3 2 9
ARTIGO/ARTICLE
Revista da Sociedade Br asileir a de Medicina Tr opical 3 7 ( 4 ) :3 2 9 -3 3 2 , jul-ago, 2 0 0 4
Establishment of HTLV-I-infected cell lines fr om per ipher al
blood mononuclear cells of Br azilian patients
Estabelecimento de linhagens celulares infectadas por HTLV-I a partir de células
mononucleares periféricas de pacientes brasileiros
Car olina V. Pannuti
1, Mar ia Lúcia S.G. Jor ge
1, Cláudia Biasutti
1,
Esper G. Kallás
2and Aluisio A.C. Segur ado
1ABSTRACT
To i n ve sti ga te e p i d e m i o lo gi c a l a n d p a th o ge n e ti c f e a tu re s o f HTLV- I i n f e c ti o n , a c o h o rt o f c a rri e rs h a s b e e n f o llo we d a t th e USP Te a c h i n g Ho sp i ta l si n c e 1 9 9 1 . Th i s stu d y d e sc ri b e s th e e sta b li sh m e n t o f c e ll li n e s f ro m p e ri p h e ra l b lo o d m o n o n u c le a r c e lls ( PBMC) o f i n f e c te d su b je c ts. Ex vivo PBMC we re c u ltu re d wi th th o se f ro m a se ro n e ga ti ve d o n o r a n d m o rp h o lo gi c e vi d e n c e o f c e ll tra n sf o rm a ti o n wa s o b ta i n e d a f te r 9 0 d a ys wi th d e te c ti o n o f m u lti n u c le a te d c e lls e x h i b i ti n g c e re b ri f o rm n u c le i . In te gra ti o n o f HTLV- I p ro vi ra l DNA a n d e x p re ssi o n o f vi ra l a n ti ge n s wa s d e m o n stra te d i n c u ltu re b y PCR a n d i m m u n o f lu o re sc e n c e . Ce ll li n e s w e re m a i n ta i n e d f o r 2 4 0 d a ys, gra d u a lly w e a n e d f ro m e x o ge n o u s IL- 2 . Im m u n o p h e n o typ i n g o f c e ll li n e s o n f lo w c yto m e try yi e ld e d e vi d e n c e o f c e ll a c ti va ti o n . Esta b li sh m e n t o f HTLV- I- i n f e c te d c e ll lin e s fro m ex vivo PBMC is fe a sib le a n d m a y b e u se fu l fo r stu die s o n lym pho c yte phe n o typic c ha n ge s a n d o n m e c ha n ism s o f HTLV- i n d u c e d c e ll p ro li f e ra ti o n . Mo re o ve r th e y m a y b e u se d wi th d i a gn o sti c p u rp o se s i n i m m u n o f lu o re sc e n c e te sts.
Ke y-words: HTLV- I. Cu ltu re . Ce ll li n e . Bra zi l.
RESUMO
Pa ra i n ve sti ga r a e p i d e m i o lo gi a e p a to gê n e se d a i n f e c ç ã o p o r HTLV- I se gu i m o s c o o rte d e p o rta d o re s d e ssa re tro vi ro se n o HC- FMUSP d e sd e 1 9 9 1 . Este e stu d o d e sc re ve o e sta b e le c i m e n to d e li n h a ge n s c e lu la re s a p a rti r d e c é lu la s m o n o n u c le a re s p e ri f é ri c a s ( CMP) d e i n d i ví d u o s i n f e c ta d o s. As CMP f o ra m c u lti va d a s c o m a s d e d o a d o r so ro n e ga ti vo , ve ri f i c a n d o - se a p ó s 9 0 d i a s e vi d ê n c i a m o rf o ló gi c a d e tra n sf o rm a ç ã o c e lu la r c o m d e te c ç ã o d e c é lu la s m u lti n u c le a d a s c o m n ú c le o s c e re b ri f o rm e s. De m o n stro u - se i n te gra ç ã o d o DNA p ro vi ra l e e x p re ssã o in vitr o d e a n tí ge n o s vi ra i s p e la PCR e i m u n o f lu o re sc ê n c i a . As li n h a ge n s c e lu la re s tra n sf o rm a d a s f o ra m m a n ti d a s p o r 2 4 0 d i a s, c o m re ti ra d a gra d u a l d e IL- 2 e x ó ge n a . A i m u n o f e n o ti p a ge m p o r c i to m e tri a d e f lu x o re ve lo u a ti va ç ã o c e lu la r. O e sta b e le c i m e n to d e li n h a ge n s c e lu la re s i n f e c ta d a s p o r HTLV- I a p a rti r d e CMP e x-vivo é e x e q ü í ve l e p o d e se r ú ti l n a i n ve sti ga ç ã o d e a lte ra ç õ e s f e n o tí p i c a s li n f o c i tá ri a s e d o s m e c a n i sm o s d e p ro li f e ra ç ã o c e lu la r i n d u zi d a p o r e sse re tro ví ru s. Po d e m a i n d a se r u ti li za d a s c o m i n tu i to d i a gn ó sti c o e m re a ç õ e s d e i m u n o f lu o re sc ê n c i a .
