imwnity, vol. 3,207-214, august, 1995, copyright 0 1995 by ... · strategies of immune evasion from...

ImWnity, Vol. 3,207-214, August, 1995, Copyright 0 1995 by Cell Press

Up-Regulation of MHC Class I by Flavivirus-Induced Peptide Translocation into the Endoplasmic Reticulum

Arno Ygllbacher and Mario Lobigs Division of Cell Biology John Curtin School of Medical Research Australian National University Canberra, ACT 2601 Australia

Summary

Flavivirus infection of mammalian cells increases the ceil SUrfaCe expression of major histocompatibility complex (MHC) class I molecules, the recognition elements for cytotoxic T cells. Here, we show that the mechanism for flavivirudnduced upregulation of class I MHC involves an increase in peptide supply to the endoplasmic reticulum. Flavivirus-mediated peptide supply for MHC class I assembly is independent of the peptlde transporters for class I antigen presentation, since infection of class I YHC peptide transport-deficient cell lines with flaviviruses results in the cell surface expression of biologically functional class I MHC peptlde complexes. The flavivirus-induced supply of antigenic peptides to the endoplasmic reticulum is not restricted to flavlvirus-encoded peptldes and independent of interferon. The data imply that peptide availability regulates surface expression of class I MHC restriction elements and suggests a mechanism for flavivirus-induced immunopathology.

Introduction

The cytotoxic T (Tc) cell response is the primary host immune defense against most viral infections. Virus-immune Tc cells recognize and lyse cells that express on the plasma membrane major hiitocompatibility complex (MHC) class I molecules presenting virus-derived peptide determinants. Strategies of immune evasion from the Tc cell response have been adopted by several virus families, which includes the down-regulation of MHC class I expression (Gooding, 1992; Maudsley and Pound, 1991). In contrast, a virus-host interaction resulting in up-regulation of MHC class I surface expression is an intriguing phenome- non that has been documented for flaviviruses (Hill et al., 1993; King et al., 1989; Liu et al., 1989) and some other virus families (Massa et al., 1987; Suzumura et al., 1986). Flavivirus-mediated up-regulation of MHC class I expression has been shown not to be mediated by interferon(s) (King et al., 1989; Liu et al., 1989).

Flaviviruses are a family of enveloped arthropod-borne positive-strand RNA viruses, which include some of the medically most important viral pathogens (Hill et al., 1993; Monath, 1990). Three members of the virus family, yellow fever virus, dengue virus, and Japanese encephalitis virus, are of global public health concern. In humans, the disease pattern associated with flavivirus infection ranges from a nondescript febrile illness to encephalitis and hemorrhagic fever. lmmunopathological effects may contrib-

Ute to the severity of some flaviviral disease and in these conditions the upregulation of MHC class I surface expression could be a contributing factor.

MHC class I antigens assemble in theendoplasmic retic- UlUtn (ER) with 82-microglobulin &m) and peptides 8-10 aa in length that are predominantly of cytoplasmic origin (Jackson and Peterson, 1993; Rammensee et al., 1993). Two recently identified gene products encoded by the MHC class II region, the proteasome components, LMPP and LMP7, and the peptide transporters, TAP1 and TAPP, appear to be important in the generation of peptide anti- gensand their import into the lumen of the ER, respectively (Monaco, 1992). Their involvement as accessory molecules in the class I antigen processing and presentation pathway was first demonstrated in complementation experiments with antigen presentation-defective cell lines (Attayaetal., 1992; Boeset al., 1994; Driscolletal., 1993; Gaczynska et al., 1993; Powis et al., 1991; Spies et al., 1992; Spies and DeMars, 1991) and later in mice with targeted TAP7 gene deletions (van Kaer et al., 1992). In the light of these new insights into the class I MHC antigen processing and presentation pathway, we examined the mechanism of flavivirus-induced increase of class I MHC surface expression.

