, the CMVpp65 epitope
by an ELISPOT assay derived from PBMCs derived from 8 HLA- A*02 restricted
infected individuals with Tax 11–19- and CMVpp65 specific
CD8+ T cells (Figure 3). We saw no correlation between IFN-γ secretion and
global PD-1 or Tim-3 expression on either the CD4+ or
CD8+ T cells, irrespective of disease status (data not
shown). The frequency of PD-1 expression on Tax-specific or CMV-specific
CD8+ T cells also did not associate with the amount of
IFN-γ secreted (r = 0.1317;
P = 0.7520 and r = 0.2245;
P = 0.594, respectively) (Figure 3B). However, we observed a
statistically significant inverse correlation between the frequency of Tim-3 on
both Tax-specific as well as CMV-specific CD8+ T cells and the
amount of IFN-γ secreted (r = −0.8982;
P = 0.0046; r = 0.9710;
P = 0.0028; Figure 3A).
10.1371/journal.pntd.0001030.g003
Association of Tax specific CD8+ T cells with effector
responses.
The graphs show the association between the frequency of Tim-3 (A) and
PD-1 (B) expression on HLA-A*02 restricted Tax11-19 or CMV pp65
specific CD8+ T cells with the number of IFN-γ secreting cells
(SFU/106) in response to Tax 11–19 peptide or the
CMV pp65 epitope. The Spearman rank test was used for correlation
analyses.
Co-Expression of Tim-3 and Tax on T Cells in HTLV-1 Infected Cells
Tax expression marks HTLV-1 viral replication in both CD4+ and
CD8+ infected T cells. We aimed to determine whether the
downregulation of Tim-3 we had observed was occurring only among infected cells,
or in bystander cells as well. We therefore co-stained for Tax and Tim-3
expression on T cells from HTLV-1 infected subjects. We also stained for PD-1
expression as a control. The culture of PBMC overnight did not alter Tim-3 or
PD-1 expression levels on the HTLV-1-infected T cells (data not shown). We
observed that Tax was expressed on PBMC from some subjects following 24 hours of
culture and was detected on both Tim-3+ as well as
Tim-3− CD4+ T cells (Figure 4A). Similarly, Tax was present on
both PD-1+ and PD-1− T cells. We further
identified a unique subset of Tax expressing CD4+ T cells that were
Tim-3hi and lacked PD-1 in most of the subjects expressing Tax
(Fig. 4A). No difference
in the pattern of co-expression between HTLV-1 seropositive asymptomatic
patients and those diagnosed with HAM/TSP was observed.
10.1371/journal.pntd.0001030.g004
Tim-3, PD-1 and Tax co-expression on T cells.
(A) Plots demonstrate representative co-staining for Tax, PD-1 and Tim-3
on CD8+ and CD4+ T cells by flow cytometry following 24 hours
incubation for the induction of Tax in two representative HTLV-1
infected patients. An isotype control was used to delineate the
measurements for Tax expression. (B, C) Plots and graph depict the
co-expression of Tim-3 and PD-1 by the indicated cytokines after 12 hr
in vitro culture of 1×106 PBMC from 4 HTLV-1 infected
patients. A representative donor is shown in B.
Elevated Tim-3 Expression by IL-2 and IL-15 Stimulated T Cells from HTLV-1
Infected Subjects
An increase in Tim-3 levels on T cells would potentially lead to a downregulation
of T cell functionality. We therefore tested several gamma-chain associated
cytokine mediators that could potentially modulate Tim-3 expression. We observed
that IL-2, and especially IL-15, led to a prominent increase in the frequency of
Tim-3 levels, specifically on the CD8+ T cell population after only 12
hours in culture (Figure
4B,C). No change in the levels of PD-1 expression were observed on
both CD8+ and CD4+ T cells (Figure 4B,C).
