0022-538X/05/$08.00⫹0 doi:10.1128/JVI.79.11.7255–7261.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Heat Shock Protein 90 Expression in Epstein-Barr Virus-Infected B
Cells Promotes
␥␦
T-Cell Proliferation In Vitro
Maria Kotsiopriftis, Jerome E. Tanner, and Caroline Alfieri*
Sainte-Justine Hospital Research Center and Department of Microbiology and Immunology, Universite´ de Montre´al, Montre´al, Que´bec H3T 1C5, Canada
Received 12 July 2004/Accepted 20 January 2005
The aim of this study was to elucidate the in vitro response of␥␦T cells to Epstein-Barr virus (EBV)-infected
B cells and to determine whether EBV-induced heat shock proteins (HSPs) might serve as␥␦T-cell stimulants.
Cytofluorometric analysis revealed HSP90 cell surface expression in 12% of the EBV-immortalized B-cell population in all four of the B-cell lines tested. HSP27, HSP60, and HSP70 were not detected on the cell surface by cytofluorometry in these same B-cell lines. HSP90 and HSP60, but not HSP70 or HSP27, were detected on
the cell surface after125
I cell surface labeling and immunoprecipitation with anti-human HSP monoclonal
antibodies. In vitro kinetic studies indicated that␥␦T cells increased at least twofold by day 11 postinfection
in cultures of EBV-seronegative peripheral blood lymphocytes infected with EBV, whereas percentages of␣
T cells in these same cultures either decreased slightly or remained relatively unchanged in response to EBV infection. Addition of anti-human HSP90 monoclonal antibody to the EBV-infected lymphocyte cultures
inhibited␥␦ T-cell expansion by 92%. The inhibition of ␥␦T-cell expansion by anti-HSP90 antibody was
reversed upon treatment with exogenous HSP90. Taken together, these results indicate that HSP90 played an
important role in the stimulation of␥␦T cells during EBV infection of B cells in vitro and may serve as an
important immunomodulator of␥␦T cells during acute EBV infection.
Epstein-Barr virus (EBV) is the cause of heterophile anti-body-positive acute infectious mononucleosis (AIM) (28, 32). Work by Tosato et al. revealed that the initial immune re-sponse to EBV during AIM is in large part dependent on non-HLA-restricted suppressor T cells (54, 60). Later, during the resolution of infection, EBV antigen-specific, HLA-re-stricted cytotoxic T lymphocytes (CTLs) arise to generate long-term protective immunity (47). Mechanistically AIM-induced suppressor T cells and CTLs appear to function as two sepa-rate T-lymphocyte populations. The suppressor T cells act to limit the spread of EBV-infected B cells by inhibiting their activation and proliferation, whereas the HLA-restricted CTLs specifically recognize and lyse EBV latent and lytic antigen-bearing B cells (42, 60). The nature or control of the suppres-sor T cells which arise during acute EBV infection is unknown; however, previous investigators have found a two- to threefold increase in the number of␥␦T cells, with a large proportion bearing the V␥9V␦2 T-cell receptor (TCR) in AIM patients at the time of maximum suppression of EBV-infected B-cell growth (19, 22, 25, 44).
The T-cell repertoire in humans consists of cells bearing the
␣TCR (greater than 90%) or the␥␦TCR (less than 10%) (15). T cells bearing the manifold combinations of␣ TCRs are the “work horses” of the immune system, serving in both helper and cytotoxic capacities.␥␦T cells, on the other hand, exhibit more-limited TCR repertoires and appear to recognize and respond to phylogenetically conserved antigens, including
heat shock proteins (HSPs), alkylamines, and small nonprotein phosphate-containing compounds (10, 26, 62).
Members of the HSP family were first identified by their marked increase in cells submitted to a heat shock stimulus (63). Besides changes in temperature, other cell-stressing con-ditions that can elevate cellular HSPs include viral and bacte-rial infection, inflammation, trauma, or cell transformation. HSPs are highly conserved throughout phylogeny, with the various members ranging in molecular mass from 10 kDa to 100 kDa. The principal functions of HSPs encompass intracel-lular transport, protein folding, and protein oligomerization, as well as the elimination of protein aggregates or improperly folded proteins (63). Recently, several members of the HSP family were found expressed on the surfaces of cells and shown to stimulate immune effector cells directly or to participate in the shuttling of whole antigen molecules to professional anti-gen-presenting cells (APC) in what is now referred to as “an-tigen cross-priming” (50, 61). Peripheral blood B cells immor-talized in vitro by EBV have been shown to express elevated levels of HSP70 and HSP90 when assayed by immunoblotting (11). However, the question of whether these two HSPs or other HSP family members are expressed on the cell surfaces of acutely infected B cells and are capable of activating the immune system remains to be determined.
Since␥␦T cells increase in vivo during the acute stage of AIM and cell surface HSPs are known to stimulate␥␦T cells in other cell systems, we investigated whether B cells obtained from EBV-seronegative individuals and infected with exogenous EBV ex-press HSPs on their cell surfaces and whether autologous␥␦T cells respond to HSP. Positive findings for these two questions may suggest an explanation of how an acutely infected B cell signals its altered state to an EBV-naive immune system and initiates an acute immune response to EBV during AIM.