Results

Flavivirus-Induced Up-Regulation of MHC Class I Surface Expression in TAP-Deficient Cells The mouse lymphoma cell line RMA-S has a greatly reduced MHC class I surface phenotype and a reduced ability to present viral antigens to CD8+ Tc cells (Ljunggren et al., 1990; Townsend et al., 1989). We tested whether flavivirus infection could also up-regulate the MHC class I surface expression in this TAP-deficient cell line and observed that following infection of RMA-S cells for 24 hr with West Nile virus (WNV) the surface expression of both H-2@ and H-2Db was augmented 2- to 4-fold (Figures 1 D, 1 E). The percentage increase in class I MHC expression was only slightly higher to that seen in RMA cells (which have the wild-type TAP phenotype) infected with WNV (Figures 1 A, 1 B). This may in part be due to the fact that class I MHC molecules in RMA-S cells were, prior to WNV infection, empty and thus the overall effect may be far greater on RMA-S cells than RMA cells. Using RMA cells stably transfected with the class I MHC molecule Kd, a shift of one log occured in the expression of Kd upon flavivirus infection (Figure 1C). Since the expression of this class I molecule is not controlled in response to interferon, this demonstrates that the effect of flavivirus infection on MHC class I surface expression is not a direct response to interferon. On the other hand, two cell surface molecules expressed on RMA-S cells, Thyl.2 and CD2, did not show increased cell surface expression after an equivalent time after WNV infection (Figures 1F and lG, respectively). Thus, flavivirus infection affects the expression of Cell surface molecules differentially.

Figure 1. Flavivirus-Induced Up-Regulation of MHC Class I Cell Sur- face Expression in RMA and the TAP-Defective Cell Line RMA-S RMA (A, B), RMA transfected with H-2Kd, and RMA-S cells (D. E, F, G) were infected with WNV for 24 hr, or left uninfected, and the cell surface expression of H-2Kb (A, D). H-2Kd (C), H-2Db (6, E), Thyl.2 (F), and CD2 (G) was determined by flow cytometry. Unbroken lines show background staining of uninfected cells, broken and dotted lines show the MHC class I-, Thyl.2-, and CD2epecific staining of uninfected and WNV-infected cells, respectively.

This increase in MHC class I expression could also be induced by infection with other encephalitic flaviviruses, Kunjin (KUN) and Murray Valley encephalitis virus (MVE), as well as dengue and yellow fever virus, but not by an- other enveloped positive-strand RNAvirus, the alphavirus, Semliki Forest virus (data not shown). Since the assembly

of MHC class I glycoproteins with peptide antigens iS cru-

cial for the export of most class I molecules from the ER (Lie et al., 1990; Ljunggren et al., 1990; Townsend et al., 1989), these results support the notion that peptide supply to the ER regulates MHC class I surface expression and suggests that flavivirus infection increases the availability of peptide determinants in the lumen of the ER.

Flavlvirus Infection Enhances Tc Cell Lysls of TAPP-Deficient RMA-S Cells To evaluate the biological significance of flavivirus-induced class I MHC up-regulation and to investigate the spectrum of peptides that become available in the lumen of the ER for assembly with class I MHC, we used Tc cell assays of RMA and RMA-S target cells infected for 24 hr and 36 hr with WNV. WNV-infected target cells were 3-fold more sensitive to H-2Kb and H-2Db alloreactive Tc cells than uninfected controls (Figures 28, 2D), and unlike mouse embryo fibroblasts (King and Kesson, 1986), maximal increases of class I MHC expression occured 24 hr after WNV infection, rather than 36-46 hr. Using RMA target cells infected with WNV, no increase in susceptibility to alloreactive Tc cells was observed over a 1 OO-fold effector to target cell ratio (Figures 2A, 2C), probably owing to the already high levels of class I MHC expression. The spontaneous %r release of both mock as well as WNV- infected RMA and RMA-S target cells ranged from 40% 6%. This, plus the fact that RMA targets did not become more susceptible to lysis by alloreactive Tc cells, excludes a WNV-induced general leakiness or increase in cell size. (We have followed RMA and RMA-S cells for over 72 hr after WNV infection and found no increase in cell death or diminished cell division capability nor an increase in cell size by changes in forward light scatter.) Interestingly, the susceptibility of RMA-S cells to lysis by VV-immune and influenza virus-immune Tc cells was also increased when the cells were infected with aflavivirus prior to super-

Figure 2. Lysis of RMA and RMA-S Target Cells by Alloreactive Tc Cells after WNV In- fection