Pal avr as-chave s: HTLV- I. Cu ltu ra . Li n h a ge n s c e lu la re s. Bra si l.
1 . Vir o lo gy Lab o r ato r y ( LIM- 5 2 ) , De par tme nt o f Infe c tio us and Par asitic Dise ase s, Sc ho o l o f Me dic ine , Unive r sity o f São Paulo , São Paulo , SP. 2 . Immuno lo gy Lab o r ato r y, De par tme nt o f Infe c tio us Dise ase s, Fe de r al Unive r sity o f São Paulo , São Paulo , B r azil.
This study was suppo r te d b y r e se ar c h gr ants fr o m Pr ó - Re ito r ia de Pe sq uisa da USP ( Pr o j e to 4 - B o lsa par a Tr e iname nto de Estudante s de Gr aduaç ão e m Té c nic as Espe c ializadas– Pr o c e sso 9 7 . 1 . 2 6 0 7 5 . 1 . 4 ) , CNPq ( B o lsa de Me str ado – Pr o c e sso 1 3 7 4 7 4 /1 9 9 9 -7 ) and FAPESP ( B o lsa de Do uto r ado – Pr o c e sso 1 9 9 7 /0 1 6 8 2 -3 ) .
Addr e ss to: Dr. Aluisio Augusto Co tr im Se gur ado . Lab o r ató r io de Vir o lo gia ( LIM- 5 2 ) , De par tame nto de Mo lé stias Infe c c io sas e Par asitár ias/FMUSP. Av. Dr. Ené as de Car valho Aguiar 4 7 0 , 0 5 4 0 3 - 0 0 0 São Paulo , SP, B r azil
Te l: 1 1 3 0 6 2 - 2 6 4 5 ; Fax: 1 1 3 0 8 8 - 4 9 4 5 e -mail: se gur ado @ usp. b r
Re c e b ido par a pub lic aç ão e m 8 /8 /2 0 0 3 Ac e ito e m 3 1 /5 /2 0 0 4
Hu m a n T- l ym p h o tr o p i c vi r u s typ e I ( HT LV- I ) , a n
e xo ge no us r e tr o vir us, is e tio lo gic ally link e d with two maj o r
dise ase s, adult T-c e ll le uk e mia/lympho ma and a c hr o nic ally
pr o gr e ssive ne ur o lo gic dise ase , k no wn as HTLV-I-asso c iate d mye lo pathy/tr o pic al spastic par apar e sis ( HAM/TSP)9 1 9. This
r e tr o vir al infe c tio n is e nde mic in se ve r al ge o gr aphic r e gio ns
o f the glo b e , that inc lude so uthe r n Japan, Ce ntr al Afr ic a, the
Caribbean basin and So uth Americ a, as well as the Melanesian
is la n ds4. I n B r a zil, in fe c te d in dividua ls h a ve b e e n m o r e
3 3 0
Pa nutti CV e t al
serosc reening of blood donors was implemented. Overall,
HTLV-I seroprevalenc e rates among Brazilian blood donors vary from 0 .0 8 to 1 .3 5 % , ac c ording to geographic origin8. Even though
markers of disease progression are presently unavailable for the
follow-up of HTLV-I asymptomatic c arriers, the lifetime risk of
developing an assoc iated disease has been estimated to vary between 0 .2 % and 4 %1 2 1 4 1 5. Both ATL/L1 7 and HAM/TSP2 have
been reported in nationwide surveys of Brazilian patients from
distinc t geo gr aphic o r igins, b ut par tic ular patter ns o f vir al
transmission, as well as the host’s genetic bac kground and loc al environmental fac tors may possibly ac c ount for differenc es in
the epidemio lo gic al pr o file o f B r azilian c o ho r ts o f
HTLV-I-assoc iated disease6.