* Corresponding author. Mailing address: Sainte-Justine Hospital Research Center, 3175 Coˆte Sainte-Catherine Road, Montre´al (Que ´-bec), Canada H3T 1C5. Phone: (514) 345-4931, ext. 6135. Fax: (514) 345-4801. E-mail: [email protected].
7255
on November 8, 2019 by guest
http://jvi.asm.org/
Surface expression of HSPs in EBV-immortalized
lympho-blastoid cell lines (LCLs).HSP60, HSP70, and HSP90 have
been shown to be important in stimulating␥␦T cells in vitro and in vivo (20, 40, 53). HSP60 has also been found on the surface of EBV-infected Burkitt tumor cells (31). In order to determine whether HSPs are expressed on the surface of EBV-immortalized LCLs, we performed both flow cytometric anal-ysis and immunoprecipitation of125I-radiolabeled cell surface
proteins using anti-human HSP monoclonal antibodies and EBV-immortalized LCLs from four adult donors. As seen in a representative fluorescence-activated cell sorting (FACS) pro-file, donor LCL 1618 exhibited 16% cell surface immunofluo-rescence reactivity following anti-HSP90 monoclonal antibody (Stressgen, Inc., Victoria, BC, Canada) staining but was non-reactive (less than 2%) to HSP27, HSP60, and anti-HSP70 monoclonal antibodies (Stressgen, Inc.) (Fig. 1A). Analysis of three additional donor LCLs revealed that HSP90 surface immunopositivity was comparable to that for LCL 1618. LCLs were on average 11.5%⫾4 standard errors of the mean (SEM) immunopositive when stained with anti-HSP90 monoclonal antibody, while anti-HSP70 and anti-HSP60 monoclonal antibodies were undetectable and anti-HSP27 monoclonal antibody exhibited 0.8%⫾1 SEM positivity when assayed by flow cytometry, indicating that HSP90 cytofluores-cence was unlikely to be a cell line-specific phenomenon.
In order to substantiate our HSP cytofluorometric findings, LCL 1618 cells were surface radiolabeled with Na125I and
HSPs immunoprecipitated with our series of HSP-specific monoclonal antibodies. Briefly, 107 logarithmically growing
LCL cells were banded on Ficoll-hypaque to remove dead cells and debris, washed with phosphate-buffered saline (PBS), and resuspended in 300 l PBS for iodogen-mediated (Pierce Chemicals, Rockford, IL)125I surface radiolabeling (500Ci
Na125I/ml, 50 mCi125I/mmol, New England Nuclear,
Missis-sauga, ON) (24). After a 15-min incubation at room temper-ature, cells were washed three times with PBS containing 0.25 mM NaI and lysed with 1 ml lysis buffer (50 mM Tris [pH 8.0], 0.5% NP-40, 140 mM NaCl, 5 mM EDTA, 5 mM phenylmeth-ylsulfonyl fluoride). Cell lysates were centrifuged at 4°C for 10 min at 30,000⫻gto remove cell debris and nuclei and incu-bated on ice for 30 min with 5g each of anti-human HSP27, anti-human HSP60, anti-human HSP70, or anti-human HSP90 monoclonal antibody. As a control to measure unintended125I
labeling of internal cell proteins and to identify proteins which may nonspecifically bind to our immunoprecipitate complex, we utilized the isotype-matched monoclonal antibody S-12, which recognizes an intracellular carboxy-terminal epitope in the 63-kDa EBV latent membrane protein 1 (LMP-1) (36, 37). LMP-1 does not have a significant extracellular protein se-quence, nor is LMP-1 predicted to expose an external tyrosine residue for potential125I labeling. As shown in Fig. 1B,
anti-HSP60 and anti-HSP90 antibodies were able to immunopre-cipitate125I-radiolabeled bands that migrated at 60 kDa and 90
kDa, respectively. No bands for the predicted molecular mass of HSP27 or HSP70 were observed when125I-radiolabeled cell
protein lysates were incubated with anti-human HSP27 or HSP70 antibodies (Fig. 1B). Other bands detected by autog-raphy were considered nonspecific, since these resulted from the immunoprecipitation process and appeared in all gel lanes irrespective of the antibody used. In reference to the
anti-LMP-1 monoclonal antibody, S-12, it was noteworthy that no band was observed migrating at 63 kDa (Fig. 1B), thus provid-ing assurance that the HSP bands detected followprovid-ing125I
sur-face labeling and immunoprecipitation were present originally on the cell surface and not the result of an inadvertent labeling of internal HSPs during experimental manipulation. The in-ability to detect cell surface expression of HSP70 was not due to a lack of expression in LCL 1618, since HSP70 along with HSP60 and HSP90 were easily detected when LCL 1618 pro-tein lysates were resolved by sodium dodecyl sulfate-polyacryl-amide gel electrophoresis and immunoblotted with our series of anti-human HSP antibodies (Fig. 1C). Although no HSP27 was detected by FACS, immunoprecipitation, and immuno-blotting, the antibody used was reported to recognize HSP27 by immunoblotting (Stressgen, Inc.), which suggests that HSP27 was either not expressed or was expressed at a level below the detection limits of the assay.