- Mock - WNV124h - WNV136h

Lysis of RMA (A, C) and RMA-S (6, D) cells by BlOA(2R) antiC57BU6, Kb alloreactive (A, B) and B10.A(5R)anti-C57BU6Dballoreactfve(C, D) Tc cells. The target cells were mock-infected (closed square), infected with 20 pfu/cell WNV for 24 hr (open square) or 36 hr (open circle), and labeled with ‘Cr. Assay time was 4 hr. - f 2jff@y ._______, _..- ::k-

10 100 .l 1 10 100 Each point constitutes the mean of J .l 1

percent-

E specific lysis of three separate wells. SEM was

g 100 always (3%. Spontaneous release ranged from

50 4%-6%x % t 80 40 - Mock -

WNV124h - WN”136h

60 30

40 20

20 10

0 0 .l 1 10 100 .l 1 10 100

wr

Flavivirus-Mediated MHC Class I Peptide Transport 209

10 100 FLUORESCENCE INTENSITY

Figure 3. Lysis of RMA-S Target Cells by Wand Influenza Virus Im- mune Tc Cells after WNV Infection Lysis of RMA-S cells by C57BU6 W-immune (A) and C57BU6 influenza virus immune (Et) Tc cells. The target cells were infected with 20 pfulcell WNV (closed symbols) or mock-infected (open symbols); 24 hr later the targets were left uninfected or infected with influenza virus AIWSN (IO3 HAUlP x IO cells) (open triangle, closed triangle) or W (10 pfu/cell) (open circle, closed circle) and labeled with sCr. Assay time was 6 hr. Each point constitutes the mean of percent- specific lysis of three separate wells. SEM was always (3%.

infection with VV or influenza virus (Figures 3A, 36, respectively). Infection of RMA-S cells with VV or influenza virus alone also gave low but significant virus-specific lysis as observed by others (Esquivel et al., 1992), and is consis- tent with the partial nature of the antigen presentation defect in this cell line.

Flavivirus Infection Augments Mouse MHC Class I Expression in TAP-Deficient Hamster Cell Lines We have recently shown that the Syrian hamster cell lines, BHK and NIL-2, are defective in the peptide supply for presentation by several transiently expressed mouse MHC class I restriction elements (Lobigs et al., 1995; Lobigs and Miillbacher, 1995). The H-2Kk molecule is internally sequestered in BHK cells and the transport defect can be repaired by xenogeneic TAP1 and TAPP. In Figure 4A, we show that flavivirus infection also induces surface expression of recombinant H-2Kk in BHK cells. The effect of flavivirus infection in this H-2Kk antigen presentation- defective cell line again seems to compensate for the lack of functional peptide transporters. In contrast, the cell surface expression of VV-encoded influenza virus haemag- glutinin was reduced in WNV-infected BHKcellscompared with mock-infected cells, as detected by fluorescence- activated cell sorter (FACS) analysis (Figure 48). Thus, there is no general increase of expression of VVsncoded recombinant cell surface molecules after flavivirus infection.

We could also demonstrate the profound effect of flavivirus infection on the class I MHC antigen presentation pathway using a biochemical assay. The H-2Kd restriction element, when expressed in BHK cells via VV-Kd, can be detected at the plasma membrane by flow cytometry and alloreactive Tc cell recognition but fails to present a range of viral peptide determinants required for virus-immune Tc cell recognition (Lobigs et al., 1995). This suggests that in contrast with the H-2Kk glycoprotein, a small fraction of “empty” H-2Kd is expressed at the cell surface of BHK cells,

Figure 4. Flavivirus-Induced Up-Regulation of MHC Class I on BHK Cells BHK cells were infected with 20 pfu per cell WNV (solid lines) or mock infected (broken line). After 24 hr. cells were superinfected with either 10 pfu W-KL (A) or 10 pfu W-HA (B). Cells were FITC stained after incubation with primary (bold and broken lines) or absence (solid line) antibody. MAb HE-159 was used for H-2KQpecific staining and rabbit anti-hemagglutinin hyperimmune serum for HA.

