To determine whether poor antigen presentation by HPV-infected keratinocytes KCs contributes to prolonged HPV infection, KCs and KCs expressing HPV 16 E7 protein E7-KCs were compared for
Trang 1interferon-c-mediated enhancement of keratinocyte
antigen processing and T-cell lysis
Fang Zhou, Graham R Leggatt and Ian H Frazer
The University of Queensland Diamantina Institute, Princess Alexandra Hospital, Brisbane, Qld, Australia
Introduction
Persistent infection of the cervical epithelium with one
of a range of oncogenic human papillomaviruses
(HPVs) can initiate cervical cancer The majority of
high-risk papillomavirus infections of
immunocompe-tent individuals are cleared, although this can take up
to 4 years, and a minority of apparently
immunocom-petent individuals will develop persisting infection [1]
These data suggest that HPV may have evolved
mech-anisms to enable infected epithelial cells to escape from
immune surveillance in vivo [2] We established a skin
graft model to study the role in immune evasion of the
HPV 16 nonstructural protein E7, which is
overexpres-sed in premalignant lesions associated with HPV 16
infection In this model, murine skin expressing
HPV 16 E7 as the product of a transgene in
keratino-cytes (KCs) from a keratin 14 promoter is grafted into naı¨ve, otherwise syngeneic, mice These mice fail to reject such grafts, either spontaneously or after immu-nization with E7 [3] In contrast, skin grafts similarly expressing ovalbumin (OVA) as a transgene product are spontaneously rejected [4] In vitro, HPV 16 E7-specific cytotoxic T lymphocytes (CTLs) fail to kill HPV 16 E7 transgenic KCs efficiently, but can kill KCs pulsed with the dominant H-2Db restricted E7 peptide epitope [5] or with OVA [6] A number of studies have shown that HPV gene expression can interfere with various components of antigen process-ing by uncertain mechanisms [7–9] Taken together, these results suggest that the HPV 16 E7 may inhibit cellular antigen processing and presentation to enable
Keywords
antigen processing; CD8 T cells; host–virus
interactions; papillomavirus
Correspondence
I Frazer, The University of Queensland
Diamantina Institute, Princess Alexandra
Hospital, Ipswich Road, Woolloongabba,
Brisbane, Qld 4102, Australia
Fax: +61 7 3240 5946
Tel: +61 7 3240 5310
E-mail: i.frazer@uq.edu.au
(Received 14 July 2010, revised 2
December 2010, accepted 10 January 2011)
doi:10.1111/j.1742-4658.2011.08011.x
Infection of epithelium with human papillomavirus (HPV) 16 is generally prolonged, suggesting an ineffective virus-specific immune response, and prolonged infection promotes anogenital cancer To determine whether poor antigen presentation by HPV-infected keratinocytes (KCs) contributes
to prolonged HPV infection, KCs and KCs expressing HPV 16 E7 protein (E7-KCs) were compared for susceptibility to T-cell-mediated lysis directed
to ovalbumin (OVA) processed for presentation by the KCs Interferon (IFN)-c efficiently enhanced susceptibility to lysis of KCs presenting OVA, but not of E7-KCs similarly presenting OVA E7-KCs also exhibited impaired IFN-c-induced upregulation of transcription of major histocom-patibility complex class I antigen processing and presentation-associated genes, and of membrane SIINFEKL–H-2Kb complexes Thus, expression
of HPV 16 E7 protein in KCs may inhibit enhancement by IFN-c of KC sensitivity to T-cell lysis, by impairing antigen presentation
Abbreviations
CTL, cytotoxic T lymphocyte; E7-KC, keratinocyte expressing HPV 16 E7; HPV, human papillomavirus; IFN, interferon; IRF, interferon regulatory factor; KC, keratinocyte; MFI, mean fluorescence intensity; MHC, major histocompatibility complex; SD, standard deviation; 2-ME, 2-mercaptoethanol.