Induction of␥␦T cells following acute EBV infection and
their suppression following treatment with anti-HSP90
mono-clonal antibody.␥␦T cells have been shown to increase during
the acute phase of EBV infectious mononucleosis (19, 25). In order to determine whether EBV was capable of stimulating
␥␦ T cells in vitro, we measured the change in ␥␦ T cell numbers over time following acute EBV infection of periph-eral blood mononuclear cells (PBMCs). Briefly, PBMCs from EBV-seronegative donors were seeded at 5 ⫻ 105 cells/ml,
infected with a 200-fold-concentrated preparation of EBV (strain B95-8, CCL 1621; American Type Culture Collection, Rockville, MD) or mock infected (52). PBMC aliquots were taken just prior to virus exposure (day 0) and on days 3, 7, 11, and 15 postinfection for staining with fluorescein isothiocya-nate (FITC)-conjugated monoclonal antibodies specific for CD3 (pan T cell),␣TCR, or␥␦TCR (Pharmingen Canada, Inc., Mississauga, ON). As shown in a representative experi-ment, depicted in Fig. 2A,␥␦T cells increased from an initial 4.8% on day 3 postinfection to a peak of 10.2% on day 11 and remained elevated throughout the remainder of the experi-ment. Analysis of␣T cells indicated no significant change in their percentage of the T-cell population compared to mock-infected cells (Fig. 2B). Analysis of three additional EBV-seronegative blood donors revealed the same trend, with a maximum␥␦T-cell increase of 15.1%⫾4.3 SEM on days 11 to 15 (Fig. 2C) and less than a 4% change in the EBV-treated
␣T-cell population compared to mock-treated PBMCs (Fig. 2C). Mock-treated PBMCs also failed to show an increase in
␥␦T cells during the 15-day time course (Fig. 2C).
Kaur et al. (1993) have shown that the Burkitt lymphoma cell line Daudi expressed HSP60 on its cell surface, which in turn was capable of stimulating␥␦T-cell clones to proliferate (31). By contrast, when antibodies specific to HSP60 were added to cocultures consisting of V␥9V␦2 anti-Daudi T-cell clones, they observed an abrogation of␥␦T-cell proliferation (31). To test whether the expression of␥␦T cells seen in vitro on days 11 through 15 was in response to B-cell surface HSP, EBV-infected PBMC cultures were treated, starting on day 8 and subsequently on days 10 and 13, with a monoclonal anti-body cocktail (5g of each anti-HSP antibody/ml) composed of anti-human HSP27, HSP60, HSP70, and HSP90 or with an equal amount of control monoclonal antibody S-12. Day 8 was selected as the first day of treatment, since it was a point of
on November 8, 2019 by guest
http://jvi.asm.org/
time noted as being just prior to the onset of␥␦T-cell increase in our cultures (Fig. 2A). The percentage of␥␦TCR-positive cells was assessed on days 9, 11, and 14 by flow cytometry. As shown in Fig. 3A, addition of all four human HSP monoclonal anti-bodies resulted in an 86% reduction in the level of␥␦T cells (P
⫽0.01) compared with results with an equal amount of anti-S12
antibody. The inhibition of␥␦T cells by the anti-human HSP monoclonal antibody cocktail appeared not to be due to nonspe-cific cell toxicity. Cell viability in both the human HSP anti-body-treated and anti-S12 antianti-body-treated cultures remained at more than 70% when tested by trypan-blue exclusion throughout the course of the experiments.
FIG. 1. Expression of cell surface HSPs in EBV-immortalized B cells. (A) Representative FACS analysis of HSP cell surface expression for LCL 1618. Cells were incubated at 4°C for 45 min in PBS containing 2% fetal bovine serum and 0.1% sodium azide with 20g/ml of individual anti-human HSP monoclonal antibody or the isotype-matched anti-keyhole limpet hemocyanin monoclonal antibody. Cells were then stained with FITC-conjugated goat anti-mouse antiserum (Jackson ImmunoResearch, BioCan Scientific, Misssissauga, ON), fixed with 2% paraformaldehyde (pH 7.4), and analyzed for fluorescence using a FACScan cytofluorometer (Becton-Dickinson Immunocytometry Systems, Inc., Mountain View, CA). Cells treated with anti-keyhole limpet hemocyanin monoclonal antibody and stained with FITC-conjugated goat antimouse antiserum are indicated by the dashed-line histogram and the M1 bar. The percentage of stained cells residing beyond the M1 region (i.e., M2) is indicated. (B) Autography of125I-surface-labeled proteins immunoprecipitated with anti-LMP-1 (LMP-1) or anti-HSP monoclonal antibodies and resolved in a 12% sodium dodecyl sulfate-polyacrylamide gel. (C) Immunoblots of HSP expression in LCL 1618 at times zero (T0), 3 h after heat treatment (T8), and 24 h after the addition of 20-ng/ml phorbol 12-myristate 13 acetate (PMA).
on November 8, 2019 by guest
http://jvi.asm.org/
In order to determine which of the four HSPs was respon-sible for reducing␥␦T-cell expansion, anti-human HSP mono-clonal antibodies were added individually to separate EBV-infected PBMC cultures. As shown in Fig. 3B, addition of anti-HSP90 to PBMCs resulted in a 91.5% reduction in the percentage of ␥␦ TCR-positive cells (P ⫽ 0.03). The three other anti-human HSP monoclonal antibody preparations also caused some decline in the ␥␦ T-cell stimulation following EBV infection, but their effect was not as dramatic as that seen with anti-HSP90 (28% inhibition by anti-HSP60 and less than 5% inhibition for anti-HSP70 and anti-HSP27 antibodies) (Fig. 3B).