a situation similar to the MHC class I surface phenotype in RMA-S cells and other cell lines deficient in peptide supply to the ER (Cerundolo et al., 1990; Hosken and Bevan, 1990; Ljunggren et al., 1990). To investigate the effect of flavivirus infection on the intracellular location of recombinant H-2Kd in BHK cells, its resistance to endoglycosidase H (endo H) digestion was examined. Acquisition of resistance to endo H digestion reflects the transit of secreted and membrane-anchored proteins through the cis-Golgi compartment, where oligosaccharide moieties are modi- fied to complex endo H-resistant forms(Kornfeld and Korn- feld, 1985). Figure 5 shows that when BHK cells were infected for 24 hr with KUN prior to superinfection with VV-Kd, at multiplicities of infection of 2 or 20 pfu per cell, a large proportion of metabolically labeled H-2Kd molecules were chased to an endo H-resistant form. This was in contrast with only a minor proportion of pulse-labeled H-2Kd glycoproteins that gained resistance to endo H digestion in the absence of KUN infection and shows that flavivirus infection induces efficient export of the mouse MHC class I molecule from the ER. A similar experiment was performed with RMA-S cells. However, owing to much greater leakiness in ER sequestration of class I MHC molecules in RMA-S cells compared with BHK cells, a dominant proportion of pulse-labeled H-2Db and H-2Kb molecules in RMA-S cells became endo H-resistant, such that a flavivirus-induced increase in surface transport was not apparent using the biochemical assay (data not shown).

Flavivirus-Induced and TAP-Independent Augmentation of Cell Surface Peptide Antigen Presentation To verify that the augmentation in surface expression of MHC class I induced by flavivirus infection correlated with an increase of peptide antigen presentation, we acid- eluted radiolabeled peptides from the surface of an equal number of intact metabolically labeled KUN-infected or uninfected RMA and RMA-S cells. The acid extracts were fractionated by reverse-phase high pressure liquid chro- matography (HPLC) and the radioactivity in individual frac-

Immunity 210

Chase E

VV-Kd (mob2) VV-Kd (nxoi=20) Kunjin Kunjin

(min) 5 120 5 120 5 120 5 120 -- ndoH. +y -+-+-+-+-+-+

tions, which reflects the relative peptide content, counted. Figure6shows that in KUN-infected RMA-S cells the radioactivity eluted in the gradient between 10% and 35% acetonitrile was greatly increased, compared with that eluted from uninfected cells. Flavivirus infection of RMA-S cells resulted in a significantly larger increase of surface- expressed peptides, sensitive to acid elution, than in RMA cells where the increase was only marginal (Figure 6). This difference was not apparent in the increase of cell surface- expressed class I restriction elements observed by flow cytometry (see Figure 1) and may be a reflection of a significant proportion of empty class I molecules on RMA-S cells detected by flow cytometry.

Cell surface acid elution from intact cells is a rapid and mild procedure for the isolation of peptide determinants presented by cell surface MHC (Franksson et al., 1993; Langlade-Demoyen et al., 1994; Storkus et al., 1993). We applied this approach rather than the traditional procedure of immunopurifying MHC molecules following detergent lysis of cells to avoid the problem of possible association of empty MHC with cytosolic peptides after detergent lysis (Elvin et al., 1991; Townsend et al., 1969).

Flavivirus-and VV Specific Tc Cell Lysis of TAP-Defective Hamster Cells The nonspecific WNV-mediated peptide supply for class I MHC assembly and transport could be demonstrated more elegantly in the antigen presentation-deficient Syr- ian hamster cell lines BHK and NIL-2. Despite the failure of both cell lines to present a number of endogenous virus- derived Tc cell determinants restricted by H-2Kd and H-2Kk (including those from W and influenza virus; Lobigs et al., 1995; Lobigs and Miillbacher, 1995) BALBlc WNV- immune Tc cells efficiently recognized NIL-2 cells when infected with WNV for 24 hr and superinfected with W- Kd to provide the mouse restriction element (Figure 7A). Importantly, recognition of NIL-2 cells by BALBlc VV- immune Tc cells was induced by infection of the target cells with the flavivirus prior to infection with W-Kd but not seen when cells were infected with W-Kd alone. The flavivirus-mediated VV peptide transport was more pro- nounced than complementation achieved with superinfection with VV encoding the rat peptide transporters TAP1

-69

-46

-30

-18

Figure 5. Acquisition of Endo H Resistance of Recombinant H-2Kd in SHK Cells Following Flavi Virus Infection

