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Copyright(C 1986, AmericanSociety for Microbiology
Enhanced Susceptibility
to
Cytotoxic T Lymphocytes of Target
Cells Isolated from
Virus-Infected
or
Interferon-Treated Mice
JACK F. BUKOWSKI ANDRAYMOND M. WELSH*
Departments of Pathology and Molecular GeneticsandMicrobiology, University ofMassachusetts MedicalSchool,
Worcester,
Massachusetts 01605Received 4April 1986/Accepted 12 June 1986
Bonemarrowcells and thymocytesisolated from virus-infectedorinterferon(IFN)-treated mice had marked
increases in sensitivity to lysis by allospecific cytotoxic T lymphocytes (CTL) and in expression of class I
histocompatibility antigens. Culturedfibroblasts treated withIFNinvitroyielded similar findingsinaddition
to having increased sensitivity to lysis by virus-specific CTL. This indicates that virus-induced IFN-may
conditiontargetcells invivo for surveillance by CTL.
Virus infections and the interferon (IFN) they induce can haveprofound effects on cells in the body, such as activation ofmacrophages (2, 14), activation (8, 10, 19, 20) and division
cytes (CTL). IFN induces the expression of histocompati-bility (H-2) antigens (9), andtreatmentof virus-infected skin fibroblasts in vitro with IFN markedly enhances their
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FIG. 1. H-2expression of thymocytes. Three- tosix-week-old BALB/corC57BL/6 micewereacutely infectedintraperitoneally with 8 x 104 PFU ofLCMV(Armstrong strain)3days before thymocyteisolation.BALB/c mice persistently infected with LCMVwere congenital carriers (17). Partially purified IFN-f (Lee Biomolecular, San Diego, Calif.) was given intravenously at 105 U per mouse 1 day before thymocyte isolation. Poly(I:C) (Sigma Chemical Co., St. Louis,Mo.) (100 ,ug) wasgiven intraperitoneally 1daybeforethymocyteisolation. Thymocyteswerewashed, and2 x 106cellsweretreatedwitha ratmonoclonalantibody (cloneMI/42;reference16) to a mouseframework classIH-2antigenic determinant for45min at4°C.Thecellswerewashed and then treated withfluorescein isothiocyanate-conjugatedF(ab')2
mouseanti-ratimmunoglobulin G for45minat4°C. The cellswerewashed, suspendedto2x 106cellsperml, and analyzed byflowcytometry
on aBecton Dickinson (Paramus, N.J.) FACS IV. Thegainwas set at 2.5(96channels per
loglo
fluorescence intensity). (A) BALB/c: a, control; b,poly(I:C) treated;c, acuteLCMV infection. (B)C3H/St:a,control; b, poly(I:C) treated; c, acuteLCMVinfection. (C)C57BL/6: a,control; b, poly(I:C) treated; c, acute LCMV infection. (D)BALB/c: a, control; b, IFN treated. (E) BALB/c: a, control; b, persistent LCMVinfection;c,acuteLCMVinfection.(F)BALB/c:a,control; b,persistent LCMV infection; c, persistent LCMV infection treated withpoly(I:C).
(1) of natural killer (NK) cells, and generation of both virus-specific (23) and allospecific (22) cytotoxic T
lympho-* Correspondingauthor.
ceptibilitytolysis by virus-specificCTL (4), whichrecognize
viralantigens in the context of class I H-2 determinants (23). Wereport here thatfreshly isolated bone marrow cells and
thymocytes from virus-infected or IFN-treated mice had
735
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[image:1.612.124.480.319.560.2]736 NOTES
240.0-E~~~~~~~~~~~~~~~~
192.0
j a
a.0
0.0 64.0 128.0 192.0 256.0 64.0 1280 19a 296.0 64.0 128.0 192 2560
[image:2.612.132.480.74.317.2]Channel Number
FIG. 2. H-2 expressiononbone marrowcells. Miceweretreatedasdescribed in the legendtoFig. 1,and bonemarrowcellswerealso
stained in thesame manner. Flow cytometric analyseswereruntwice for each cell sample; high andlowlightscattercorrespondedtolarge
andsmall-sized cells, respectively. The gainwasset at2.5 (96channelsperlogl0fluorescence intensity)unless otherwise indicated.Whenthe
gain was set at 1.9, this equalled 80 channels perlogl0 fluorescence intensity. (A) BALB/c small cells: a, control; b, persistent LCMV
infection;c,acuteLCMV infection (gain= 1.9). (B)BALB/clarge cells:a,control; b, persistent LCMV infection;c,acuteLCMVinfection
(gain= 1.9). (C) BALB/c small cells:a,control; b, IFN treated.(D)BALB/c large cells:a,control; b, IFN treated. (E) C57BL/6 small cells:
a,control; b,acuteLCMVinfection. (F) C57BL/6 large cells:a,control; b,acuteLCMVinfection (gain = 1.9).