In order to verify that our observed decrease in the percent-age of␥␦TCR-positive cells was due to the direct interaction of the HSP90 antibody and cellular HSP90 and not due to the activation of an alternative mechanism or the presence of an unknown component within our antibody preparations, we performed a set of competition assays in which purified HSP90 (25g/ml; Sigma-Aldrich, Oakville, ON.) was incubated for 1 h with the anti-HSP90 or anti-LMP1 antibody prior to its addition to our EBV-infected PBMC cell cultures on days 8, 10, and 13. Results indicated that the preincubation of HSP90 with anti-HSP90 antibody increased the percentage of ␥␦ TCR-positive cells from 3% (Fig. 3C, EBV⫹HSP90 Ab) to 17% (Fig. 3C, EBV⫹HSP90 Ab⫹HSP90) on day 14 postin-fection. This compared to cells treated with EBV, EBV plus LMP1 antibody, or EBV plus LMP1 antibody plus HSP90, which exhibited 20%, 17%, and 20.5%␥␦TCR-positive cells, respectively, on day 14 postinfection (Fig. 3C). Inclusion of HSP90 reversed an inhibition of 82% (Fig. 3C) (P⫽0.02; EBV versus EBV plus HSP90 antibody) to only 17% (Fig. 3C) (P⫽
0.001; EBV plus HSP90 antibody versus EBV plus HSP90 antibody plus HSP90). These results indicated that the
anti-HSP90 antibody binding to exogenous anti-HSP90 directly blocked the increase of␥␦TCR-positive cells in our cultures. Future studies will need to be performed to determine whether the inhibition displayed with the anti-HSP90 antibody was due to its site of action on EBV-infected B cells, antigen-presenting cells, including precursor dendritic cells and monocytes, or dying cells which released intracellular HSP90 (59). However, these results, coupled with the data obtained for surface ex-pression of HSPs in EBV-infected B cells (Fig. 1A and B), suggest that expression of HSP90 on the surface of EBV-immortalized LCLs can stimulate␥␦T-cell proliferation.
Although the immunoprotective mechanisms operating dur-ing the early stages of acute EBV infection are likely to be multifactoral (35), it is known that the initial immune protec-tive response is partially dependent upon large numbers of non-antigen-specific, non-HLA-restricted CTLs that are thought to control EBV spread through growth suppression of the infected B cell (54, 60). The rise of these suppressor T cells occurs concomitantly with a two- to threefold increase in␥␦ T-cell numbers (19). In vitro EBV immunology studies docu-mented that maximum regression of EBV-infected B cells in PBMC cultures occurred between days 11 and 14 postinfection with a concomitant rise in␥␦T cells (44, 54). This was not due to resident EBV-specific memory T cells, since the PBMCs used in those studies were derived from EBV-seronegative donors. Given the nonspecific nature of the in vitro suppres-sion and a large-scale expansuppres-sion of␥␦T cells following EBV exposure, one could postulate that the␥␦T cells were respond-ing to a broadly stimulative factor present durrespond-ing AIM and also found following EBV infection of B cells in the in vitro cell cultures. Our results indicate that HSP90 and HSP60 were expressed on the surface of EBV-immortalized B cells (Fig. 1) and that the addition of anti-HSP90 antibody effectively
re-0 5 10 15
0 3 7 11 15
Day %γδ
P
osit
ive
0 25 50 75 100
0 3 7 11 15
Day
%
Po
si
tiv
e
γδ T cell αβ T cell 0
10 20 30 40 50 60 70 80 90
MK EBV MK EBV
%
P
osi
tive
A
B
[image:4.585.136.454.70.281.2]C
FIG. 2. Expansion of␥␦T cells following acute EBV infection or mock infection of PBMCs. PBMCs from healthy EBV-seronegative donors were infected with EBV (filled symbol) or mock infected (open symbol) and cultured for 15 days. On days 3, 7, 11, and 15, cell aliquots were immunostained with (A) FITC-conjugated anti-␥␦ TCR monoclonal antibody or (B) FITC-conjugated anti-␣ TCR monoclonal antibody (Becton-Dickinson, Bedford, MA) and analyzed by FACS. (C) Represents the percentage of␥␦- or␣-TCR positive cells⫾SEM for three independent experiments wherein cells were analyzed on days 11 to 15 to measure the maximum percent increase.
on November 8, 2019 by guest
http://jvi.asm.org/
versed the observed increase in the␥␦ T-cell population fol-lowing EBV exposure by days 11 to 14 postinfection (Fig. 3B). Further, we could reverse the effects of anti-HSP90 antibody by addition of exogenous HSP (Fig. 3C). Thus, our in vitro results confirm and extend earlier studies by showing that an
increase in in vitro␥␦T-cell proliferation may be due in large part to a response to HSP90. Further work will be needed to determine whether the expanded␥␦T-cell population directly or indirectly suppresses the growth of EBV-infected B cells by serving as cytolytic T cells or provides some assistance in the generation of suppressor T cells (30).