Mock-infected (MI) and KUN-infected SHK cells were superinfected with W-Kd at a multiplicity of infection of 2 or 20 pfu per cell, metabolically labeled for 1 hr. and then label chased. H-2Kd glycoproteins were immunoprecipitated, treated with endo H (plus) or mock-treated (mi- nus), and analyzed by SDS-PAGE. Molecular weight standards were coelectrophoresed, with sizes indicated on the right. The endo H-sensitive form of the Kd glycoprotein has a slightly faster electrophoretic mobility than the endo H-resistant form, and the band core sponding to the endo H-sensitive Kd molecule shifts to a lower electrophorettc mobility (- 39 kDa) folowing endo H digestion.

and TAP2 (Figure 78). Similar data were obtained using W encoding H-2@ and BHK target cells (data not shown). In addition, NIL-2 and BHK cells also became more sensitive to lysis by the appropriate mouse alloreactive Tc cells when infected with a flavivirus prior to infection with W- Kd or W-Kk (data not shown). The induction of TAP- independent supply of a variety of peptides for assembly of class I MHC molecules of different haplotypes was also seen following infection with other encephalitic flaviviruses. BHK cells infected with WNV, KUN, and MVE and superinfected with W-Kd were lysed by BALBlc W- immune Tc cells but not when infected by W-Kd alone (Figure 6). Preliminary studies indicate that this effect holds true for all members of the Flaviviridae, including yellow fever virus and dengue virus.

The effect of WNV on peptide transport and class I MHC

0 20 40 60 80 loo

Fraction Number

Figure 6. Acid Extraction of Peptides from the Cell Surface of Flavi- virus or Uninfected RMA and RMA-S Cells

Uninfected (line a) or KUN-infected (line b) RMA and RMA-S cells were metabolically labeled and cell surface acid extracts separated by reverse-phase HPLC. Solid lines, radioactivity in reverse-phase HPLC fractions; dotted line, acetonitrile gradient.

Flavivirus-Mediated MHC Class I Peptide Transport 211

A

--B- MOCK -Cl- W-Kd --.- WNV -0- WNV/VV-Kd

1 10 1 10

E/T E/T

up-regulation becomes first apparent 6 hr postinfection and is maximal 16-26 hr after infection. Longer infection (- 40 hr) shows extensive decline in specific target cell lysability by Tc cells, probably owing to the flavivirus- induced cytopathic effects in these Syrian hamster cell lines (data not shown).

Flavivirus-Mediated MHC Class I Up-Regulation Is Independent of Interferon To eliminate a further possible interferon effect on flavivirus-induced up-regulation of MHC class I, we tested whether the interferon inducer polycytidylic-inosinic acid (poly[lC]) or supernatents of BHK cells infected with WNV or treated with poly(lC) for 24 hr could induce Kk expression after infection with VV-Kk. Figure 9 shows one such experiment. The only treatment that can facilitate KL expression is infection with WNV or transfection with TAP1 and TAP2 (see Figure 78; Lobigs and Miillbacher, 1995). Neither poly(lC) treatment nor conditioned supernatants such as from WNV-infected cell cultures was able to facilitate Kk cell surface expression as indicated by Kk-restricted W- immune Tc cells lysis. Thus, a role for interferon in this class I MHC up-regulation can be ruled out.

40 1

UY !F 30 I Pp P -I I / / 0 G 20 z

!z

10 x

0 E

c-,o ___-- ---0’‘-- --- 0

_A- --em

1 10 100

E/T Figure 6. Flavivirus-Mediated Peptide Transport in BHK Target Cells

Lysis of BHK target cells by BALBlc W-immune Tc cells. BHK ceils were infected for 24 hr with 20 pfu/cell of either WNV (open square), MVE (closed circle), KUN (open circle), or left uninfected (closed square). Target cells were mock infected (broken line) or superinfected (solid line) with 20 pfu W-K” for 3 hr. Assay as for Figure 5.

Figure 7. WNV-Induced Peptide Transport in TAP-Deficient Hamster Cell Line NIL-2

Lysis of Nil-2 target cells by BALB/c WNV- immune and BALE/c W-immune Tccells. Nil-2 cells were infected with 20 pfu WNV/cell or mock infected. After 24 hr, WNV-infected cells were supsrinfected with 10 pfu W-Kd and mock-infected cells infected wtth 10 pfu W-Kd, or with 10 pfu W-Kd, 10 pfu W-mtpl plus 10 pfu W-mtp2. The assay time was 6 hr. Each point constitutes the mean percent-specific lysis of three separate wells. SEM was always >3%.