Trang 2HPV to evade viral antigen-specific host immune
responses
Several viruses have been demonstrated to interfere
with antigen processing and presentation by infected
cells, through inhibition of peptide recruitment to and
processing by the proteasome, or through blocking the
production and transport of major histocompatibility
complex (MHC) class I complexes [10–13] Interferon
(IFN)-c plays an important role in facilitating
CTL-mediated immune effector function, through induction
of multiple genes associated with MHC class I antigen
presentation [14], and also contributes to the
elimina-tion of HPV infecelimina-tion [15]
HPV 16 E7 has been shown to interfere with the
transduction of IFN signaling [16–19] HPV 16 E7
binds to the C-terminal transactivation domain of
interferon regulatory factor (IRF)-1 in vitro, and
cul-tured fibroblasts transduced with HPV 16 E7
demon-strate reduced transcription of some IFN-c-inducible
gene products following overexpression of IRF-1 when
compared with untransduced cells [20] In HPV 18
E6⁄ E7 transgenic mice, there is some evidence for
reduced transcription of the same gene products in the
cervix, as compared with nontransgenic animals [21]
We therefore investigated, using OVA as a model
anti-gen, whether HPV 16 E7, when expressed in KCs at
levels similar to those found in HPV infection, could
inhibit enhancement by IFN-c of antigen processing
and presentation, and CTL-mediated killing of KCs expressing non-self-antigen
Results
E7 does not affect the ability of IFN-c to upregulate MHC class I expression and exogenous CTL epitope presentation on KCs
We first wished to investigate whether expression of HPV 16 E7 in KCs would alter the expression of mem-brane-associated MHC, the presentation of processed endogenous antigen as a peptide in association with MHC, or CTL-mediated lysis directed at cell mem-brane MHC–peptide complexes As IFN-c can upregu-late MHC class I expression on keratinocytes [22] and enhances CTL epitope presentation and CTL-mediated lysis of KCs [6], we also wished to investigate whether E7 interfered with IFN-c-induced enhancement of anti-gen processing and presentation Therefore, KCs and KCs from H-2b mice transgenic for HPV 16 E7 expressed from a keratin 14 promoter (E7-KCs) were exposed in vitro to IFN-c We first examined the effect
of E7 on IFN-c-mediated induction of MHC expres-sion Induction by IFN-c of the expression of MHC class I on cultured E7-KCs was similar to induction on nontransgenic KCs (Fig 1A) We then exposed KCs and E7-KCs to IFN-c and SIINFEKL, a peptide
A
B
Fig 1 Lysis of KCs and E7-KCs treated with IFN-c Mouse KCs were exposed to SIINFEKL and IFN-c as shown (A) Expression of MHC class I on KCs and E7-KCs after exposure to IFN-c, as shown, was assessed by flow cytometry with an H-2K b -specific antibody (B) Expres-sion of SIINFEKL–H-2K b peptide complexes on KCs and E7-KCs after incubation with 10 l M SIINFEKL, and IFN-c, as shown, was assessed with mouse antibody against SIINFEKL–H-2Kb (C) Expression of SIINFEKL–H-2Kbcomplexes on KCs and E7-KCs after incubation with IFN-c
at 100 UÆmL)1and SIINFEKL, as shown, was assessed (D) Susceptibility of SIINFEKL-exposed KCs to lysis by SIINFEKL-specific CTLs at an effector ⁄ target ratio of 1 : 20 was compared for KCs and E7-KCs pulsed with three concentrations of SIINFEKL The experiments in (A), (B) and (C) were repeated twice, and those in (D) were repeated three times, with similar results In (D), mean and SD from triplicate determina-tions of percentage lysis for one experiment are shown.