Much like their␣CD4⫹and CD8⫹T-cell brethren,␥␦T cells exhibit both helper and cytotoxic functions in vitro (1); however, the role of these cells in in vivo immunity is still unclear. It has been suggested that␥␦T cells act as both early sentinels of the immune system by providing immediate pro-tection during the acute phase of infection and as bridging elements between the innate immune system and the adaptive immune arm to promote the emergence of long-term antigen-specific, HLA-restricted T cells (7, 16, 17, 23, 49). The nature of the signaling molecule(s) which initiates the innate or adap-tive immune response in␥␦T cells will vary depending upon the individual pathogen but can include conventional protein antigens or phylogenetically conserved molecules, such as the HSPs (7, 10, 16, 21, 26, 49, 62). In the experiments reported here, it is likely that HSP90 signaled␥␦ T cells through the HSP receptor molecule CD91 (2, 6) in conjunction with mem-bers of the scavenger receptor family (18, 46) and the Toll-like receptor family (56–58). Support for this comes from the find-ing that␥␦T cells express members of the Toll-like receptor family (38) and the observation that ␥␦ T cells respond to HSP60 in Behcet’s disease by the generation of suppressor and effector cells (20). Interestingly, the literature appears to indi-cate that ␣ T cells do not express members of the HSP receptor family (6), which may explain in part why the per-centage of␣ T cells did not change significantly during the course of the 14-day EBV infection. We also recognize that the PBMC population from our seronegative blood donors should have no␣memory T cells that would respond to the various EBV antigens, and this may also explain why we failed to observe a significant stimulatory response by this set of T cells (15).
[image:5.585.44.283.66.399.2]Although antigens that elicit classic CTL responses during the convalescence phase of infectious mononucleosis are prin-cipally comprised of the EBV nuclear antigen 3 (EBNA-3) and several lytic-cycle antigens (43), one group of investigators has observed a very significant T-cell response to the Gly:Ala re-peat domain of EBNA-1 (8). This finding was quite surprising, since the EBNA-1 domain in question is well noted for its ability to block conventional antigen processing and is thereby thought to promote the escape of B cells harboring latent EBV from immune recognition (34). Subsequently, this same group of investigators found that CTL recognition of EBNA-1 oc-curred after internalization of exogenous EBNA-1 through a process termed cross-priming (7). Through use of this biolog-ical process, exogenous EBNA-1 would be captured initially by professional antigen-presenting cells (APC), which in turn rep-resent EBNA-1 to CD8⫹T cells via the HLA class I pathway (27). HSPs play a crucial role in antigen cross-priming by acting both as shuttle molecules for exogenous antigen and as direct stimulants of T cells by prompting APC cytokine secre-tion (50). Based upon several in vitro model systems which measured the response of memory T cells to ovalbumin pep-tide or tumor-related peppep-tides, it would appear that HSPs are capable of presenting peptide epitopes during lytic infection
FIG. 3. Expansion of␥␦T cells in the presence of EBV and anti-HSP monoclonal antibodies. (A) PBMCs from healthy EBV-seroneg-ative blood donors were mock infected (MK) or infected with EBV and cultured in the presence of anti-LMP-1 monoclonal antibody (LMP1 Ab) or a cocktail of anti-HSP monoclonal antibodies (HSP Abs). Mock-infected cells shown here were cultured in the presence of HSP monoclonal antibodies to monitor PBMC response to anti-HSP monoclonal antibodies. (B) PBMCs from healthy EBV-seroneg-ative blood donors were mock infected or cultured in the presence of EBV and the addition anti-LMP-1 monoclonal antibody or individual anti-HSP monoclonal antibodies. Cells were stained with FITC-conju-gated anti-␥␦ TCR monoclonal antibody and analyzed by FACS. (C) PBMCs from healthy EBV-seronegative blood donors were mock infected (MK) or cultured in the presence of EBV and anti-LMP-1 monoclonal antibody or anti-HSP90 monoclonal antibody. In parallel cultures, PBMCs infected with EBV were grown in the presence of anti-LMP1 or anti-HSP90 antibodies preincubated with exogenous HSP90. Cells were stained with FITC-conjugated anti-␥␦ TCR or anti-keyhole limpet hemocyanin antibody and analyzed by FACS. Pos-itive percentages⫾SEM were obtained using 10,000 FACS events from three independent experiments following subtraction of anti-keyhole limpet hemocyanin antibody reactivity. The asterisk indicates
Pvalues ofⱕ0.05 using Student’sttest for EBV cultures versus EBV cultures and the presence of anti-HSP antibody.⫹indicatesPvalues ofⱕ0.05 using Student’sttest for EBV cultures containing anti-HSP90 antibody versus EBV cultures containing anti-HSP90 antibody and treated with exogenous HSP90.
on November 8, 2019 by guest
http://jvi.asm.org/
through the mechanism of antigen cross-priming (4, 39, 48), which results in a strong proliferative and effector response in these T cells. Further, it was noted in these various studies that the peptides must be bound to HSPs for maximal response by the memory T cells. From our in vitro finding that anti-HSP90 antibody abrogated ␥␦ T-cell proliferation in EBV-infected cultures and the knowledge that HSP90 is a principal chaper-one involved in antigen cross-priming, proinflammatory re-sponses, and cell targeting by both NK and␥␦T cells in vitro and in vivo (5, 13, 29, 33, 40, 45, 50, 51), we hypothesized that HSP90 may also be involved in cross-priming of EBV antigens, particularly EBNA-1 (12, 64). The finding that anti-HSP90 blocked the EBV-induced increase in␥␦T cells suggests that HSP90 is important in the stimulation of␥␦T cells and serves as an immune sentinel trigger during acute virus infection or as an aid in the generation of EBV-specific T cells during AIM convalescence (3). This conclusion follows directly from data suggesting that HSPs were expressed on the surfaces of EBV-infected tumors and other tumor types and were found to augment APC maturation and growth factor expression (9, 14, 21, 41, 50, 55).