Discussion

Presentation of endogenously derived peptides via class I MHC in TAP-deficient cells has been demonstrated by others (Esquivel et al., 1992; Hosken and Bevan, 1992; Zhou et al., 1993a, 1993b; Zweerink et al., 1993) but differs fundamentally from our data in that the antigen presentation was peptide specific, in contrast with the apparently general effect on peptide supply to the ER induced by flavivirus infection. Sendai virus Tc cell determinants were presented by class I MHC molecules on the surface of RMA-S and T2 cells, but appeared to enter the antigen presentation pathway by a TAP- and Brefeldin A-independent mechanism, and no effect on the presentation of peptides not encoded by Sendai virus was noted (Zhou et al., 1993a, 1993b). Brefeldin A inhibited MHC class I surface expression in flavivirus-infected cells, suggesting that the effect of flavivirus infection on MHC class I expression was via the normal antigen presentation pathway (data not shown). The vesicular stomatitis virus H-2Kb-restricted immunodominant Tc cell determinant was also presented on the surface of RMA-S cells infected with the virus or transfected with the vesicular stomatitis virus nucleopro-

I NIL WNV poty-IC WNVlSUP poly-ICISUP

TREATMENT OF BHK TARGET CELLS

Figure 9. Lysis of BHK Cells after Treatment with Interferon Inducers

Lysis of BHK target cells by CBA VV-immune Tc cells. Values given are from a 4fold titration curve resolved by logarithmic regression analysis at an effector to target cell ratio of 1O:l. Prior to assay (24 hr), the target cells were either infected with 20 pfu WNV, treated with 25 uglml poly(lC), or cultured in culture supernatent of 24 hr WNV-infected or poly(lC)-treated BHK cells. Assay time was 6 hr.

Immunity 212

tein gene (Esquivel et al., 1992; Hosken and Bevan, 1992). However, contrary to our results with flavivirus-infected RMA-S cells, no increase in surface expression of the MHC class I glycoproteins was found, and vesicular stomatitis virus infection did not augment the MHC class l-restricted presentation of other peptide antigens. These data and those of others (Zweerink et al., 1993) may suggest that high concentrations of cytoplasmic peptide ligands for class I restriction elements can enter the class I antigen presentation pathway by a TAP-independent route.

We were unable to identify significant sequence homolo- gies between the genomes of flaviviruses and member of the ATP binding cassette family of transporter protein genes (Higgins, 1992). It therefore appears unlikely that flaviviruses encode a protein that functions specifically in the transport of peptides across the ER membrane. We were also unable to induce TAP-independent peptide sup ply in RMA-S and antigen processing-deficient Syrian hamster cells by transient expression of large fragments of MVE (Lobigs et al., 1994) or KUN (Hill et al., 1993) polyproteins, which, in combination, span the entire translation products. No increase in class I MHC expression could be observed in any cell line tested (either with TAP defects or low MHC class I expression), even after pro- longed infection time (36 hr) in the presence of arabinose C to prevent W-mediated cell pathology. This indicates that a stage in flavivirus replication following translation of the flaviviral gene products is required for this effect. Flavivirus assembly takes place in close association with the ER membranes (Westaway, 1967). The virus-encoded polyprotein traverses the membrane numerous times, and virus maturation takes place by budding into the lumen of the ER, by which the acquisition by the virus of a host cell-derived lipid envelope is achieved. Flavivirus-mediated peptide supply to the lumen of the ER demonstrated here could thus be a consequence of virus-inflicted pertur- bation of ER membrane integrity or the proliferation of ER membranes that is noted in flavivirus-infected cells. Flavivirus infection of vertebrate cells does not induce significant cytopathic effects until late after infection. Virus replication involves a long latent period and only margin- ally reduces host macromolecular synthesis. The functions of the ER related to protein folding, assembly, and intracellular targeting are not affected to a significant de- gree by flavivirus infection (Hill et al., 1993). It is during this early period of flavivirus infection that we observe the TAP-independent supply of peptides to the ER. It is inter- esting to note that some members of the Coronaviridae, which also assemble by budding through the membranes of the ER (Holmes, 1990) are reported to up-regulate MHC class I cell surface expression (Suzumura et al., 1966).