Trang 3derived from OVA that is able to associate with
mem-brane H-2KbMHC complexes without further
process-ing KCs and E7-KCs showed similar dose-dependent
increases in the density of membrane SIINFEKL–
H-2Kb complexes after IFN-c exposure (Fig 1B)
Furthermore, KCs and E7-KCs exposed to a fixed
concentration of IFN-c and to increasing
concentra-tions of SIINFEKL displayed similar increased density
of SIINFEKL–H-2Kb complexes (Fig 1C) By
select-ing appropriate concentrations of peptide, we could
then compare the susceptibility of KCs and E7-KCs
expressing similar densities of SIINFEKL–H-2Kb
com-plexes to lysis by a set number of E7-specific T cells,
to establish whether E7 expression might impact on
the sensitivity of KCs to T-cell lytic mechanisms For
any given level of expression of MHC–peptide
com-plex, KCs and E7-KCs were equally susceptible to lysis
in vitro by SIINFEKL-specific CTLs (Fig 1D) Thus,
endogenous expression of E7 has no effect on
IFN-c-induced enhancement of MHC expression by KCs, or
on the susceptibility of KCs pulsed with exogenous
SIINFEKL to CTL-mediated lysis
E7 inhibits IFN-c-dependent upregulation of
presentation of endogenous antigen by KCs
As there was no observed effect of endogenous E7 on
MHC expression or on the susceptibility of KCs
expressing MHC–peptide complexes to T-cell-mediated
lysis, we next wished to test the hypothesis that
endog-enous E7 expression in KCs might inhibit the
process-ing and presentation of endogenously expressed
protein We therefore compared KCs and E7-KCs,
each also expressing OVA endogenously as a transgene
product, for susceptibility to lysis by OVA-specific
T-cells, both with and without IFN-c pretreatment E7
and OVA double-transgenic KCs and OVA
single-transgenic KCs, if not treated with IFN-c, were
simi-larly susceptible to lysis by OVA-specific T cells
(Fig 2A) However, lysis of OVA transgenic KCs by
OVA-specific T cells was significantly increased
follow-ing IFN-c exposure, whereas lysis of E7 and OVA
double-transgenic KCs was not (Fig 2A) These
results, together with those showing that E7 has no
effect on the presentation of exogenous peptide or on
T-cell-mediated lysis of cells sensitized by exogenous
peptide, with or without IFN-c exposure, allow the
conclusion that E7 inhibits the ability of IFN-c to
enhance the processing of endogenous antigen for
pre-sentation To confirm these findings in an independent
system, we loaded KCs osmotically with OVA, using
previously established techniques KCs and E7-KCs
loaded with OVA were treated with IFN-c, or left
untreated, and assessed for susceptibility to T-cell-med-iated lysis CTLs specific for SIINFEKL were equally able to kill untreated KCs or E7-KCs when osmoti-cally loaded with OVA, as expected As predicted from the findings with OVA transgenic KCs, lysis of KCs osmotically loaded with OVA was similar whether the cells were E7 transgenic or not, but after exposure to IFN-c, substantially increased lysis of OVA-loaded KCs was observed (Fig 2B), whereas no such enhance-ment was seen for osmotically loaded E7-KCs, con-firming the findings with double-transgenic KCs that endogenous E7 inhibits processing of endogenous anti-gens by KCs for presentation to antigen-specific T cells (Fig 2B)
To further confirm this finding, we used an antibody against SIINFEKL–H-2Kb complexes to measure MHC-associated presentation of SIINFEKL, derived from osmotic loading of KCs with OVA For KCs, expression of SIINFEKL–H-2Kb complexes was sub-stantially upregulated in response to IFN-c, whereas for E7-KCs, exposure to IFN-c failed to upregu-late expression of SIINFEKL–H-2Kb complexes (Fig 2C–E) Thus, endogenously expressed E7 inhibits IFN-c-mediated enhancement of processing of endoge-nous antigen by KCs, without inhibiting IFN-c-mediated upregulation of MHC class I expression
HPV 16 E7 blocks the ability of IFN-c to upregulate the transcription of MHC class I antigen processing and presentation-associated genes in keratinocytes
As HPV 16 E7 expression in KCs impairs IFN-c-medi-ated processing of endogenous OVA for presentation
of SIINFEKL (Fig 2D), and HPV 16 E7 has been suggested to inhibit IFN-c signal transduction, we hypothesized that endogenously expressed E7 might inhibit transcription of IFN-c-dependent genes that are necessary for MHC class I antigen presentation
To investigate whether HPV 16 E7 could reduce the ability of IFN-c to upregulate transcription in KCs of genes relevant to the processing and presentation
of endogenous antigen, we assessed the transcription
of three MHC class I antigen processing and presenta-tion-associated genes (pa-28, tap-1 and irf-1) in KCs and E7-KCs both before and after exposure of the cells to IFN-c, using quantitative RT-PCR (Fig 3) Basal levels of expression of pa-28 and irf-1 were somewhat higher in E7-KCs than in KCs (Fig 3) However, the increase in level of expression induced