The resolution of the viral infection and the arrest of tumor cell growth are two important functions of the immune system. Our identification of intercellular signals and T-cell responses which occur after in vitro EBV infection will hopefully lead to a greater understanding of how␥␦T cells respond and possibly limit the spread of virally transformed cells.
This work was supported by an award from the J. A. DeSe`ve Foun-dation and a grant from CAFIR to C.A. and through a grant from the Canadian Foundation for AIDS Research (CANFAR) to J.T. M.K. has been supported by Pediacom Canada.
We are grateful to Marie Blagdon for the FACS analysis.
REFERENCES
1.Aljurf, M., A. Ezzat, and O. Musa.2002. Emerging role of gamma delta T-cells in health and disease. Blood Rev.16:203–206.
2.Basu, S., R. J. Binder, T. Ramalingam, and P. K. Srivastava.2001. CD91 is a common receptor for heat shock proteins gp96, hsp90, hsp70, and calre-ticulin. Immunity14:303–313.
3.Basu, S., and P. K. Srivastava.2000. Heat shock proteins: the fountainhead of innate and adaptive immune responses. Cell Stress Chaperones5:443– 451.
4.Berwin, B., R. C. Reed, and C. V. Nicchitta.2001. Virally induced lytic cell death elicits the release of immunogenic GRP94/gp96. J. Biol. Chem.276:
21083–21088.
5.Binder, R. J., K. M. Anderson, S. Basu, and P. K. Srivastava.2000. Cutting edge: heat shock protein gp96 induces maturation and migration of CD11c⫹
cells in vivo. J. Immunol.165:6029–6035.
6.Binder, R. J., R. Vatner, and P. Srivastava.2004. The heat-shock protein receptors: some answers and more questions. Tissue Antigens64:442–451. 7.Blake, N., T. Haigh, G. Shaka’a, D. Croom-Carter, and A. Rickinson.2000.
The importance of exogenous antigen in priming the human CD8⫹T cell response: lessons from the EBV nuclear antigen EBNA1. J. Immunol.165:
7078–7087.
8.Blake, N., S. Lee, I. Redchenko, W. Thomas, N. Steven, A. Leese, P. Steiger-wald-Mullen, M. G. Kurilla, L. Frappier, and A. Rickinson.1997. Human CD8⫹T cell responses to EBV EBNA1: HLA class I presentation of the (Gly-Ala)-containing protein requires exogenous processing. Immunity
7:791–802.
9.Botzler, C., R. Issels, and G. Multhoff.1996. Heat-shock protein 72 cell-surface expression on human lung carcinoma cells is associated with an increased sensitivity to lysis mediated by adherent natural killer cells. Cancer Immunol. Immunother.43:226–230.
10.Bukowski, J. F., C. T. Morita, and M. B. Brenner.1999. Human gamma delta T cells recognize alkylamines derived from microbes, edible plants, and tea: implications for innate immunity. Immunity11:57–65.
11.Cheung, R. K., and H. M. Dosch.1993. The growth transformation of human B cells involves superinduction of hsp70 and hsp90. Virology193:700–708. 12.Crotzer, V. L., R. E. Christian, J. M. Brooks, J. Shabanowitz, R. E. Settlage,
J. A. Marto, F. M. White, A. B. Rickinson, D. F. Hunt, and V. H. Engelhard.
2000. Immunodominance among EBV-derived epitopes restricted by HLA-B27 does not correlate with epitope abundance in EBV-transformed B-lymphoblastoid cell lines. J. Immunol.164:6120–6129.
13.Csermely, P., T. Schnaider, C. Soti, Z. Prohaszka, and G. Nardai.1998. The 90-kDa molecular chaperone family: structure, function, and clinical appli-cations. A comprehensive review. Pharmacol. Ther.79:129–168.
14.Dai, J., B. Liu, M. M. Caudill, H. Zheng, Y. Qiao, E. R. Podack, and Z. Li.
2003. Cell surface expression of heat shock protein gp96 enhances cross-presentation of cellular antigens and the generation of tumor-specific T cell memory. Cancer Immun.3:1.
15.Davis, M. M., and Y.-H. Chien.1999. T-cell antigen receptors, p. 341–366.In W. E. Paul (ed.), Fundamental immunology. Lippincott-Raven, Philadel-phia, Pa.
16.Dechanet, J., P. Merville, V. Pitard, X. Lafarge, and J. F. Moreau.1999. Human gammadelta T cells and viruses. Microbes Infect.1:213–217. 17.De Libero, G.1997. Sentinel function of broadly reactive human gamma
delta T cells. Immunol. Today18:22–26.
18.Delneste, Y., G. Magistrelli, J. Gauchat, J. Haeuw, J. Aubry, K. Nakamura, N. Kawakami-Honda, L. Goetsch, T. Sawamura, J. Bonnefoy, and P. Jean-nin.2002. Involvement of LOX-1 in dendritic cell-mediated antigen cross-presentation. Immunity17:353–362.