A biological consequence of a flavivirus-induced augmented MHC class I surface phenotype could be the breakdown of self-tolerance. Unapparent flavivirus infections could prime a host for later autoimmune disorders by a quantitative increase in presentation of self-Tc cell determinant previously below the threshold for Tc cell induction (Blanden et al., 1987) a mechanism distinct from “molecular mimicry” by viral and bacterial antigens of host T cell determinants thought to be involved in the break-

down of tolerance. We have observed that at least in vitro a large fraction of murine secondary flavivirus-immune Tc cells are self-reactive. The immunopathology seen in dengue hemorrhagic fever/shock syndrome and flaviviral encephalitis may also relate to increased MHC surface expression.

Experimental Procedures

Animals BALB/c (KdDd), CBA/H (ICDL), C57BU6 (KbDb), BlO.A(PR) (KLDb), BlOA(5Ft) (KbDd), and C3H.H~2” (KdD’) were bred under pathogen-free conditions at the John Curtin School of Medical Research breeding facilities. Only female animals were used at 3 12 weeks of age.

Cell Lines The mouse tumor cell lines RMA, its TAP2 gene mutant derivative (Townsend et al., 1969) RMA-S. the H-2Kd-transfected RMA cell line (provided by K. Flynn, Canberra, Australia), and the Syrian hamster cell lines BHK and NIL-2 (American Type Culture Collection [ATCC], Bethesda, Maryland) were grown in Eagle’s minimal essential medium (EMEM) supplemented with 10% fetal calf serum (FCS).

Viruses and lmmunfxstlon The vaccinia viruses WR strain (VV-WR), the thymidine kinase deletion mutant (W-f/r) and the W-recombinant viruses encoding the mouse MHC class I heavy chain Kd (VV-Kd) (provided by Drs. J. Ben- nink and J. Yewdell, National Institutes of Health, Bethesda, Mary- land), KL (W-w) (a gift of Dr. B. Arnold, Deutsches Krebsforschungs- zentrum, Heidelberg, Federal Republic of Germany), and the W recombinants encoding the rat class I MHC peptide transporter TAPI’ (VV-mtpl) and TAP2* (VV-mtp2) (the genes for the rat transporters were provided by Drs. J. C. Howard and 0. W. Butcher, Cambridge, England), the W recombinants encoding the hemagglutinin gene of influenza virus (W-HA) (provided by Dr. D. Boyle. Geelong, Australia), were grown and titrated on CV-1 cell monolayers. Influenza virus A/WSN was prepared and titrated as has been described (Miillbacher et al., 1991). The flaviviruses Kunjin virus (KUN). West Nile virus (WNV), and Murray Valley encephalitis virus (MVE) were grown and titrated as described (Kesson et al., 1966).

Animalswereimmunized with IO’pfuofW-WR, lo’hemagglutinat- ing units of AWSN, or 20 pfu of WNV, intraperitoneally.

Ertdo H Assay BHK cells (5 x Iv cells) were infected with KUN at a multiplicity of 10 pfu per cell for 1 hr at 37OC. Infected or mock-infected cells were left at 37OC for 24 hr prior to superinfection with W-Kd at a multiplicity of 2 or 20 pfu per cell for 1 hr. The W-infected cells were incubated for a further 4 hr at 37OC, starved for 30 min in minimal essential medium without methionine (GIBCO), labeled for 1 hr by incubation in 2 ml of methioninefree medium containing [“Sjmethionine (100 uCi/ ml; 1160 Cilmmol; ICN), and the label chased for the times indicated by replacing the labeling medium with EMEM containing 5% FCS. lmmunoprecipitation with the H-2KQeactive monoclonat antibody (MAb) Hb159 (ATCC designation), endo H digestion, electrophoresis, and fluorography were as described (Lobigs et al., 1990).