marked increases in surface H-2 antigen expression witha
concomitant increase in their sensitivities to lysis by al-lospecific CTL. In addition, the sensitivity of cultured fibro-blasts to lysis by virus-specific and allospecific CTL was
markedly increased by in vitro treatment with IFN. These results suggest that the IFN induced during virus infection enhances T-cell-dependent processes such as viral
clear-ance, virus-induced immunopathology, and graft rejection
by altering the susceptibility oftargetcellstoT-cell surveil-lance.
Four- to six-week-old mice were injected with
lympho-cytic choriomeningitis virus (LCMV), poly(I:C), orpurified
IFN-P
and sacrificed 1 to 3 days later. Mice persistently infected with LCMV werecongenital carri,ers (17). Thymo-cytesand bonemarrowcellswere isolated and treated witha ratmonoclonal antibody (16) directed against framework determinants ofmouse class I histocompatibility antigens,
followed by treatment with fluorescein isothiocyanate-conjugated F(ab')2 mouse anti-rat immunoglobulin G. The cells were then analyzed by flow cytometry. The results showed that thymocytes isolated from acutely (Fig. 1A, B, C, and E) orpersistently (Fig. 1E and F) infected mice had
upto fivefoldgreatersurface expression of class I
antigens
than those from uninfected mice. Thymocytes from mice
TABLE 1. Thymocytes and bonemarrowcells isolatedfromvirus-infected and IFN-treated micewere moresensitivetolysis by
allospecificCTL thanthose isolated from controlmicea
%Specific51Cr release by cells from:
Target cells Effector cells
Bontrol
LCMV-infectedAcutely LCMV-infectedPersistently IFN-treatedCotrIlAcutely
C57
LCMV-infectedBALB/c
BALB/cBALB/c
BALB/c C57 C5Thymus C57anti-BALB/c 12 31 28 24 -3.4 -1.4
BALB/canti-C57 0.2 2.1 1.2 1.2 15 28
ActivatedNK 20 6.1 16 14 12 6.2
Bone marrow C57 anti-BALB/c 11 26 23 17 0.5 1.2
BALB/canti-C57 0.2 0.8 1.2 1.1 6.5 25
ActivatedNK 0.2 0.6 1.2 -2.0 2.0 0.2
a Three-tosix-week-oldBALB/corC57BL/6 micewereacutelyinfectedintraperitoneally with8x104PFUof LCMV(Armstrongstrain)3daysbeforetarget
cellisolation. BALB/c micepersistently infected withLCMVwerecongenitalcarriers(17).Partiallypurified IFN-P (Lee Biomolecular)wasgiven intravenously
at10'U per mouse 1day beforetargetcell isolation.Thymocytesand bone marrow cellswereisolatedfromthesemice and labeled with100 .CiofNa25lCr]04
for 1 h at 37'C.Targetcells were washed anddispensedinto round-bottom microtiter wellsat4x104 cellsper well(7).EffectorcellswereeitheractivatedNK
cellswhichwereisolated from C57BL/6 mice3daysafter acuteLCMV infection(20)orallospecificCTL from6-day-old,one-way,mixedleukocytecultures(5).
Cytotoxicityassays were 4hlong,and theresultsarereportedas%specific"Crrelease(20)withaneffector-to-targetcell ratioof20:1.Spontaneousreleasewas
between17 and31%.