Sinc e 1 9 9 1 , a c ohort of HTLV-I-infec ted individuals has been
followed at the outpatient c linic of the Department of Infec tious and Parasitic Diseases, University of São Paulo School of Medicine
fo r c linic al, e pide m io lo gic al and lab o r ato r y inve stigatio n
purposes. HTLV-I infec tion in these individuals is more often
diagnosed by rec ognition of seroreac tivity to spec ific ga g and
e nv viral antigens in serologic tests that include screening enzyme immunoassays and c onfirmatory and disc riminative Western blot
tests. Additionally detec tion of proviral DNA in peripheral blood
mononuc lear c ells by polymerase c hain reac tion provides further evidenc e of persistent retroviral infec tion1 0. The present study,
approved by the Institutional Review Board ( CAPPesq) , desc ribes
the establishment of HTLV-I-infec ted c ell lines from peripheral
b lo o d mo no nuc le ar c e lls ( PB MC) o f se r o po sitive patie nts. Tho ugh time-c o nsuming and labo r-intensive, vir al c ultur es,
c ar r ie d o ut unde r pr o pe r b io safe ty r e q uir e me nts, pr o vide
valuable information on the biological behavior of HTLV-I-infected
c ells and may eventually lead to the establishment of infec ted lymphoc ytic c ell lines.
MATERIAL AND METHODS
After informed consent, blood specimens were collected from
two seropositive women, assisted at the outpatient clinic of the
University hospital in São Paulo, Brazil. The first patient was a 4 7
-year-old white housewife, who complained of progressive weakness
of her lower limbs and urinary retention for 3 years. At first medical
visit, she already required a walking aid, but no other abnormality was notic ed on c linic al examination. Spastic paraparesis with
pyramidal signs was detected on neurologic examination and CSF
analysis revealed mild mononuclear pleocytosis ( 1 0 cells/mm3)
with normal protein levels ( 3 7 mg/dl) . Spec ific anti-HTLV-I/II
antib o die s and HTLV-I pr o vir al DNA we r e de te c te d in CSF,
confirming the diagnosis of HAM/TSP1 8. The second patient was a
3 4 -year-old saleswoman, who reported diffuse alopec ia and
infiltrated skin plaques on the anterior abdominal wall. A skin
b io ps y wa s pe r fo r m e d in th e a ffe c te d a r e a a n d yie lde d
dermatotropic cutaneous T-cell lymphoma. Her total white blood cell and lymphocyte counts were normal ( 6 ,7 0 0 and 2 ,0 3 7 cells/
mm3, respectively) . Cytometric immunophenotypic analysis of her
peripheral blood mononuclear cells ( PBMC) , using monoclonal
antibodies was normal for CD4 + and CD8 + subsets, as well as for
CD2 5 expression. Serum lactic dehydrogenase ( LDH) and calcium
levels were within normal limits and therefore, she was diagnosed with smoldering ATL/L1 3.
PBMC from both patients were separated from EDTA-treated blood
samples by Ficoll gradient centrifugation and 2x106 cells were cultured
in RPMI 1640 medium, supplemented with 20% heat-inactivated fetal
bovine serum and 1 0 % partially purified rec ombinant human interleukin 2 ( IL-2 ) , in the presence of penicillin ( 1 0 0 IU/ml) ,
streptomycin ( 100
µ
g/ml) , amphotericin B, and glutamine ( 2nmol/l) on 24-well plates. After 72-hour incubation at 37oC in a 5 % CO 2atmosphere, an equal number of lymphocytes previously stimulated with phytohemagglutinin ( 2
µ
g/ml) from a seronegative donor were added to HTLV-infected PBMC. Cocultures were then maintained underthese experimental conditions and fed, every 3 to 4 days, with fresh
medium to provide appropriate expansion, according to in vitro cell growth. Subsequently cultures were added with PHA-stimulated PBMC
from the same seronegative donor every 2 weeks.