19.De Paoli, P., D. Gennari, P. Martelli, V. Cavarzerani, R. Comoretto, and G. Santini.1990. Gamma delta T cell receptor-bearing lymphocytes during Epstein-Barr virus infection. J. Infect. Dis.161:1013–1016.
20.Direskeneli, H., and G. Saruhan-Direskeneli.2003. The role of heat shock proteins in Behcet’s disease. Clin. Exp. Rheumatol.21:S44–S48.
21.Fisch, P., M. Malkovsky, S. Kovats, E. Sturm, E. Braakman, B. S. Klein, S. D. Voss, L. W. Morrissey, R. DeMars, W. J. Welch, R. L. H. Boluhis, and P. M. Sondel.1990. Recognition by human V gamma 9/V delta 2 T cells of a GroEL homolog on Daudi Burkitt’s lymphoma cells. Science250:1269– 1273.
22.Fisch, P., K. Oettel, N. Fudim, J. E. Surfus, M. Malkovsky, and P. M. Sondel.
1992. MHC-unrestricted cytotoxic and proliferative responses of two distinct human gamma/delta T cell subsets to Daudi cells. J. Immunol.148:2315– 2323.
23.Gao, Y., W. Yang, M. Pan, E. Scully, M. Girardi, L. H. Augenlicht, J. Craft, and Z. Yin.2003. Gamma delta T cells provide an early source of interferon gamma in tumor immunity. J. Exp. Med.198:433–442.
24.Goding, J. W.1996. Analysis of antigens, p. 234–326. Monoclonal antibodies: principles and practice. Academic Press, New York, N.Y.
25.Hassan, J., C. Feighery, B. Bresnihan, and A. Whelan.1991. Elevated T cell receptor gamma delta⫹T cells in patients with infectious mononucleosis. Br. J. Haematol.77:255–256.
26.Hayday, A. C.2000. Gamma delta cells: a right time and a right place for a conserved third way of protection. Annu. Rev. Immunol.18:975–1026. 27.Heath, W. R., and F. R. Carbone.1999. Cytotoxic T lymphocyte activation by
cross-priming. Curr. Opin. Immunol.11:314–318.
28.Henle, W., and G. Henle.1973. Epstein-Barr virus and infectious mononu-cleosis. N. Engl. J. Med.288:263–264.
29.Hoos, A., and D. L. Levey.2003. Vaccination with heat shock protein-peptide complexes: from basic science to clinical applications. Expert Rev. Vaccines
2:369–379.
30.Kabelitz, D.1999. Effector functions and control of human gamma delta T-cell activation. Microbes Infect.1:255–261.
31.Kaur, I., S. D. Voss, R. S. Gupta, K. Schell, P. Fisch, and P. M. Sondel.1993. Human peripheral gamma delta T cells recognize hsp60 molecules on Daudi Burkitt’s lymphoma cells. J. Immunol.150:2046–2055.
32.Kawa, K.2000. Epstein-Barr virus-associated diseases in humans. Int. J. He-matol.71:108–117.
33.Lehner, T., L. A. Bergmeier, Y. Wang, L. Tao, M. Sing, R. Spallek, and R. van der Zee.2000. Heat shock proteins generate beta-chemokines which function as innate adjuvants enhancing adaptive immunity. Eur. J. Immunol.
30:594–603.
34.Levitskaya, J., M. Coram, V. Levitsky, S. Imreh, P. M. Steigerwald-Mullen, G. Klein, M. G. Kurilla, and M. G. Masucci.1995. Inhibition of antigen processing by the internal repeat region of the Epstein-Barr virus nuclear antigen-1. Nature375:685–688.
35.Levitsky, V., and M. G. Masucci.2002. Manipulation of immune responses by Epstein-Barr virus. Virus Res.88:71–86.
36.Liebowitz, D., D. Wang, and E. Kieff.1986. Orientation and patching of the latent infection membrane protein encoded by Epstein-Barr virus. J. Virol.
58:233–237.
37.Mann, K. P., D. Staunton, and D. A. Thorley-Lawson.1985. Epstein-Barr virus-encoded protein found in plasma membranes of transformed cells. J. Virol.55:710–720.
38.Mokuno, Y., T. Matsuguchi, M. Takano, H. Nishimura, J. Washizu, T. Ogawa, O. Takeuchi, S. Akira, Y. Nimura, and Y. Yoshikai.2000. Expression of toll-like receptor 2 on gamma delta T cells bearing invariant V gamma 6/V delta 1 induced by Escherichia coli infection in mice. J. Immunol.165:931– 940.
39.More, S. H., M. Breloer, and A. von Bonin.2001. Eukaryotic heat shock
on November 8, 2019 by guest
http://jvi.asm.org/
proteins as molecular links in innate and adaptive immune responses: Hsp60-mediated activation of cytotoxic T cells. Int. Immunol.13:1121–1127. 40.Moseley, P.2000. Stress proteins and the immune response.
Immunophar-macology48:299–302.
41.Multhoff, G., C. Botzler, M. Wiesnet, E. Muller, T. Meier, W. Wilmanns, and R. D. Issels.1995. A stress-inducible 72-kDa heat-shock protein (HSP72) is expressed on the surface of human tumor cells, but not on normal cells. Int. J. Cancer61:272–279.