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[image:2.612.59.557.554.670.2]TABLE 2. Mouse fibroblasts isolated from liverswere more sensitive tolysis by CTL when pretreated with IFN" %Specific"'Crreleased by livercells:
Effector cells IFN-treated LCMV-'ntected LCM V-infected LCMV-infected,
C3H IFFraeCM-netd lN-treated C-57 INraeFCVnetd lN-treated
C3H C3H C3H C57 C-57 C57
C3H anti-C57 2.3 1.0 0.2 0.0 39 60 35 65
C57anti-C3H 47 68 40 69 0.0 2.7 0.1 -0.1
C3H anti-LCMV 0.5 0.1 20 47 5.2 0.2 4.2 4.5
C57anti-LCMV 2.4 0.9 2.7 3.0 2.0 0.3 22 49
Fibroblasts were grown from cultures ofminced, trypsin-treatedlivers isolated from 3-day-old C3H/St or C57BL/6 mice. The cells wereeitherleft untreated
orinfectedwithLCMVat amultiplicityof 0.05.Ontheday3postinfection,103Uof IFN-,B per ml was added to some of the cultures. After 18 to 24h,thecells
were labeled withNal[5'Cr]O4for 1 h at 37'C and then washed. The cells were dispensed at104 cells per well in round-bottom microtiter wells. Spleen cells
containing LCMV-specificCTL were isolated from mice infected intraperitoneally 7 to 9 days earlier with LCMV(20). AllospecificCTL were from6-day-old,
one-way,mixed leukocyte cultures (5). Cytotoxicity assays were 6 to 8 h long, and results are reported as % specific5'Crrelease: effector-to-targetcell ratios
were 100:1,and spontaneous releaseoflabel was between23and40%.
treated with poly(I:C), an IFN inducer (Fig. IA, B, C, and
F), and purified IFN-3 (Fig. 1D) also had enhanced class I
antigen expression. Poly(I:C) treatment of mice persistently infected with LCMV and already having chronic, low-level interferonemia (32 to64 U/ml ofplasma) (3, 13) resulted in
thymocytes whose surface H-2antigen expression was even
further enhanced comparedwith those from untreated,
per-sistently infected mice, correlating with the enhanced IFN levels (256to 512 U) in these poly(I:C)-treated persistently infected mice (3) (Fig. IF).
Bone marrow cells isolated from these same mice were also analyzed for surface class I antigen expression. The resultswere similartothoseobtained forthymocytes. Acute
LCMV infection resulted in bone marrow cells with a dramatic (up to 12-fold) increase in surface class I antigen expression (Fig. 2A, B, E, and F). Persistent LCMV infec-tion (Fig. 2A and B), IFN-1 treatment(Fig. 2C and D),and
poly(I:C) treatment (data not shown) also resulted in such increases. Owing to the heterogeneity of the bone marrow cell population, each sample was analyzed twice, and
sepa-rateprofileswereobtained forlargeand small(highand low
light scatter, respectively) cells. These separate analyses
revealed that both large and small cell populations isolated
from virus-infected or IFN-treated mice had increased sur-faceexpression of class Iantigencompared withthose from control mice. The virus infections and IFN treatments did notdecrease the cellyieldsfrom the bonemarrow (datanot
shown) or therelative number oflargeversus smallcells at
this timeposttreatment.
We tested whether the increase in class Iantigen
expres-sion in these leukocyte populations would correlate with increased sensitivity to CTL, which recognize class I
anti-gens. Freshly isolated thymocytes from LCMV-infected or
IFN-treated mice were two tothree times more susceptible
to lysis by allospecific CTL than were cells isolated from
control mice (Table 1). Thymocytes isolated from mice
acutely infected with LCMV were also more resistant to
lysis by activated NK cells (Table 1), confirming previous
results which have shown that IFN protects thymocytes from NK cell-mediated lysis (7). Whereas thymocytesfrom
persistently infected mice having lowplasma IFN titers(16
to 32 U) displayed relatively normal sensitivity to lysis by
NK cells (7) (Table 1), they were much more sensitive to
lysis by allospecific CTL(Table 1).This result is consistent
with those obtained using culture cells in which treatment
with lowconcentrations ofIFN hadapronounced effecton
sensitivity to T-cell-mediated lysis (4) but no significant effectonsensitivitytoactivated NK cell-mediatedlysis (data
not shown). However, poly(I:C)-treated, persistently in-fected mice had higher IFN titers (256 to 512 U) (3),
enhanced class I antigen expression on thymocytes (Fig.
1B), and decreased sensitivity of thymocytes to NK
cell-mediated lysis (6.3% lysis) compared with untreated persis-tently infected mice (24% lysis).
IFN treatment and virus infection of mice also rendered
freshly isolated bone marrow cells up to four times more
susceptible than control cellsto lysis by allospecific T cells
(Table 1). However, control bone marrow cells were not
lysed invitrobyactivated NKcells, and virusinfectionsor IFN treatmenthad noeffect on this result(Table 1).