Discussion
CD8+ T cell dysfunction and/or exhaustion are common features of many
chronic viral infections, including HIV-1 and HCV infections [29], [30], [31], [32], [33], [34], [35], [36]. The mechanisms of T cell
dysfunction are complex, but are in part mediated by a distinct set of inhibitory
receptors [27],
[51]. A
high, and sustained, expression of Tim-3 and PD-1, have emerged as hallmarks of T
cell exhaustion in human viral infections, and blockade of these pathways can
reinvigorate immune responses during persisting viral infections [29], [30], [33], [34], [36]. In this
study, we report that CD8+ and CD4+ T cells in
HTLV-1 infection express lower levels of Tim-3, and this was more pronounced in
patients with HAM/TSP. Phenotypically, we observed that Tax HTLV-1-specific,
HLA-A*02 -restricted CD8+ T cells consistently retain a lower
frequency of Tim-3. We propose that this low expression of Tim-3 on HTLV-1
Tax-specific T cells may lead to a persistent and deleterious effector T cell pool
leading to more inflammation.
The pattern of expression of PD-1 in HTLV-1 infection has recently been shown to be
elevated on T cells in HTLV-1 carriers and also on CMV and EBV specific T cells in
asymptomatic carriers compared to healthy controls [52]. This opposing relationship of
PD-1 and Tim-3 expression on T cells in patients with HTLV-1 infection suggests that
the downregulation of Tim-3 expression potentially leads to more vigorous T cell
activity in the HTLV-1-infected individual, whereas PD-1 may not fully reflect T
cell dysfunction, but rather an activated status of the T cell response to
infection. Indeed the association between the frequency of Tim-3 and PD-1 levels
with IFN-γ secretion in response to either Tax or CMVpp65 epitopes show
remarkably different correlations. In a study by Petrovas and colleagues, it was
apparent that PD-1 expressing T cells are able to secrete cytokines in response to
viral peptides [39]. Our data suggests that PD-1 and Tim-3 on antigen
specific CD8+ T cells are functionally different, and this may
reflect a distinct stage of differentiation. PD-1 appears to mark early T-cell
activation and exhaustion, while Tim-3 represents a more terminal stage of
impairment.
The positive association between the frequency of HTLV-1's Tax-specific
CD8+ T cells and HTLV-1's Tax mRNA load and proviral load
is well documented [8], [53], [54]. Studies evaluating the phenotype of CD8+
T cells in HTLV-1 infection have been largely limited to characterizing the
expression of T cell maturation and differentiation markers (CD28, CD45RO) [14]. Our data
suggest that downregulation of Tim-3, rather than PD-1, marks global and
Tax-specific CD8+ T cells, which are hyperfunctional. This contrasts
with HIV-1 and HCV infections, where the expression of Tim-3 is increased, leading
to a population of CD8+ T cells that are rendered dysfunctional both
in terms of proliferative capacity and cytokine release as well as release of
cytolytic granules [29], [36].
Surface receptors known to regulate T cell function like CD244 and PD-1 have been
shown to be upregulated either directly due to Tax or indirectly due to the cytokine
milieu [52], [55]. We
postulate that either direct HTLV-1 viral components led to a downregulation of
Tim-3, or as yet to be defined cytokine(s), suppress Tim-3 expression. In several
human and murine studies, the manifestation of autoimmune diseases such as multiple
sclerosis, have been attributed as a result of downregulated Tim-3 expression on T
cells [56].
It still remains unclear how HTLV-1 infection sustains low levels of Tim-3 on T cells
in infected patients and whether this is a cause or a consequence of disease
progression. Multilayered mechanisms for this regulation may be occurring in the
context of HTLV-1 infection. One strategy to reduce the T cells response would be
through enhancement of the Tim-3 receptor for engagement with its cognate ligand.
This could serve as a novel strategy to dampen the inflammatory inducing T cells.
From our results, PD-1 engagement may not be as effective since both
PD-1− and PD-1+ cells retain the potential for
CD8+ T cell lytic function.
A novel strategy to reverse or prevent the onset of neurological complications would
be through dampening effector T cell functions. From our results, it appears the
γ-chain cytokines elicited higher levels of Tim-3 on specifically on
CD8+ T cells, and such a strategy could be harnessed to dampen T
cell function in the HTLV-1 infected individual. Further work to understand the
mechanisms for HTLV-1 disease progression and devise strategies to effectively
prevent neurological complications will be needed. Targeted modulation of the Tim-3
pathway provides a viable model for this intervention.
The content is solely the responsibility of the authors and does not necessarily
represent the official views of the National Institute of Allergy and Infectious
Diseases or the National Institutes of Health.
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