by exposure to IFN-c was significantly blunted in E7 KCs, as compared with KCs, for pa28, tap-1 and irf-1 (Fig 4) The maximal level of expression achieved
Trang 4after IFN-c exposure was also significantly diminished
in E7-KCs, as compared with KCs, for tap-1 and irf-1
(Fig 3)
Discussion
In this study, we show that expression of HPV 16 E7
as a transgene product in epithelial cells does not
directly impair, but rather slightly increases, MHC
class I expression E7 expression is nevertheless
associ-ated with impairment of IFN-c-induced enhancement
of presentation of endogenous antigen to CTLs For
E7-KCs, IFN-c treatment is less able to enhance the
transcription of genes regulating antigen presentation,
including tap-1, irf-1 and pa28 The reduction in gene
transcription is from 5- to 10-fold, which is sufficient
to reduce antigen presentation about five-fold and impair T-cell-mediated killing in vitro, and may there-fore be sufficient to explain the failure of E7-expressing skin to be rejected in vitro
Viruses use multiple strategies to make infected cells
of less interest to virus protein-specific immune effector responses Papillomavirus nonstructural viral proteins have been shown to interact with several cellular pro-cesses in a manner that could impair MHC class I expression HPV E5, when overexpressed as a trans-gene product, can trap MHC class I molecules in the Golgi [23] HPV 16 E7, when overexpressed, can repress the MHC class I heavy chain promoter, as well
as the promoters of tap1 and lmp2 [7], and can also
P < 0.05
C
E
D
Fig 2 HPV E7 impairs the enhancement by IFN-c of the presentation of endogenous antigen (A) OVA transgenic KCs (KC-OVA) and KCs expressing E7 and OVA (E7-KC-OVA) were compared for susceptibility to lysis by OVA-primed effector T cells, with or without IFN-c expo-sure (B) KCs and E7-KCs were loaded osmotically with OVA (OSM ⁄ OVA) or myoglobin (MYO), and, where indicated, exposed to IFN-c (100 UÆmL)1for 48 h) OVA-loaded KCs with or without an E7 transgene were compared for susceptibility to lysis with and without IFN-c pretreatment Nontransgenic KCs treated with IFN-c (KC + IFN-c) are shown as a control (C, D) Expression of SIINFEKL–MHC class I pep-tide complexes on KCs (C) and E7-KCs (D) osmotically loaded with OVA and exposed to IFN-c (100 UÆmL)1) for 48 h was assessed with spe-cific antibody by flow cytometry (E) Processing of OVA for presentation by MHC as SIINFEKL, according to the protocols for (C) and (D) OVA-loaded KCs exposed to IFN-c (KC + IFN + OVA) had significantly higher expression of SIINFEKL–MHC complexes than OVA-loaded E7 transgenic KCs exposed to IFN-c (E7-KC + IFN + OVA) (P = 0.02, t-test, n = 4) For the positive control (E7-KC + IFN + OVA + SIINFEKL), MHC complexes were saturated with SIINFEKL added to the culture medium For the negative controls, no IFN-c (KC + OVA; E7-KC + OVA)
or no OVA (E7-KC + IFN) was added Means and SD values for MFI are shown.
Trang 5bind to TAP1 and inhibit peptide transport [24], and
reduce the expression of MHC in cultured murine
fi-broblasts [25] However, these reported effects of E7
do not seem, from our current study, to impact on the
ability of KCs expressing E7 at levels more typical of
those seen in HPV infection to present exogenous
peptide, or on the level of MHC class I expression
Papillomavirus-associated cervical cancers express high
levels of E7, and demonstrate impaired membrane
expression of MHC class I complexes, which would be expected to impair antigen presentation However, in cancer cells, reduced MHC class I display is associated with low levels of TAP1 or TAP2 [26], as a result of gene mutations associated with transformation, and the contribution, if any, of overexpression of HPV 16 E7 to reduced MHC class I expression in these cells is unclear
We have recently shown that E7-specific CTLs that are well able to kill E7-expressing transplantable tumors fail to efficiently kill KCs expressing E7 as a transgene product at levels commensurate with those in infected cervical epithelium [5] As impaired recognition could be overcome by exposure to exogenous E7, it probably reflects either low availability of E7 for pro-cessing for presentation, or impaired antigen propro-cessing
in E7-expressing cells To distinguish these possibilities,
we studied the processing and presentation of OVA expressed as a transgene product in E7 transgenic and control KCs, using antibody against SIINFEKL–H-2Kb OVA presentation appeared to be normal in this system, as cells with or without E7 were equally suscep-tible to killing, although the fixed level of OVA expres-sion did not exclude the possibility that high-level OVA expression could overcome any restriction on process-ing We therefore tested cells loaded osmotically with OVA, where lesser levels of OVA loading were still
C
Fig 3 Transcription of genes associated
with antigen processing and presentation in
KCs and E7-KCs after IFN-c treatment.
mRNA was extracted from KCs and E7-KCs.