Fibroblasts growing from primary cultures of
trypsin-treated skin, brains, livers, and hearts were either infected with LCMV orleftuntreated. IFN-4 was added to someof the cultures for 15 to 24 h before use as targets in
cytotoxicityassayswithactivated NK cells, LCMV-specific CTL, or allospecific CTL as effector cells. IFN treatment
increases the surface H-2 expression of fibroblasts (4). All
typesoffibroblasts, whenpretreated with IFN, were
signif-TABLE 3. Mousefibroblasts isolated from brains and hearts were more sensitive tolysisby CTL when pretreated withIFN'
%Specific51Crreleased by cells from:
Brain: Heart:
Effectorcells LCVIfce,LCM
V-infected.
EfsC57
IFN-treated
LCMV-infected IFN-treatedLCMV-infected
E-aC57C57 C57 IF~~~lN-treated C57 C57 C57 CF57eae
C57 C57
~~~~C57C5
C3H anti-C57 30 65 26 50 19 39 20 46
C57 anti-C3H 0.0 2.3 -2.5 -0.1 0.2 0.1 -1.5 -1.1
C3H anti-LCMV 2.6 8.2 5.2 0.0 4.2 4.6 0.0 2.6
C57anti-LCMV 0.1 -1.9 18 40 2.6 -2.6 21 50
"Fibroblasts were grown from cultures ofminced,trypsin-treated brains and hearts isolated from3-day-oldC3H/St or C57BL/6 mice and treated asdescribed
inthefootnote to Table 2.
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TABLE 4. Mouse fibroblasts isolated from skin were more sensitive tolysis by CTL when pretreated with IFN"
%Specific5Cr releasedby skin cells:
Effectorcells IFN- IFN-
IFN-C57 treated C3H treated BALB/c treated
C57 C3H BALB/c
C3H anti- NDb ND 1.0 0.4 11 38
BALB/c
BALB/c ND ND 31 62 0.1 0.8
anti-C3H
C3H 18 33 4.8 8.9 4.2 3.7
anti-C57
C57 -1.2 2.3 11 31 -0.9 -0.2
anti-C3H
aFibroblasts were grown from cultures of minced, trypsin-treated skin
isolated from3-day-oldC3H/StorC57BL/6mice and treated as described in
the footnoteto Table 2.
bND,Notdone.
icantly more sensitive than untreated fibroblasts to lysis by allospecific CTL, whether or not the fibroblasts were
in-fected with LCMV (Tables 2 and 3) (4). IFN pretreatment renderedvirus-infected, but not uninfected, fibroblasts more sensitive to lysis by virus-specific, H-2-restricted CTL. Thus, the increased susceptibility of target cells to the relevant allospecific CTL correlated with the increased
susceptibility of virus-infected targets to the appropriate virus-specific CTL. Conversely, IFN pretreatment de-creasedthesensitivityoffibroblaststolysis by activated NK cells by an average of86%, confirming and extending our
previous results (4) and those of others (18).
These results and those of others (9, 15, 21) suggest that
differenttypesofcells from various organs in thebodyeither freshly isolated or recently cultivated express relatively low levels of class I antigens on their cell surfaces under normal
conditions.Virus infection (Fig. 1and2)orprophylactically administered IFN (Fig. 1 and 2) (9, 15, 21) is capable of
enhancing surface class Iantigen expression onthese cells,
and this may result in either beneficialordeleterious
conse-quences for the host. On one hand, enhancement of
sensi-tivitytovirus-specific killingcould facilitate the clearance of
virus, but on the other hand, the result could be
enhance-ment ofvirus-specific, T-cell-dependent immunopathology, which is responsible for death during LCMV infection (6).
Indeed,administration of IFNto miceinfected withastrain
of LCMV which induces low levels of IFN results in an
increased mortality rate (12).
The involvement of allospecific CTL in mediation of
graft-versus-host disease or graft rejection could be ampli-fiedduringvirus infectionorIFNtherapy. Milton and Fabre (11) have recently noted that heart allografts undergoing
rejection have increased histocompatibility antigen expres-sion on the surface of cells isolated from the transplanted
hearts compared with those isolated fromgrafted
syngeneic
hearts. Further, virus infections have been shownto stimu-latepolyclonal allospecificCTLgeneration (22). Knowledge
of the fact that virus infection and IFN maycausemarkedly
increasedhistocompatibility antigen expression in vivo and demonstration of the possible consequences may lead to
increased understanding ofimmunoregulation, viral immu-nology, and transplantation immunology.
This research was supported by Public Health Service grants
AI-17672andCA34461 from the National Institutes of Health. WethankKathleenDenehyand LindaColbyfortechnical
assist-anceand Dottie Walsh for preparationof themanuscript. We also thank David Parker and NancyPhilips for the monoclonal anti-H-2 antibody.
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