RESULTS
Afte r 9 0 days in c ultur e , mo r pho lo gic e vide nc e o f c e ll
transformation was obtained in cocultures developed from both
patients’ PBMC, with identification of multinucleated cells ( Figure
1 ) that exhibited cerebriform nuclei on Giemsa staining. Long-term cell lines were maintained for up to 2 4 0 days, after being
gradually weaned from exogenous IL-2 . In order to demonstrate
pr o vir al inte gr atio n in e stab lishe d c e ll line s, ne ste d PCR amplific atio n o f HTLV-I ta x sequenc es was c ar r ied o ut, as previously described1 0. Briefly, cultivated cell lysates were obtained
by proteinase K digestion and subsequently underwent genomic
amplification, using consensus oligonucleotide primers ( SK 4 3 and SK4 4 ) , that allow detection of both HTLV-I and HTLV-II proviral
sequences. A second round of amplification was then performed,
with pr im e r s n t 7 3 7 5 - 7 3 9 4 a n d n t 7 4 8 6 - 7 5 0 2 , th a t a r e
complementary to sequences that lie internally to the edges of
amplicons generated in the first round, producing a DNA fragment of 1 2 8 bp ( Figure 2 ) . Discrimination between HTLV-I and HTLV-II
sequences was achieved by restriction enzyme digestion of nested
PCR produc ts, with Ta q I and restric ted length polymorphism ( RFLP) analysis was carried out visually, after electrophoresis in
2 % agarose gels2 0. In both studied patients, RFLP analysis yielded
a 1 2 2 bp DNA fragment, compatible to HTLV-I infection ( data not
shown) . Cells from the seronegative donor, used in cocultures, yielded in contrast no HTLV proviral sequences.
In vitro production of viral antigens was sought after by direct immuno fluo resc enc e ( IF) , as previo usly repo rted1. Fo r this
purpose, c ultures were harvested and submitted to 2 0 0 0 rpm
c entrifugation for 1 0 minutes. Cell pellets were subsequently
resuspended in sterile PBS, spotted onto IF slides and c ells fixed with c old 1 :1 ac etone/methanol solution for 1 5 minutes. PBMC
from the same HTLV seronegative donor that provided PBMC for
c oc ultures were used as negative c ell c ontrols. Dried slides were kept at -2 0oC until the detec tion step was c arried out with a 1 :1 0
diluted serum sample from a HTLV-I seropositive patient. PBS
3 3 1 Revista da Sociedade Br asileir a de Medicina Tr opical 3 7 ( 4 ) : 3 2 9 -3 3 2 , jul-ago, 2 0 0 4
additional negative c ontrols. Slides were then inc ubated with a
1 :1 0 0 dilution of sheep anti-human IgG fluorescein isothiocyanate
c onjugate at 3 7oC for 4 5 minutes and examined for detec tion of
viral antigens. Immunofluoresc enc e, using polyc lonal antiserum,
revealed expression of viral antigens in a large proportion of
c ultivated c ells from both established c ell lines ( Figure 1 ) .
Lymphoc yte immunophenotyping of established c ell lines was
performed after 9 0 days in c ulture, using a FACSCalibur flow
c ytometer. Briefly, for 4 -c olor multiparameter flow c ytometric
analysis, c ells were stained in separate tubes, using monoc lonal
antibodies ( BDIS) to c ell surfac e markers CD3 , CD4 , CD8 , CD1 9 ,
CD2 5 ( IL-2 r e c e pto r ) , CD5 6 and HLA-DR, in a 1 5 -minute
inc ubation in the dark. After washing in PBS, c ells were run in
flow c ytometry and data analysis performed with the CellQuest
c omputer software. A predominanc e of CD3 + CD4 + lymphoc ytes
was verified in HTLV-I-infected cell lines and increased expression
o f sur fac e mar k e r s o f c e ll ac tivatio n ( HLA-DR+) c o uld b e
demonstrated ( mean fluoresc enc e intensity of 9 2 7 and 5 3 7 for
HTLV-I-infec ted c ell lines and of 2 1 2 and 1 1 8 for respec tive
c ontrols) . No differenc e was notic ed in CD2 5 expression.