Acid Elutlon snd HPLC Sepsrstlon of Radlolsbeled Peptldes from Intact Cells RMA and RMA-S cells (1 x 10’ cells) were infected with KUN at a multiplicity of 20 pfu per cell, or left uninfected. Following incubation at 37OC for 12 hr. cells were pelleted and suspended in 5 ml EMEM containing 10% FCS and 100 uCi/ml of a PHI-amino acid mixture (1 mCi = 37 MBq; Amersham). The cells were metabolically labeled for 12 hr, washed twice in phosphate-buffered saline, and suspended in 3 ml acid elution buffer (500 mM NaCI, 200 mM acetic acid [pH 2.4j) for 2.5 min at 20°C to extract MHC class l-presented peptides from the cell surface. Acid-treated cells were pelleted by centrifugation and the low molecular weight fraction of the acid extract recovered by centricon- centrifugation. The material was dried by vacuum centrifugation, dissolved in 0.3 ml of 0.1% trifluoroacetic acid in water, and analyzed by HPLC on a reverse-phase column (Beckman; 150 x 4.6

FlavivirusMediated MHC Class I Peptide Transport 213

mm). The 5%-40% acetonitrile gradient was made from solution A (H?o with 0.1% trifluoroacetic acid), and solution B (acetonltrlle with 0.1% trifluoracetic acid). Flowrate. 1 mllmin; 0.5 ml fractions collected. Radioactivity in each fraction was counted by liquid scintillation counting.

Target Cells and %I Release Cytotoxicity Assay The mouse cell lines RMA(H-29, the TAP2 mutant RMA-S, and the Syrian hamster cell lines NIL-2 (embryo), and BHK-21 (kidney) were grown in EMEM supplemented with 10% FCS. The Syrian hamster cell lines were obtained from ATCC. Target cells were infected with flaviviruses 24 hr previously. For the cytotoxicity assays, cells were labeled with SICr for 1 hr and infected with W at a multiplicity of 10 pfu per cell for 3 hr. In double and triple superinfection experiments, target cells were infected at a multiplicity of 10 pfu for each virus simultaneously. Targetcellswere infectedwith 103 hemagglutinin units of influenza virus AMlSN per IO7 cells.

The assay methods used for cell line targets have been described in detailelsewhere(Miillbacheret al., 1991). Theduration oftheassays was 6 hr. Percentage specific lysis was calculated by the following formula: percent specific lysis = ([experimental release - medium release] I [maximum release - medium release]) x 100. Data given are the means of triplicate determinations. SEM values were always <5%.

Tc Cell Culturea For the generation of alloreactive Tc cells, 6 x IO7 responder splenc- cytes were cocultured with 4 x IO’ irradiated (2000 rads) allogeneic stimulator cells for 5 days. The generation of secondary WNV-, influenza-, or W-immune Tc cells has been described (Kesson et al., 1968; Milllbacher et al., 1991).

FACS Analysis Infection of RMA and RMA-S cells (5 x IO6 cells in 1 ml EMEM containing 10% FCS) with flaviviruses was at a multiplicity of 20 pfu per cell for 1 hr at 37OC. Cell surface staining was performed as described (Lobigs and MGlbacher, 1993) using the H-2Kb-specific MAb Hb176 (ATCC), H-2Db-specific MAb Hb27 (ATCC), rat anti-mouse CD2 MAb (Pharmingen, San Diego, California), or rat anti-mouse Th1.2 MAb (Olac, Bicester, England), and FlTCconjugated sheep anti-mouse im- munoglobulin (Serotec, Melbourne, Australia). Propidlum iodide was added to a final concentration of 1 ug/ml and surface fluorescence of life cells analyzed with a Beckton and Dickinson FACScan. Infection of BHK cells with WNV was at a multiplicity of 20 pfu per cell. At 24 hr after infection, the cells were superinfected with W-KL or W-HA at IO pfu per cell for 1 hr at 37OC, followed by a further incubation for 12 hr at 37OC. Staining and flow cytometry were as above using the H-2KL-specific MAb Hbl60 (ATCC). In the case of W-HA infection, the cells were incubated with polyclonal hyperimmune rabbit anti- hemagglutinin antiserum (agift of Dr. G. Lever, Cannberra, Australia), followed by biotinilated mouse anti-rabbit MAb (Immunotech, Mar- seille, France) followed by streptavidin-FITC (Pierce, Rockford, Illinois).

We would like to thank Dr. R. V. Blanden for many helpful discussions, Dr A. Gibbs for sequence data base search, R. Tha Hla for excellent technical assistance, and G. Osborne and S. Gruninger for help with FACS analysis.

Received January 5, 1995; revised June 22, 1995.

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