KCs and E7-KCs were treated or not, as
shown, with IFN-c (IFN) at 100 UÆmL)1for
48 h Expression levels of mRNA assessed
by RT-PCR with specific primers are shown
relative to a reference gene, rRNA adenine
dimethylase Transcription after IFN-c
expo-sure was higher for KCs than for E7-KCs for
irf-1 (P = 0.02, n = 7, t-test) and tap-1
(P < 0.01, n = 7) Differences for pa28 and
pias1 were nonsignificant by unpaired t-test
(n = 3) Error bars represent mean and SD
(n = 3).
Fig 4 Upregulation of gene expression in KCs and E7-KCs
follow-ing IFN-c exposure For each tested gene, the ratio of expression
level between cells exposed or not exposed to IFN-c is shown for
E7-KCs, and also for KCs Significant differences in magnitude of
the IFN-c-induced change in expression between KCs and E7-KCs
were seen for irf-1 (P = 0.01, n = 3, t-test), tap-1 (P = 0.05, n = 3),
and pa-28 (P = 0.05, n = 3) The change for pias1 was
nonsignifi-cant by unpaired t-test (n = 3) Error bars represent mean and SD.
Trang 6equally able to sensitize cells to OVA, whether
express-ing E7 or not However, in both the osmotic loadexpress-ing
model and the transgene model, induction by IFN-c of
increased expression of MHC–peptide complexes and
susceptibility to T-cell-mediated lysis was significantly
impaired if KCs expressed E7 as a transgene product
Looking for a mechanism, we assessed levels of
tran-scription of genes whose protein products participate in
antigen expression HPV 16 E7 attenuated the ability
of IFN-c to induce the transcription of several genes,
and also of IRF-1, a master regulator of IFN-inducible
genes IFN-c is a potent inducer of antigen processing
and of MHC class I expression for many cell types [14]
Signal transduction occurs via the JAK–STAT
path-way, and upregulation of the expression of the
down-stream genes relevant to antigen processing and
presentation is mediated by members of the IRF family
[27], particularly IRF-1[28] E7 blocked the ability of
IFN-c to efficiently induce irf-1 transcription to the
level observed in nontransgenic KCs Thus, E7 may
block the ability of IFN-c to induce IRF-1 expression,
in turn inhibiting the expression of downstream genes
related to MHC class I antigen processing and
presentation E7 could potentially also interfere with
IFN-c-mediated upregulation of IRF-1 expression by
inhibiting upstream transduction of IFN signaling
[16,17,29,30] Furthermore, HPV 16 E7 can also
inter-fere with IRF-1 function without affecting IRF-1
tran-scription and translation This occurs through
alteration of the DNA-binding capacity and promoter
transactivation of IRF-1 without alteration of IRF-1
level [20,21,31], probably by direct binding to E7 [20]
Differences between the effects of E7 on induction by
IFN-c of IRF-1 mRNA in our study and on the
steady-state levels of IRF-1 measured by others may
reflect the different experimental systems, or effects of
E7 on post-transcriptional regulation of IRF-1
produc-tion or destrucproduc-tion
HPV 16 is a member of the mucosotropic
a-papillo-mavirus clade Papilloa-papillo-maviruses from the genetically
and functionally distinct skin tropic b-clade use
alter-native means to impair antigen presentation HPV 38
E6 inhibits STAT1 expression and phosphorylation
induced by IFN-b and IFN-c in human KCs, and
inhibits IRF-1, TAP1 and MHC class I expression in
host cells [29] An impaired response of E7-KCs to
IFN-c has significant implications for immunotherapy
of HPV-associated skin lesions, which have proven
refractory to induced antigen-specific immunotherapy
[32,33] IFN-c, secreted by CD8 T cells, by activated
NK and NKT cells and by dendritic cells [34,35], is