Fi gu re 1 - Di re c t i m m u n o f lu o re sc e n c e , u si n g p o lyc lo n a l a n ti - HTLV- I se ru m , d e te c ts HTLV- I a n ti ge n e x p re ssi o n i n c u ltu re d e x vivo p e ri p h e ra l b lo o d m o n o n u c le a r c e lls ( PBMC) f ro m a p a ti e n t w i th sm o ld e ri n g a d u lt T- c e ll le u k e m i a ( A) a n d i n lym p h o c yte s f ro m a n HTLV- I- i n f e c te d e sta b li sh e d c e ll li n e – MT- 2 ( B) ; n o a n ti ge n e x p re ssi o n i s se e n i n PBMC f ro m th e HTLV- I- se ro n e ga ti ve d o n o r th a t we re u se d to f e e d c o c u ltu re s ( C) ; HTLV- I- i n d u c e d m u lti n u c le a te d c e lls i n e x vivo PBMC o f a p a ti e n t wi th HAM/TSP a f te r 9 0 d a ys i n c u ltu re .
3 3 2
DISCUSSION
I n fe c ti o n wi th HTLV- I i s k n o wn to i n du c e i n vi tr o
spontaneous lymphoc yte proliferation in the absenc e of mitogen
or antigen stimulation and this proliferative response is believed
to be dependent on the transac tivation proprieties of the proviral
ta x gene produc t ( p4 0ta x) . Lymphoc yte proliferation eventually
evolves to c ell immortalization and in vitro transformation1 6.
However HTLV-I-induc ed leukemogenesis in vivo is so far not fully understood. HTLV-I-mediated T-c ell transformation in
in fe c te d in dividua ls pr e s um a b ly a r is e s fr o m a m ultis te p onc ogenic proc ess resulting in ac c umulation of genetic defec ts
and dysregulated growth of infec ted c ells leading to development
of ATL/L in a minority of HTLV-I c arriers7 1 1. Nevertheless further
researc h is still warranted for a better understanding of ATL/L pathogenetic mec hanisms.
The establishment of HTLV-I-infec ted c ell lines from e x vivo
PBMC of HTLV-I-infec ted individuals provides useful investigative
tools for studies on phenotypic c hanges of infec ted lymphoc ytes
and may thus help in further eluc idation of the mec hanisms involved in HTLV-induc ed c ell proliferation.
I n th e pr e s e n t s tudy we h a ve de m o n s tr a te d th a t th e e stab lishme nt o f HTLV-I-infe c te d CD4+ c e ll line s fr o m e x vi vo
PB MC is fe asib le unde r pr o pe r b io safe ty r e q uir e m e nts in
B r azilian lab o r ato r ie s, as pr e vio usly de sc r ib e d ab r o ad5.
Apart from their potential use in pathogenetic studies of HTLV-I-induc ed c ell transformation, these infec ted c ell lines may also
be helpful in the c onfirmatory diagnosis of HTLV-I infec tion.
Cur r e nt se r o lo gic al algo r ithms fo r the diagno sis o f HTLV-I
infe c tio n usually r e c o mme nd the use o f sc r e e ning e nzyme im m uno assays, fo llo we d b y se r o lo gic al c o nfir m atio n and
disc rimination based on seroreac tivity to viral ga g and e nv-coded antigens on Western blot assays3 2 1. However given the overall
h igh c o s t o f c o m m e r c ia lly a va ila b le We s te r n b lo t k its ,
serodiagnostic algorithms in resource-poor settings may consider the possibility of using HTLV-I infec ted c ell lines with diagnostic
purposes in c onfirmatory immunofluoresc enc e tests.
ACKNOWLEDGEMENTS
The autho r s wo uld lik e to thank Dr. Mar ia Jo sé Andr ada-Se r pa fo r having k indly pr o vide d te c hnic al suppo r t to this inve stigatio n.
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