42.Murray, R. J., M. G. Kurilla, J. M. Brooks, W. A. Thomas, M. Rowe, E. Kieff, and A. B. Rickinson.1992. Identification of target antigens for the human cytotoxic T cell response to Epstein-Barr virus (EBV): implications for the immune control of EBV-positive malignancies. J. Exp. Med.176:157–168. 43.Ohga, S., A. Nomura, H. Takada, and T. Hara.2002. Immunological aspects
of Epstein-Barr virus infection. Crit. Rev. Oncol. Hematol.44:203–215. 44.Orsini, D. L., P. C. Res, J. M. Van Laar, L. M. Muller, A. E. Soprano, Y. M.
Kooy, P. P. Tak, and F. Koning.1993. A subset of V delta 1⫹T cells proliferates in response to Epstein-Barr virus-transformed B cell lines in vitro. Scand. J. Immunol.38:335–340.
45.Panjwani, N. N., L. Popova, and P. K. Srivastava.2002. Heat shock proteins gp96 and hsp70 activate the release of nitric oxide by APCs. J. Immunol.
168:2997–3003.
46.Peiser, L., S. Mukhopadhyay, and S. Gordon.2002. Scavenger receptors in innate immunity. Curr. Opin. Immunol.14:123–128.
47.Rickinson, A. B., M. F. Callan, and N. E. Annels.2000. T-cell memory: lessons from Epstein-Barr virus infection in man. Philos. Trans. R. Soc. Lond. B. Biol. Sci.355:391–400.
48.Rivoltini, L., C. Castelli, M. Carrabba, V. Mazzaferro, L. Pilla, V. Huber, J. Coppa, G. Gallino, C. Scheibenbogen, P. Squarcina, A. Cova, R. Camerini, J. J. Lewis, P. K. Srivastava, and G. Parmiani.2003. Human tumor-derived heat shock protein 96 mediates in vitro activation and in vivo expansion of melanoma- and colon carcinoma-specific T cells. J. Immunol.171:3467– 3474.
49.Sciammas, R., and J. A. Bluestone.1999. TCR gamma delta cells and viruses. Microbes Infect.1:203–212.
50.Srivastava, P.2002. Interaction of heat shock proteins with peptides and antigen presenting cells: chaperoning of the innate and adaptive immune responses. Annu. Rev. Immunol.20:395–425.
51.Suto, R., and P. K. Srivastava.1995. A mechanism for the specific
immu-nogenicity of heat shock protein-chaperoned peptides. Science269:1585– 1588.
52.Tanner, J. E., and C. Alfieri.1999. Epstein-Barr virus induces Fas (CD95) in T cells and Fas ligand in B cells leading to T-cell apoptosis. Blood94:3439– 3447.
53.Todryk, S. M., M. J. Gough, and A. G. Pockley.2003. Facets of heat shock protein 70 show immunotherapeutic potential. Immunology110:1–9. 54.Tosato, G., and R. M. Blaese.1985. Epstein-Barr virus infection and
immu-noregulation in man. Adv. Immunol.37:99–149.
55.Trieb, K., B. Grubeck-Loebenstein, T. Eberl, and R. Margreiter.1996. T cells from rejected human kidney allografts respond to heat shock protein 72. Transpl. Immunol.4:43–45.
56.Vabulas, R. M., P. Ahmad-Nejad, C. Da Costa, T. Miethke, C. J. Kirschning, H. Hacker, and H. Wagner.2001. Endocytosed HSP60s use toll-like receptor 2 (TLR2) and TLR4 to activate the toll/interleukin-1 receptor signaling pathway in innate immune cells. J. Biol. Chem.276:31332–31339. 57.Vabulas, R. M., P. Ahmad-Nejad, S. Ghose, C. J. Kirschning, R. D. Issels,
and H. Wagner.2002. HSP70 as endogenous stimulus of the Toll/interleu-kin-1 receptor signal pathway. J. Biol. Chem.277:15107–15112.
58.Vabulas, R. M., H. Wagner, and H. Schild.2002. Heat shock proteins as ligands of toll-like receptors. Curr. Top. Microbiol. Immunol.270:169–184. 59.Wallin, R. P., A. Lundqvist, S. H. More, A. von Bonin, R. Kiessling, and H. G. Ljunggren. 2002. Heat-shock proteins as activators of the innate immune system. Trends Immunol.23:130–135.
60.Wang, F., R. M. Blaese, K. C. Zoon, and G. Tosato.1987. Suppressor T cell clones from patients with acute Epstein-Barr virus-induced infectious mono-nucleosis. J. Clin. Investig.79:7–14.
61.Wells, A. D., and M. Malkovsky.2000. Heat shock proteins, tumor immu-nogenicity and antigen presentation: an integrated view. Immunol. Today
21:129–132.
62.Wilson, I. A., and R. L. Stanfield.2001. Unraveling the mysteries of gamma delta T cell recognition. Nat. Immunol.2:579–581.
63.Young, J. C., J. M. Barral, and H. F. Ulrich. 2003. More than folding: localized functions of cytosolic chaperones. Trends Biochem. Sci.28:541– 547.
64.Zheng, H., J. Dai, D. Stoilova, and Z. Li.2001. Cell surface targeting of heat shock protein gp96 induces dendritic cell maturation and antitumor immu-nity. J. Immunol.167:6731–6735.