a key intermediate mediator of CD8 T-effector cell
function, enhancing antigen presentation as well as
polarizing the immune response to the Th1 type Impaired antigen presentation may thus be one of the reasons why HPV infection is slow to clear in the face
of adequate cellular immunity, and why immunother-apy has proven ineffective for persisting HPV infec-tion It may also explain why local administration of supraphysiological concentrations of IFNs can contrib-ute to the clearance of HPV-associated genital warts [36] Administration of proinflammatory mediators that can enhance antigen presentation by an IFN-independent pathway, perhaps through toll-like recep-tor signaling [34], may therefore facilitate the immune clearance of HPV-associated disease
Experimental procedures
Immunogen, peptide and mice
An 8-mer peptide (SIINFEKL) corresponding to the major CTL epitope of OVA was synthesized by AusPep (Park-ville, Vic., Australia) to bind to MHC class I H-2Kb(amino acids 258–266 of OVA) C57BL⁄ 6J mice, C57BL ⁄ 6J mice expressing SIINFEKL [37] or HPV 16 E7 [38] from the keratin 14 promoter, and C57BL⁄ 6J mice expressing OVA from the keratin 5 promoter [4], were bred under conven-tional conditions in specific pathogen-free holding rooms in the Princess Alexandra Hospital biological resources facility (Brisbane, Qld, Australia) The protocols of these experi-ments were approved by the institutional Animal Ethics Committee
Generation of effector cells C57BL⁄ 6J female mice (6–8 weeks of ages) were immunized once with 100 lg of SIINFEKL⁄ 30 lg of keyhole limpet hemocyanin (Sigma Pharmaceuticals, Melbourne, Victoria, Australia) and 30 lg of QuilA (Spikoside; ISCOTEC AB, Lulea, Sweden) Lymph node cells were collected from immunized mice 4 days after immunization Lymphocytes were cultured in filtered Click’s medium [50% Eagle’s⁄ Ham’s amino acids (Sigma), 50% RPMI-1640 (Gibco; Invitrogen, Carlsbad, CA, USA), 10% heat-inactivated fetal bovine serum, containing 1 ngÆmL)1 mouse inter-leukin-2 (Pharmingen, San Diego, CA, USA) and 0.05 lm SIINFEKL, 2· 105
UÆmL)1 penicillin⁄ 2 · 105
UÆmL)1 streptomycin, 200 mm l-glutamine and 5· 10)5m 2-mercap-toethanol (2-ME)] for 4 days
Generation of target cells Isolation and culture of KCs from mouse skin has been described previously [5] In brief, KCs were cultured in epidermal cell culture 3 : 1 medium [for 500 mL of 3 : 1
Trang 7l-glutamine⁄ penicillin ⁄ streptomycin, 200 mm ⁄ 2 · 105
U⁄ 2
· 105 UÆmL)1, 125 mL of Ham’s F12, 50 mL of fetal
bovine serum, 500 lL of transferrin (Sigma), 5 mgÆmL)1 in
0.1% BSA in NaCl⁄ Pi, 500 lL of insulin (Sigma),
5 mgÆmL)1 in 1 mm HCl, 500 lL of cholera toxin (Sigma),
8.4 mgÆmL)1 in NaCl⁄ Pi, 100 lL of hydrocortisone
(Sigma), 1.2 mgÆmL)1in 90% ethanol in water, 1000 lL of
184 mm adenine (Sigma), 17 mgÆmL)1 in 0.1% BSA in
NaCl⁄ Pi, 500 lL of gentamicin, 20 mgÆmL)1, 500 lL of
2-ME stock (35 lL of 2-ME in 10 mL of RPMI-1640
med-ium for 2-ME stock)] for 48 h, and then transferred into
serum-free KC medium (Gibco) for 2 days KCs were
seeded in 96-well plates at 2· 104cells per well for CTL
assays, or suspended at 5· 105cells per tube for FACS
experiments
Osmotic loading of KCs
The techniques have been described previously [39] In
brief, 5· 106
KCs were suspended in 1 mL of RPMI-1640
osmotic loading buffer [25 mm Hepes, 0.5 m sucrose (w⁄ v),
10% polyethylene glycol, pH 7.2] containing 10 mgÆmL)1
OVA (Sigma) or myoglobin (Sigma), and incubated at
37C for 10 min The cells were then diluted into 14 mL of
a mixture of 60% RPMI-1640 medium and 40% water,
and held at 37C for 2 min The loaded KCs, pelleted at
300 g for 7 min, were resuspended in RPMI-1640 medium
and pelleted at 300 g for 5 min Finally, the cells were
resuspended in culture medium and incubated with or
with-out IFN-c at 100 UÆmL)1for 24 h [40]
CTL assay (51Cr release)
A standard 5-h 51Cr release assay was conducted as
described previously [5] CTL assay data were expressed as
percentage specific lysis according to the following formula:
The data were analyzed by t-test, and results were
regarded as significantly different when P < 0.05
Flow cytometry
KCs (5· 105) were incubated with the first antibody (mouse
anti-SIINFEKL–H-2Kb clone D25.1.1.16 [41], 50 lL per
sample, provided by D Purcell, University of Melbourne),
mouse anti-H-2Kb(clone AF6-88.5; Pharmingen), or mouse
anti-(rat IgM) (Pharmingen), an isotype control, for 1 h at
4C Cells were then washed twice and incubated with the
secondary antibody [fluorescein isothiocyanate-conjugated
rabbit anti-mouse IgG (Dako Cytomation, Copenhagen,
Denmark)] for 1 h at 4C, washed, and fixed with 5% formaldehyde Data collected on a FACSCalibur (Becton Dickinson, San Diego, CA, USA) were analyzed with winmdi2.8 (Joseph Trotter, Scripps Research Institute, La Jolla, CA, USA) Viable KCs were selected preferentially by excluding small particles The change in mean fluorescence intensity (MFI) was calculated as the difference in MFI between test and isotype control samples
Real-time PCR mRNA was extracted from KCs and converted to cDNA with the use of random primers and PowerScript RT (Gene-Works, Hindmarsh, SA, Australia), according to the manu-facturer’s protocol cDNA samples dissolved in the PCR mix buffer (FastStart SYBR Green Master; Roche Applied Science, Mannheim, Germany) were used to conduct quanti-tative PCR under the following conditions: 50C for 2 min;
95C for 10 min; and 40 cycles of 94 C for 1 min, 55 C for
1 min and 72C for 1 min The following primers were used: TAP1, forward, 5¢-ACC TGG CTA CGG TAC ACC TG-3¢; TAP1, reverse, 5¢-CCT CTG AGC TCC CAC TTG AC-3¢; IRF-1, forward, 5¢-CCT GGG TCA GGA CTT
G-GA TA-3¢; IRF-1, reverse, 5¢-TTC GGC TAT CTT CCC
T-TC CT-3¢; PA28, forward, 5¢-CCG CT-TC CT-TC CTT CT-TC
ATG TGT TC-3¢; JAK1, forward, 5¢-TCA ACC TTC CCA AAG TGA CC-3¢; JAK1, reverse, 5¢-CAT GAC TCG CTG CAT GAA CT3¢; PIAS1, forward, 5¢AAG TGC TCA -CAG CCT TGG AT-3¢; PIAS1, reverse, 5¢-TCC CTA GGT GCA TGT TCT CC-3¢; rRNA adenine dimethylase, for-ward, 5¢-GGA GGG CCC ATC AGT TTA AT-3¢; rRNA adenine dimethylase, reverse, 5¢-AAA CAA TTG CAT TGC ATA GTGC-3¢ The data were analyzed with rotor-gene6000
Statistical analysis All experimental data, including DMFI of FACS data, were analyzed with unpaired t-tests Error bars represent mean and standard deviation (SD) Results were regarded as showing significant differences if P-values were < 0.05
Acknowledgements
The authors are grateful to the staff of the biological research facility at the Princess Alexandra Hospital for their assistance This work was funded by program grant No 352439 from the National Health and
%specific lysis ¼ mean sample release (c.p.m.) mean spontaneous release
mean maximum release (c.p.m.) mean spontaneous release (c.p.m.) 100
Trang 8Medical Research Council of Australia, and grants
from the Lions Medical Research Foundation, the
Australian Cancer Research Foundation, the Cancer
Council Queensland, and the Princess Alexandra
hos-pital Foundation Scholarship funding to F Zhou was
from the Cancer Collaborative Group, Princess
Alex-andra Hospital and from ANZ Trustees I Frazer was
recipient of a Queensland Government Premier’s
fel-lowship The authors declare that they have no conflict
of interest or financial interests regarding the research
findings described in this article
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